EP1763537A2 - Peptides for inhibiting the interaction of protein kinase a and protein kinase a anchor proteins - Google Patents

Abstract

The invention relates to a nucleic acid sequence that codes for peptides which inhibit the interaction of protein kinase A (PKA) and protein kinase A-anchor proteins (AKAP), a host organism, which has the nucleic acid sequence and, optionally, expresses the peptides. The invention also relates to the use of the peptides and of the host organism when studying diseases, which are associated with the AKAP PKA interaction, and to the use of the peptides as a pharmaceutical agent for treating diseases of this type.

Description

Peptides for inhibiting the interaction of protein kinase A and protein kinase A anchor proteins

The invention relates to nucleic acid sequences which encode peptides which inhibit the interaction of protein kinase A (PKA) and protein kinase ^'A anchor proteins (AKAPs), a host organism which comprises the nucleic acid sequence and peptides of the invention expressed as well as the use of the peptides as well as the host organism in the therapy and experimental study of diseases associated with a modified AKAP-PKA interaction and the use of the peptides as a pharmaceutical agent for the treatment of such diseases, in particular diabetes insipidus, duodenal ulcer, hypertension and diabetes mellitus.

The biological action of hormones and neurotransmitters is mediated through the activation of signaling cascades that alter the phosphorylation status of effector proteins. At this reversible process are two classes of ^enzymes' involved: protein kinases and phosphoprotein phosphatases. The phosphorylation is carried out by kinases which catalyze the transfer of the terminal phosphate group of ATP to specific serine or threonine residues; the dephosphorylation is mediated by phosphoprotein phosphatases. One mechanism for controlling and regulating these enzyme activities is to compartmentalize these enzymes by association with anchor proteins located near their substrates. Protein kinase A (PKA) is one of the multifunctional kinases with a broad substrate specificity, which is characterized by the so-called protein kinase anchoring proteins (AKAPs) is anchored to subcellular structures.

In many important cellular processes such as contraction, secretion, metabolism, gene transcription, cell growth and proliferation extracellular signaling via G-protein coupled receptors, G protein G _s , activation of adenylyl cyclase and formation of secondmessenger cyclic adenosine monophosphate (cAMP) occur , The effects of cAMP are mediated by the cAMP-dependent PKA.

The protein kinase A (PKA) -holoenzyme consists of a dimer of regulatory (R) subunits, to each of which a catalytic (C) subunit is bound. Activation of the kinase by the binding of two molecules of cAMP to each R subunit induces the dissociation of the C subunits that phosphorylate the nearby substrates. According to the presence of type I (RI) or type II (RII) regulatory subunits, the PKA holoenzyme is referred to as type I or type II PKA. Rice and RI / 3 exist in the RI subunits, in the RII subunits RIIa and RII /? and in the C subunits Coi, Cβ and Cγ. The different PKA subunits are encoded by different genes (Klussmann, 2004, Tasken and Aandahl, 2004).

The regulatory subunits ^• show a different expression patterns. While RIa and RIIa are ubiquitous in the tissues, the regulatory subunit RI is primarily found in the brain.

The association of the two R subunits with intracellular compartments is mediated by AKAPs. The anchor proteins are a group functionally related molecules characterized by the interaction with type I or type II regulatory subunits (RI and RII, respectively) of the PKA holoenzyme. The first anchor proteins were isolated in the affinity chromatographic purification of the R subunits via cAMP-Sepharose. These associated proteins also displayed RII binding after transfer to a nitrocellulose membrane. This observation is also based on the most common method (RII overlay) for the detection of AKAPs. It is a modified Western blot that uses radioactively labeled RII subunits instead of a primary antibody as a probe.

Little is known about the functional importance of the RI-AKAP interaction. Although RIa is mainly localized cytosolic, several studies show anchoring in vivo. The dynamic anchoring of the Rlα subunits, in contrast to the static anchoring of the RII subunits, seems to be of crucial importance for the cell. Thus, the association of the RI subunits with the plasma membrane of erythrocytes and activated T lymphocytes has been described. In cAMP-mediated inhibition of T-cell proliferation by PKA type I, the localization of the enzyme might also be mediated by AKAPs. In knockout mice expressing no type II regulatory subunits in skeletal muscle tissue, the Rlα subunits bind to a calcium channel-associated AKAP and thus maintain normal, cAMP-dependent channel conductivity through the proper availability of the catalytic subunits of PKA.

In vivo it was further shown that the catalytic subunits in the cell preferentially interact with the RII subunits associate and type I PKA holoenzyme is formed when the amount of free catalytic subunits exceeds the amount of free RII subunits.

Specificity in PKA anchoring is achieved by the targeting domain, a structural motif that, unlike the anchoring domain, is conserved in neither the sequence nor the structure of the AKAPs. Thus, AKAPs are anchored to structural elements in the cell through protein-protein interactions and to membranes through protein-lipid interactions.

The literature describes various AKAPs that associate with different cellular compartments, such as the centrosomes, the mitochondria, the endoplasmic reticulum and Golgi apparatus, the plasma and nuclear membranes, and vesicles.

The exact anchoring mechanisms are so far only known for some AKAPs. Thus, the cardiac muscle-specific anchor protein mAKAP is anchored to the perinuclear membrane of cardiomyocytes by a region with three spectrin-like repeats. Two isoforms of AKAP15 / 18 are anchored to the plasma membrane by lipid modifications (myristoylation and palmitoylation). Three polybasic regions in the targeting domain of AKAP79 are involved in the localization of the protein to the inner postsynaptic membrane (PSD, postsynaptic density).

The AKAPs were first characterized by the interaction with the PKA. However, some of these proteins may also bind other enzymes involved in signal transduction. By simultaneously anchoring enzymes that catalyze opposing reactions, such as kinases and phosphatases, these AKAPs, also known as scaffolding proteins, can localize entire signal complexes in the vicinity of specific substrates, thus providing specificity and regulation of the cellular response Contribute extracellular signals. AKAP79 was the first AKAP to detect interaction with multiple enzymes. This protein binds protein kinase A, protein kinase C and the protein phosphatase calcineurin (PP2B), each enzyme being inhibited in the bound state. Since different signals are required for the activation of each enzyme, different second messengers such as cAMP, calcium and phospholipids can be found at this point. Further examples are the AKAP220 which locates the PKA and protein PPl to the peroxisomes and AKAP yotiao, in addition to PKA, also the protein phosphatase ^■ PPl binds. The AKAP CG-NAP binds not only the PKA and the protein phosphatase PP1, but also, the Rho-dependent kinase PKN (nerve growth factor (NGF) -activated protein kinase) and the protein phosphatase PP2A.

Other proteins can also associate with AKAPs, so Ezrin, a member of the cytoskeleton-associated ERM family Ezrin / radixin and moesin, identified as AKAP, binds to a protein (EBP50 / NHERF) that is involved in the regulation of sodium Proton transport is involved in the apical membrane of epithelial cells. AKAPs mediate the modulation of ion channel conductance through the localization of protein kinases and phosphatases near specific channel subunits, which are likely to be regulated by phosphorylation and dephosphorylation. The activity of the NMDA receptor is modulated by the AKAP Yotiao, which also binds the protein phosphatase PP1. The bound-state active phosphatase limits the channel conductivity of the NMDA receptor until the PKA is activated by cAMP and phosphorylates the ion channel or an associated protein, thereby rapidly increasing the conductivity. It has also been shown that myristoylated Ht31 peptides, which inhibit the interaction between PKA and ^■ AKAP, abolish cAMP-dependent inhibition of interleukin 2 transcription in Jurkat T cells, and that S-Ht31 peptides limit sperm motility.

Also in the important complex biological processes, such as by the hormone GLP-I {glucagon-like peptide) -ver¬ mediated enhancement of insulin secretion in the ß-cells of the pancreas and in RINm5F cells (clonal ß-cell line of the rat) are AKAPs involved. The activation of PKA by GLP-I leads to the phosphorylation of L-type calcium channels and favors the exocytosis of insulin from secretory granules. Ht31 peptide-mediated inhibition of PKA anchoring significantly reduced insulin secretion. Neither the cAMP formation nor the activity of the catalytic subunits of PKA were affected by the peptides. Furthermore, after expression of wild-type AKAPISCK in RINm5F cells, an increase in insulin secretion after GLP-I administration could be detected compared to control cells which did not express AKAP18a.

The redistribution of aquaporin-2 water channel from intracellular vesicles, dependent on the antidiuretic hormone arginine-vasopressin (AVP), into the plasma membrane of major cells of the renal collecting tube, the molecular basis of vasopressin-mediated water reabsorption is another example of a process that requires the interaction of PKA with AKAP ^■ proteins (Klussmann et al., 1999). If the interaction is prevented, the redistribution can not take place. However, the interaction plays an important role in numerous ^"other operations in a variety of different. Cell types, such as the interaction increases the Herzmuskelkontraktilitat (Hulme et al., 2003).

To analyze the effect of the PKA-AKAP interaction, it is necessary to modify the interaction efficiently and selectively, in particular to inhibit or decouple. At present, an Ht31 peptide is available for the decoupling of PKA from AKAP proteins. ^'The peptide Ht31 can be coupled to stearate to be present membrane-permeable. However, the Pepdid Ht31 decouples PKA and AKAP in a way that is inadequate for many studies or even for therapeutic use. In particular, the peptide Ht31 is not to selectively interact in a position with the regulatory subunits of PKA RIIa or RIIß, so that the meaning ^'of the subunits for selected processes can not be analyzed.

The object of the invention is therefore to overcome the disadvantages mentioned and, in particular, to provide new nucleic acid sequences which code for peptides which efficiently and specifically modify, in particular decouple, the interaction of AKAP and PKA and which can furthermore be used as over-expressing substances in host organisms with the help of these host organisms - spielsweise of mice - model to analyze diseases that are associated with the AKAP-PKA interaction, preferably diabetes insipidus, but also duodenal ulcer, hypertension and diabetes mellitus. The present invention solves this technical problem by providing an isolated nucleic acid sequence selected from the group comprising

a) a nucleic acid molecule comprising a nucleotide sequence encoding at least one amino acid sequence selected from the group comprising SEQ ID Nos. 1 to 39,

b) a nucleic acid molecule according to a) under stringent conditions hybridizes with a nucleotide sequence ^■,

c) a nucleic acid molecule ^■ comprising a nucleotide sequence which has sufficient homology to be to a nucleotide sequence according to a) or b) functional analog,

d) a nucleic acid molecule which is degenerate as a result of the genetic code to a nucleotide sequence according to a) - c) and / or

e) a nucleic acid molecule according to a nucleotide sequence according to a) - d) which is modified by deletions, additions, substitutions, translocations, inversions and / or insertions and is functionally analogous to a nucleotide sequence according to a) to d).

It was surprising that the nucleic acid sequences of the invention can be used to peptides according to Table 1 ^■. (SEQ ID NO. 1 - 39) to encode that modify the interaction of AKAP and PKA, preferably inhibit, particularly preferably decouple. Advantageously, the nucleic acid molecules according to the invention are suitable for coding peptides which bind selectively to regulatory subunits of PKA, in particular to RICH or RI1 / 3. Furthermore, it is possible - through the Nucleic acid molecules according to the invention coded - peptides to perform a modification, inhibition or decoupling of AKAP and PKA depending on the species used. The nucleic acid molecules or peptides derived from these ^'.eignen with advantage for the production of transgenic organisms, such as mice in which the AKAP-PKA interaction tissue- and / or modified cell-specific.

In a preferred embodiment of the invention, the nucleic acid sequence having sufficient homology to become one. Nucleotide sequence to be functionally analogous, at least 40% homologous. For the purposes of the invention, in order to be functionally analogous to the stated nucleic acid sequences or the sequences hybridizing to these nucleic acid sequences, the coded homologous structures enable an efficient and selective decoupling of the PKA-AKAP interaction and a high affinity for the binding to RII. Possess subunits of PKA.

In a further advantageous embodiment of the invention, the nucleic acid molecule. at least 60%, preferably 70%, preferably 80%, most preferably 90% homology to the nucleic acid molecules of the invention.

In a further preferred embodiment of the invention, the nucleic acid molecule is a genomic DNA and / or an RNA; more preferably, the nucleic acid molecule is a cDNA.

The invention also relates to a vector comprising at least one nucleic acid molecule according to the invention. Furthermore, the invention also relates to a host cell comprising the vector. The invention . also concerns one Polypeptide which is encoded by at least one nucleic acid molecule according to the invention.

In a preferred embodiment of the invention, the polypeptide comprises an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 39, or at least one polypeptide according to these sequences. The present invention ^'relates to a polypeptide which is modified by deletion, addition, substitution, translocation, inversion and / or insertion and functionally analogous to a polypeptide of SEQ ID Nos. 1 to 39. and / or a polypeptide comprising a polypeptide having sufficient homology to be functionally analogous to a polypeptide of SEQ ID Nos. 1 to 39 or their mutations (deletion, addition, substitution, translocation, inversion and / or insertions).

The following peptides according to the invention are particularly preferred:

SEQ ID NO: 1 PEDAELVRLSKRLYENAVLKAVQQY (Akapl8δ-wt) SEQ ID NO: 2 PEDAELVRTSKRLVENAVLKAVQQY (AKAP18δ-L304T) SEQ ID NO: 3 PEDAELVRLSKRDVENAVLKAVQQY (AKAP18δ-L308D) SEQ ID NO: 4 PEDAELVRLSKRLVENAVEKAVQQY ^• (AKAP18Ö-L314E) SEQ ID NO: 5 PEDAELVRLSKRLPENAVLKAVQQY (AKAP185-P) SEQ ID NO. 6 PEDAELVRLSKRLPENAPLKAVQQY (AKAP18Ö-PP) SEQ ID NO. 7 PEDAELVRLDKRLPENAPLKAVQQY (AKAPl8δ-phos) SEQ ID NO. 8 EPEDAELVRLSKRLVENAVLKAVQQYLEETQ (Akapl8δ-RI) SEQ ID NO. 9 NTDEAQEELAWKIAKMIVSDIMQQA SEQ ID NO. 10 SEQ ID NO: 11 NGILELETKSSKLVQNIIQTAVDQF SEQ ID NO: 12 TQDKNYEDELTQVALALVEDVINYA SEQ ID NO: 13 LVDDPLEYQAGLLVQNAIQQAIAEQ SEQ ID NO: 14 QYETLLIETASSLVKNAIQLSIEQL SEQ ID NO: 15 LEKQYQEQLEEEVAKVIVSMSIAFA SEQ ID NO. 16 EEGLDRNEEIKRAAFQIISQVISEA SEQ ID NO. 17 ETSAKDNINIEEAARFLVEKILVNH SEQ ID NO. 18 ADRGSPALSSEALVRVLVLDANDNS SEQ ID NO. 19 SDRGSPALSSEALVRVLVLDANDNS SEQ ID Nr..20 TDRGFPALSSEALVRVLVLDANDNS SEQ ID NO. 21 FLAGETESLADIVLWGALYPLLQDP SEQ ID NO. 22 SELLKQVSAAASWSQALHDLLQHV SEQ ID NO. 23 SEQ EKESLTEEEATEFLKQILNGVYYLH ID NO. 24 EKGYYSERDAADAVKQILEAVAYLH SEQ ID NO. 25 WLYLQDQNKAADAVGEILLSLSYLP SEQ ID NO. 26 LKISPVAPDADAVAAQILSLLPLKF SEQ ID NO. 27 SKTEQPAALALDLVNKLVYWVDLYL SEQ ID NO. 28 VLASAYTGRLSMAAADIVNFLTVGS SEQ ID NO. 29 VKLSNLSNLSHDLVQEAIDHAQDLQ SEQ ID NO. 30 APSDPDAVSAEEALKYLLHLVDVNE SEQ ID NO. 31 QMKAKRTKEAVEVLKKALDAISHSD SEQ ID NO: . 32 KDKLKPGAAEDDLVLEWIMIGTVS SEQ ID NO. 33 EKRVADPTLEKYVLSWLDTINAFF SEQ ID NO. 34 QENLSLIGVANVFLESLFYDVKLQY SEQ ID NO. 35 HQSWYRKQAAMILNELVTGAAGLE SEQ ID NO. 36 QQLQKQLKEAEQILATAVYQAKEKL SEQ ID NO: 37 HSVMDTLAVALRVAEEAIEEAISKA SEQ ID NO. 38 RQVQETLNLEPDVAQHLLAHSHWGA., SEQ ID NO. 39 DIPSADRHKSKLIAGKIIPAIATTT.

The peptides of the invention are either (i) derived from AKAP18 (delta) (SEQ ID NOs: 1 to 7) or (ii) proteins not associated with AKAP molecules (SEQ ID NOs: 8 to 39).

Common to the peptides according to (i):

Akapl8δ-wt AKAP185-L304T AKAP18Ö-L314E Akapl8δ-RI

that they bind the RIIalpha subunits of PKA more strongly than any other peptide derived from natural AKAPs. This is explained by hydrogen bond bonding (H-bonding) between the peptide and the RII dimer (see Fig. Hydrogen bonds in dashed lines). Accordingly, the minimum number of (8) amino acids forming H-bonds is common to the peptides.

The following peptides are also derived from AKPA18 (delta) but are characterized by the fact that, despite high amino acid similarity, they do not bind RII subunits of the PKA (negative controls, if necessary, patenting can be omitted). Common to them is that they no longer bind due to structural (1,2) or due to charge differences (3,4).

1 AKAP18δ-P 2 AKAP185-PP 3 AKAP185-L308D 4 AKAP18δ-phos

The peptides of the invention, derived from proteins other than AKAPs, have a defined size that surprisingly contributes to the ability of the peptides to modify an interaction between AKAP and PKA, as it enhances the affinity of the peptides for the RII (alpha) subunits the PCA influences. The peptides consist of 25 amino acids and are according to the 25mere. If the peptides are chosen to be shorter or longer (eg, 17mers), their activity will be changed. The common structural feature of the length of the peptides, together with the functional feature of the AKAEP / PKA decoupling, defines the structures of the invention. These peptides of the invention are characterized by the general formula:

xxxxxxxxx [AVLISE] xx [AVLIFJ [AVLI] xx [AVLI] [AVLIF] xx [AVLISE] xx XX

where x represents any amino acid; in particular, in each case one of the 20 biogenic amino acids (represented in the one-letter code) is represented by x each of these are: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, ^'R, S, T, V, - W, Y). Claimed is any amino acid described in Alberts et al. (2004) Molecular Biology of the Cell, pp. 8, 73, 79 ff., 150 ff. Or 1717G, in Römpp (1999) Biotechnology and Genetic Engineering, p. 45 ff, and in Römpp (2000) Lexicon Biochemistry and Molecular Biology, p 28 ff, or is disclosed in another standard work of biology. These particularly preferred peptides have either a positively charged amino acid (H, K or R) - in first or second position (position is the number of the amino acid of the N-terminus) or leucine in positions 19, 18 or 14 or serine in position 4th

A functionally analogous peptide is a peptide that is capable of modifying, preferably decoupling, the PKA-AKAP interaction.

The invention also relates to an organism which overexpresses a nucleic acid molecule according to the invention or comprises a vector according to the invention and / or has a polypeptide according to the invention. This may be, for example, a transgenic mouse or rat or a cow, horse, donkey, sheep, camel, goat, pig, rabbit, guinea pig, hamster, cat, monkey or dog, in the tissue and / or Cell-specific, the PKA-AKAP interaction is disturbed. Such organisms, for example mice, may be used in particular ¹ to develop drugs that modify, preferably decouple, the PKA-AKAP interaction.

By means of the organisms according to the invention metabolic processes can also be investigated in vivo in which the PKA-AKAP interaction plays a role or in which it is to be clarified whether the AKAP-PKA interaction is involved in a particular event.

The organism is preferably a transgenic mouse which overexpresses the strongly binding peptide AKΑP185-L304T or AKAP18δ-L314E specifically in the main cells of collecting tubes of the kidney. Advantageously, the decoupling of the PKA from AKAP proteins results in primary cultured collection tube cells, the vasopressin-induced redistribution of AQP2 is prevented, whereby the animals in particular have diabetes insipidus. This disease is characterized by high water loss (polyuria), try to compensate by ^'the absorbing large quantities of liquid, for example, the human patients (polydipsia).

With the aid of the transgenic organisms according to the invention, it can be investigated, for example, how the decoupling of the PKA or of selected subunits of AKAP proteins can be regarded and used as a therapeutic principle. In the consequence of such investigations can then advantageously optimized substances (pharmaceuticals) are analyzed, which act equally. Such optimized substances are preferred as aquaretics and can therefore be used with advantage in patients with edema, for example in heart failure or liver cirrhosis.

The invention also relates to a recognition molecule directed against the nucleic acid molecule, the vector, the host cell and / or the polypeptide. Detecting substances in the sense of the invention are molecules which can interact with the structures mentioned, such as nucleic acid molecules or sequences, vectors, host cells and / or polypeptides or their fragments; interact in particular so that a detection or a manipulation of these structures is possible. In particular, the recognition substances may be specific nucleic acids which bind to the nucleic acid molecules or polypeptides mentioned, such as antisense constructs, cDNA or mRNA molecules or fragments thereof, but also antibodies, fluorescence markers, labeled carbohydrates or lipids or chelators. It is of course also possible that the recognition substances are not proteins or nucleic acids or antibodies, but antibodies directed against them. In particular, the recognition substances may be secondary antibodies in such a case.

In a particular embodiment of the invention, the recognition molecule is an antibody, an antibody fragment and / or an antisense construct, in particular an RNA interference molecule.

The antibodies according to the invention specifically bind the peptides according to the invention. The antibodies may also be modified antibodies (for example oligomeric, reduced, oxidized and labeled antibodies). The Indian As used herein, antibody includes both intact molecules and antibody fragments, such as Fab, F (ab ') _2, and Fv, which are capable of binding to certain epitope determinants of the polypeptides. In these fragments, the ability of the antibody to selectively bind its antigen or receptor has been partially preserved, the fragments being defined as follows:

(1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule can be produced by cleavage of a whole antibody with the enzyme papain to obtain an intact light chain and a part of a heavy chain;

(2) the Fab 'fragment of an antibody molecule can be recovered by treatment of a whole antibody with pepsin and subsequent reduction to yield an intact light chain and a portion of the heavy chain; per antibody molecule, two Fab 'fragments are obtained;

(3) F (ab ') ₂ , the fragment of the antibody which can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction; F (ab ') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the light chain variable region and the heavy chain variable region, expressed in the form of two chains; and (5) Single chain antibody ("SCA") defined as a genetically engineered molecule containing the light chain variable region and the heavy chain variable region linked by a suitable polypeptide linker to a genetically fused single chain molecule ,

The invention also relates to a pharmaceutical composition which comprises the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the polypeptide according to the invention and / or the recognition molecule according to the invention, optionally together with a pharmaceutically acceptable carrier.

In a preferred embodiment of the invention, the pharmaceutical composition is an aquaretics. Aquaretics according to the invention modify the interaction between PKA and AKAP proteins, in particular they decouple the interaction between these two. Of course, it is also possible to use the recognition molecules of the invention as a pharmaceutical composition, in particular those which are directed against the peptide according to the invention or the coding nucleic acid.

In particular, the peptides according to the invention, the vectors according to the invention or the pharmaceutical compositions comprising the recognition molecules according to the invention may preferably be used in patients with edemas, in particular in heart failure or liver cirrhosis. The vectors or nucleic acid molecules according to the invention can be used according to the invention as a pharmaceutical composition at the nucleic acid level, whereas the Peptides according to the invention, but in part also the recognition molecules according to the invention, can be used on the .Aminosäureebene. Depending on whether the therapy in the decoupling of AKAP and PKA - eg by means of the peptides of the invention - or ^' in the prevention of decoupling between AKAP and PKA - for example by means of the antibodies of the invention directed against the peptides - consists, the expert can preferably Peptides according to the invention or the recognition molecules according to the invention, which are directed for example against these peptides or other structures, use as a pharmaceutical composition. The erfidnungsgemäßen peptides can be used in particular for the decoupling of AKAP / PKA us thus for example in edema. The erfidnungsgemäßen recognition molecules (eg antibodies) can be used in particular in the prevention of the decoupling of AKAP / PKA eg in diabetes insipidus.

Of course, it is possible that the peptides according to the invention comprise conventional adjuvants, preferably carriers, adjuvants and / or vehicles. The carriers may be, for example, fillers, extenders, binders, humectants, disintegrants, dissolution inhibitors, absorption accelerators, wetting agents, adsorbents and / or lubricants. In this case, the peptide is referred to in particular as a drug or pharmaceutical agent.

In a further preferred embodiment of the invention, the agent according to the invention as gel, powder, powder, tablet, sustained release tablet, premix, emulsion, infusion formulation, drops, concentrate, granules, syrup, pellet, BoIi, capsule, aerosol, spray and or inhalant prepared and / or used in this form. The tablets, dragees, capsules, pills and granules can be mixed with the usual optionally containing opacifying, coatings and shells be provided and also be composed so that they deliver the active ingredient or only optionally delayed in a ^• certain part of the intestinal tract, where used as embedding masses, for example, polymeric substances and waxes.

For example, the pharmaceutical compositions of this invention may be used for oral administration in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added.

The active substance (s) may optionally also be present in microencapsulated form with one or more of the excipients specified above.

Suppositories may beside the active compound or compounds, the customary water-soluble or water-insoluble Träger¬ substances, for example polyethylene glycols, fats, for example cocoa fat and higher esters (for example _Ci-C4 alcohol with C ₆ fatty acid) or mixtures of these substances).

Ointments, pastes, creams and gels may contain, in addition to the active substance (s), the usual excipients, for example animal and vegetable fats, waxes, paraffins, Starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.

Powders and sprays may contain, in addition to the active substance (s), the usual excipients, for example lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays may additionally contain the customary propellants, for example hydrochlorofluorocarbons.

Solutions and emulsions can, in addition to the active ingredients CHP and gemcitabine, the customary carriers such as solvents, solubilizers and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, ricinus oil and sesame oil, glycerol, glycerol formal, tetrahydofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures thereof. For parenteral administration, the solutions and emulsions may also be present in sterile and blood isotonic form.

Suspensions may, in addition to the active ingredients, the usual carriers such as liquid diluents, for example water, ethyl alcohol, propylene glycol, suspending agents, for example ethoxylated Isostearylalkohole, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures contain these substances.

The medicaments may be in the form of a sterile injectable preparation, for example as a sterile injectable aqueous or oily suspension. This suspension can also be formulated by methods known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Compatible vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are usually used as solvent or suspending medium. Any mild non-volatile oil, including synthetic mono- or diglycerides, may be used for this purpose. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol or similar alcohol as a diluent or dispersant.

The formulation forms mentioned may also contain colorants, preservatives and odour- and taste-improved additives, for example peppermint oil and eucalyptus oil and sweeteners, for example saccharin. The peptides according to the invention should preferably be present in the listed pharmaceutical preparations in a concentration of from about 0.01 to 99.9, preferably from about 0.05 to 99,% by weight of the total mixture.

The pharmaceutical ^{'preparations} listed, other than the peptide or structural homologs - eg peptides with D-amino acids - .or Funktiohsanalogen - eg peptide mimetics - other active pharmaceutical ingredients contain. The preparation of the pharma¬ ceutical listed above. Preparations are carried out in a customary manner by known methods, for example by mixing the active substance (s) with the carrier (s).

The preparations mentioned can ^'in humans and animals ent either orally, rectally, parenterally (intravenous, intramuscular, subcutaneous), intracisternal, intravaginal, intraperitoneal, local (powders, ointments, drops) and used for the treatment of these diseases. Suitable preparations are injection solutions, solutions and suspensions for oral therapy, gels, infusion formulations, emulsions, ointments or drops. For local therapy, ophthalmic and dermatological formulations, silver and other salts, ear drops, eye ointments, powders or solutions may be used. In ^animals' can also be via the feed or drinking water in suitable formulations, the ^• micrograph. Furthermore, the drugs or the combination agents can be incorporated into other carrier materials such as plastics, (plastic chains for local therapy), collagen or bone cement.

In a further preferred embodiment of the invention, the peptides are introduced in a concentration of from 0.1 to 99.5, preferably from 0.5 to 95, particularly preferably from 20 to 80,% by weight in a pharmaceutical preparation. That is, the peptides are present in the pharmaceutical compositions listed above, for example, tablets, pills, granules and others, preferably in a concentration of 0.1 to 99.5 wt .-% of the total mixture in a certain ratio. The amount of active ingredient, that is to say the amount of a compound of the invention combined with the carrier materials to produce a single dosage form, will be appreciated by those skilled in the art Depending on the patient to be treated and the particular mode of administration may vary. After improving the condition of the patient, the proportion of active compound in the preparation may be changed to provide a maintenance dose that arrests the disease. Thereafter, the dose or frequency of administration, or both as a function of the symptoms, may be reduced to a level at which the improved condition is maintained. When the symptoms have been alleviated to the desired level, treatment should cease. However, patients may require long-term discontinued treatment after any recurrence of disease symptoms. Accordingly, the proportion of the compounds, that is to say their concentration, in the overall mixture of the pharmaceutical preparation as well as their composition or combination is variable and can be modified and adapted by the person skilled in the art on the basis of his specialist knowledge.

The skilled worker is well known that peptides of the invention with an organism, preferably ^■ a human or an animal, can be placed on various routes. Furthermore, it is known to the person skilled in the art that, in particular, the pharmaceutical agents can be administered in various dosages. The application should be carried out so that the disease is combated as effectively as possible or the outbreak of such disease is prevented in a prophylactic administration. The concentration and the type of application can be determined by the skilled person through routine experimentation. Preferred applications of the compounds according to the invention are oral administration in the form of powders, tablets, juice, drops, capsules or the like, rectal administration in the form of suppositories, solutions and the like, parenterally in Forms of injections, infusions and solutions as well as locally in the form of salves, patches, envelopes, rinses and the like. The bringing into contact of the compounds according to the invention preferably takes place prophylactically or therapeutically.

The suitability of the selected application forms as well as the dose, application regimen, selection of adjuvant and the same der¬ example, by taking serum aliquots from the patient, human or animal, and testing for ^■ ie the presence of disease indicators in the course of Treatment protocol can be determined. Alternatively and concomitantly, the condition of the kidney, as well as the amount of T cells or other cells of the immune system, can be determined concomitantly in a conventional manner to obtain an overall view of the immunological constitution of the patient and in particular the constitution of metabolically important organs , In addition, the patient's clinical condition can be monitored for the desired effect. If insufficient therapeutic efficacy is achieved,. If necessary, the patient may be further treated with agents according to the invention modified with other known medicaments, from which an improvement of the overall constitution can be expected. Of course, it is also possible to modify the carriers or vehicles of the pharmaceutical agent or to vary the route of administration.

In addition to the oral intake, it may then be provided, for example, that injections, for example intramuscularly or subcutaneously or into the blood vessels, are another preferred route for the therapeutic administration of the compounds according to the invention. At the same time, the delivery via catheters or surgical tubes can be used become; For example, via catheters that lead directly to specific organs such as the kidneys.

The compounds according to the invention can be used in a preferred embodiment in a total amount of preferably 0.05 to 500 mg / kg body weight per 24 hours, preferably from 5 to 100 mg / kg body weight. This is advantageously a therapeutic amount used to prevent or ameliorate the symptoms of a disorder or respon- sive, pathological, physiological condition.

Of course, the dose will depend on the age, health and weight of the recipient, the degree of the disease, the nature of a necessary concomitant treatment, the frequency of treatment, and the nature of the desired effects and side effects. The daily dose of 0.05 to 500 mg / kg body weight can be used once or several times to obtain the desired results. Typically, pharmaceutical agents are used for about 1 to 10 times daily administration or, alternatively or additionally, as continuous infusion. Such administrations can be used as a chronic or acute therapy. Of course, the amounts of drug combined with the carrier materials to produce a single dosage form may vary depending on the host to be treated and the particular mode of administration. It is preferable to distribute the target dose to 2 to 5 applications, with 1 to 2 tablets having an active ingredient content of 0.05 to 500 mg / kg of body weight being administered for each application. Of course, it is possible to choose the active ingredient content also higher, spielsweise up to a concentration of up to 5000 mg / kg. The tablets may also be retarded, resulting in the Number of applications per day reduced to 1 to 3. The active ingredient content of the sustained-release tablets can be 3 to 3000 mg. If the active ingredient is administered by injection as described above, it is preferred that the host is treated with the compounds according to the invention 1 to 10 times per day or by continuous infusion. To bring, with amounts of 1 to 4000 mg per day are preferred. The preferred total amounts per day have proven beneficial in human and veterinary medicine. It may be necessary to deviate from the stated dosages, depending on the type and body weight of the host to be treated, the nature and severity of the disease, the method of preparation of the drug and the period or interval within which the administration takes place. Thus, in some cases it may be preferable to bring the organism into contact with less than the stated amounts, while in other cases the stated amount of active ingredient must be exceeded. The determination of the respectively required optimal dosages and the mode of administration of the active ingredients can easily be carried out by the skilled person on the basis of his specialist knowledge.

In a further particularly preferred embodiment of the invention, the pharmaceutical agent is used in an individual from 1 to 100, in particular from 2 to 50 mg / kg Kör pergewicht. As well as the total amount per day, the amount of single dose per application can be varied by the skilled person on the basis of his expertise. The compounds used according to the invention can also be used in veterinary medicine in the named individual concentrations and preparations together with the feed or with feed preparations or with the drinking water. A single dose preferably contains the amount of active ingredient administered in one application and the usual corresponds to a whole, half a daily dose or a third or a quarter of a daily dose. The dosage units may accordingly preferably contain 1, 2, 3 or 4 or more single doses or 0.5, 0.3 or 0.25 of a single dose. The daily dose of the compounds according to the invention is preferably distributed over 2 to 10 applications, preferably 2 to 7, more preferably 3 to 5 applications. Of course, a continuous infusion of the compositions of the invention is possible.

In a particularly preferred embodiment of the invention, 1 to 2 tablets are administered for each oral application of the compounds according to the invention. The tablets of the invention may be provided with the skilled person be¬ known coatings and shells and ^■ also be composed so that they preferred the active compound or compounds only, release of the host in a certain part.

In a further embodiment of the invention, it is preferred that the peptide segments are optionally associated with one another or bound to liposomes in a carrier, wherein the inclusion in liposomes in the sense of the invention need not necessarily mean that the peptides in the An inclusion in the context of the invention may also mean that the peptides are associated with the membrane of the liposomes, for example, such that they are anchored to the outer membrane. Such a representation of the peptides according to the invention in or on the liposomes is advantageous if the person skilled in the art selects the liposomes in such a way that they have an immunostimulating action. DE 198 51 282 discloses various possibilities for the skilled person to modify the im-stimulating action of liposomes. The lipids may be simple lipids, such as for example, esters and amides or complex lipids such as, for example, glycolipids such as cerebrosides or ganglionides, sphingolipids or phospholipids.

For example, it is possible to exchange individual or groups of amino acids in the peptides without adversely affecting the activity of the peptides with respect to the solution of the object of the invention. For the exchange . Such amino acids should be referred to the corresponding standard works of biochemistry and genetics.

Various ways of preparing peptides are disclosed in the prior art. Peptides which are developed from the peptides according to the invention, starting from such methods, are included in the teaching according to the invention. One possibility of generating functionally analogous peptides is described, for example, in PNAS USA 1998, Oct. 13; 9521: 12179-84, WO 99/6293 and / or WO 02/38592 be¬ wrote; these teachings are incorporated into the disclosure of the invention ^'with. That is to say, all peptides, peptide fragments or structures which comprise peptides which are generated by the abovementioned methods-starting from the peptides according to the invention-are peptides in the sense of the invention, provided that they achieve the object according to the invention. The peptides according to the invention are also lead structures for the development of peptide mimetics.

It is further known to the person skilled in the art that individual amino acids have analogous physicochemical properties which advantageously lead to these amino acids being interchangeable. These include, for example, the group of amino acids (a) glycine, alanine, valine, leucine and / or isoleucine; or the amino acids (b) serine and threonine, the amino acids (c) asparagine and Glutamine, the amino acids (d) aspartic acid and glutamic acid; the amino acids (e) lysine and, arginine and the group of aromatic amino acids (f) phenylalanine, tyrosine and / or tryptophan. Amino acids within the same group (af) can be replaced ^'with each other. Furthermore, it is possible that amino acids are exchanged by modified amino acids or specific enantiomers. Further modifications to the teaching of WO 99/62933 or WO 02/38592 are possible in accordance with the ^'of the inventive teaching in the disclosure are included.

In a further preferred embodiment, the peptide comprises a linker and / or a spacer which is selected from the group comprising: ά-aminocarboxylic acids and their homo- and hetero-oligomers, α, ω-aminocarboxylic acids and their branched homo- or hetero-oligomers, other amino acids and the linear and branched homo- or hetero-oligomers (peptides); Amino-oligoalkoxy-alkylamines; Maleimidocarboxylic acid derivatives; Oligomers of alkylamines; 4-alkylphenyl derivatives; 4-Oligoalkoxyphenyl- or 4-Oligo- alkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylamine phenyl or 4-oligoalkylaminophenoxy derivatives; (Oligo- alkylbenzyl) phenyl or 4-01igoalkylbenzyl) phenoxy derivatives and 4-oligoalkoxybenzyl) phenyl or 4-Oligo alkoxybenzyl) phenoxy derivatives; Trityl derivatives; Benzyl oxyaryl or Benzylöxyalkyl derivatives; Xanthen-3-yl-oxyalkyl derivatives; (4-alkylphenyl) or ω- (4-alkylphenoxy) alkanoic acid derivatives; Oligoalkyl-phenoxyalkyl or oligo-alkoxy-phenoxyalkyl derivatives; Carbamate derivatives; amines; Trialkylsilyl or dialkylalkoxysilyl derivatives; Alkyl or aryl derivatives and / or combinations thereof; further possible structures are described in EP 1 214 350, which are included in the disclosure of the invention.

Preferably, synthetic peptides or fragments thereof can be multimerized by chemical crosslinkers or coupled to a carrier molecule such as BSA, dextran, KLH or others. The chemical crosslinkers used for this purpose are listed in "Bioconjugate Techniques", Greg T. Hermanson, Academic Press, 1996, which are included in the disclosure content of the teaching according to the invention. Preferred crosslinkers are homobifunctional crosslinkers, preferably: NHS esters, such as DSP, DTSSP, DSS, BS, DST, sulfo-DST, BSOCOES, sulfo-BSOCOES, EGS, sulfo-EGS, DSG or DSC, homobifunctional imidoesters, such as DMA, DMP , DMS or DTBP, homobifunctional sulfhydryl-reactive crosslinkers such as DPDPB, BMH or BMOE, difluorobenzene derivatives such as DFDNB or DFDNPS, homobifunctional photoreactive crosslinkers such as BASED, homobifunctional aldehydes such as formaldehyde or glutaraldehyde, bis-epoxides such as 1,4 Butanediol diglycidyl ethers, homobifunctional hydrazides such as adipic dihydrazides or carbohydrazides, bis-diazonium derivatives such as o-tolidine, bis-diazotized benzidine or bis-alkyl halide.

Preference is also given to heterobifunctional crosslinkers, in particular amine-reactive and sulfhydryl-reactive crosslinkers, such as SPDP, LC-SPDP, sulfo-LC-SPDP, SMPT, sulfo-LC-SMPT, SMCC, sulfo-SMCC, MBS, sulfo-MBS, SIAB, sulfo -SIAB, SMPB, sulfo-SMBP, GMBS, sulfo-GMBS, SIAX, SIAXX, SIAC, SIACX or NPIA, carbonyl reactive and sulfhydryl reactive crosslinkers such as MPBH, M ₂ C ₂ H or PDPH, amine reactive and photoreactive crosslinkers such as NHS-ASA , Sulfo-NHS-ASA, sulfo-NHS-LC-ASA, SASD, HSAB, sulfo-HSAB, SANPAH, sulfo-SANPAH, ANB-NOS, SAND, SADP, sulfo-SADP, sulfo-SAPB, SAED, sulfo-SAMCA , p-Nitrophenyldiazopyruvat or PNP-DTP, sulfhydryl and photoreactive crosslinkers such as ASIB, APDP, benzophenone-4-iodoacetamide or benzophenone-4-maleimide, carbonyl reactive and photoreactive crosslinkers such as ABH, carboxylate reactive and photoreactive crosslinkers such as ASBA, arginine reactive crosslinkers, such as APG, trifunctional crosslinkers such as 4-azido 2-nitrophenylbiozytin-4-nitrophenyl ester, sulfo-SEBD, TSAT and / or TMEA. ^{• •}

In a further preferred embodiment of the invention, the peptides according to the invention and recombinantly produced structures are linked by peptide bridges with a length of 0 to 50 amino acids. This also includes recombinant proteins consisting of two N-terminal and one C-terminal sequence or hexamers consisting of three N-terminal sequences and three C-terminal sequences, or multimers of the recombinant structures listed above, wherein between the N and the C-terminal sequences depending on a peptide bridge of 0 to 50 amino acids may be present. The peptides may be provided for the purpose of ^■ purification, solubilization, and the conformational change with specific proportions fusion either at the N- or at the ^{'C-terminal',} as for example, CBP (calmodulin-binding protein), His-tag and / or others. Similar constructs can also be encoded by DNA used for therapy.

The invention also relates to a kit comprising a nucleic acid molecule according to the invention, a ^" vector ^" according to the invention, a host cell according to the invention, a polypeptide according to the invention, a recognition molecule according to the invention and / or a pharmaceutical composition, optionally together with information - for example an instruction leaflet or an internet address which on homepages with further information, etc. - about the handling resp. about the combination of the contents of the kit. The information for handling the contents of the kit may include, for example, a treatment regimen for edema, heart failure, liver cirrhosis ^■, hyperinsulinemia, hypertension, duodenal ulcer. However, the information may also include information on how to use the substances and products according to the invention within a diagnosis of diseases associated with the AKAP-PKA interaction or its decoupling. The kit according to the invention can also be used in basic research. Within basic research, the kit is preferably used to detect whether a metabolic phenomenon is associated with the interaction or non-existent interaction of AKAP and PKA. In particular, it is possible with the aid of the kit according to the invention to determine which subunits of AKAP and / or PKA are responsible for the interaction of these two molecules or for the non-occurrence of the interaction between them.

The ^■ products according to the invention, such as peptides, vectors, nucleic acid molecules, other advantageous nucleic acids, amino acids, carbohydrates and lipids can include. For example, it may be preferred that the peptides are modified with a fatty residue, such as a stearate, such that they are well membrane permeable. With such peptides attempts can ^'be carried out on cell cultures. Such peptides can be used as tools to decouple PKA most efficiently from AKAP proteins in cells, cell cultures, tissue cultures, organ cultures or organisms. The peptides in the sense of the invention can be used in cell cultures, in particular to answer the question whether a particular process of anchorage of PKA to AKAP proteins. Due to the advantageous high affinity for the human RIIa- subunits of PKA to peptides of the invention are particularly suitable ^'for studies on human systems. By comparing peptides that bind PKA with a different affinity, it will still be possible to make quantitative statements that define to what degree a PKA-AKAP interaction is necessary to ensure the progression of a physiological process. In particular, the kits of the invention may be used to study this course of the physiological process. It is advantageous here that the peptides according to the invention which bind the RII subunits of the PKA more strongly than the typical PKA binding domains of AKAPlδδ. Because the _. peptides according to the invention are advantageously RIIa or RII / 3-specific, for example, with the kit, particularly detailed knowledge about the interaction can be obtained. The decoupling of one or the other regulatory subunits of the PKA of AKAP proteins can in particular shed light on which PKA, Typllα or type II / 3, is involved in the particular process to be investigated. In particular, the peptide AlδδRIIβRnl selectively binds RUβ subunits of PKA.

The invention also relates to a method for modification, in particular an inhibition, preferably a decoupling, an AKAP-PKA interaction or the interaction of AKAP or PKA subunits comprising the steps:

a) providing a nucleic acid molecule ^{according to the} invention, a vector according to the invention, a host cell according to the invention and / or a polypeptide according to the invention and b) contacting at least one product according to a) with a cell, a cell culture, a tissue and / or a target organism.

It is preferred that the interaction is analyzed or modified on a regulatory R subunit, particularly preferably on a RIIα and / or RII / 3 subunit.

The invention also relates to the use of a nucleic molecule according to the invention, a host cell according to the invention, an organism according to the invention, a polypeptide according to the invention, a recognition molecule according to the invention, a pharmaceutical composition according to the invention and / or a kit according to the invention for the modification, in particular an inhibition, of an AKAP-PKA interaction , The invention also relates to the use of fragments or subregions. the peptides or nucleic acids according to the invention. Furthermore, it can ^'be provided to enhance the peptides or nucleic acids of the present invention to more amino acids or nucleotides. Of course, it is also possible to modify the peptides with lipid or carbohydrate structures.

In . a preferred ^'embodiment of the invention, particularly the use according to the invention, the Zelle.- for example, as cell culture - used PKA interaction or the organism as a model for tissue and / or cell-specific AKAP, in particular as a model for diabetes insipidus. Further preferred models are cell cultures or tissues which comprise nucleic acid molecules or peptides according to the invention. In a further preferred embodiment of the invention, the vasopressin-induced redistribution of AQP2 is modified, in particular prevented, by the AKAP-PKA modification.

In a further particularly ^'preferred embodiment, the polypeptide and / or the pharmaceutical composition be used as a water loss-causing agent, in particular as aquaretic.

In a further preferred embodiment of the invention, in particular the use according to the invention, the interaction of the Rllce or RIIß subunit of the PKA with AKAP is modified, in particular inhibited.

In a further preferred. Use are human subunits or ^'murine origin.

In the following, the invention will be explained in more detail by means of examples, without being limited to this.

Peptides for inhibiting the interaction of protein kinase A and protein kinase A anchor proteins

material and methods

Preparation of peptide libraries derived from the sequence of the PKA binding domain of AKAP185 on membranes

All chemicals and solvents were purchased from Fluka (Steinheim) or Sigma Aldrich (Munich) and used without further purification steps. Fmoc-protected amino acid pentafluorophenyl esters were purchased from Novabiochem Merck Biosciences GmbH (Darmstadt). Peptide libraries were synthesized by automatic SPOT synthesis on Whatman 50 cellulosic membranes according to standard protocols using Fmoc chemistry and AutoSpot Robot ASS 222 (Intavis Bioanalytical Instruments AG, Cologne). Protecting groups of the amino acid side chains were cleaved by a mixture of trifluoroacetic acid (TFA) in dichloromethane (DCM) (Frank, 1992, Kramer and Schneider-Mergener, 1998). As a control, spots (about 50 nmol peptide per spot) were excised from the cellulosic membrane, from _. the membrane removed by treatment with 0.05 M NaOH and ^■ TOF-MALDI mass spectrometry and analyzed by HPLC.

Detection of membrane-associated peptides in the RII overlay experiment with regulatory RIIa and RII / 3 subunits of PKA as a probe

material

1. Regulatory RIIα> (human) and RII / 3- (Rat) Subunits of PKA Received by Prof. Dr. med. Friedrich W. Herberg, University of Kassel

2. Catalytic Subunits, PKA, Promega, Mannheim, Cat. : V5161

3. Dy ³² P] ATP 5000 Ci / mmol Amersham Biosciences, Brunswick, order no. : AA0018

4. Sephadex G 50, medium Pharmacia, order no. : 17-0043-01

5. Phosphate buffered saline (PBS) NaCl 8 g Dissolve KCl 0, 2 g Na ₂ HPO ₄ ^■ 1.44 g KH ₂ PO ₄ 0, 24 g in 800 ml H ₂ O, adjust to pH 7.4 and make up to 1 1 with H ₂ O.

6. Tris-buffered saline with Tween 20 Tris-HCl 10mM NaCl 150mM Tween 20 0, 05% pH 7.5

Radioactive labeling of the regulatory subunit of PKA .N _C

1st reaction batch

Final concentration stock solution. in the beginning

RIIa or RII / 3 15 μg 7 μg / μl 5, 6 μl Catalytic 2 μg 0, 9 μg / μl 2 μl subunit of PKA. Potassium phosphate buffer, 25 mM 1 M 12.5 μl pH 7.0 cAMP 10 μM 1 mM 5 μl MgCl ₂ 10 mM 0.5 M 10 μl DTT 0.5 mM 50 mM 5 μl [γ ³² P] ATP / ATP 0, 1 uM radioactively: 3.3 x 10 ^ö cpm / ml = 75 uCi 5 uCi / ul 15 ul nonradioactive: 10 .mu.M 5 ul H ₂ O 434.9 ul of 10 min incubation at 0 ⁰ C (on ice). 2. Setting the ATP concentration

The concentration of ATP ^'was prepared by addition of non-radioactive ATP set at 10 uM (addition of 5 ul of a 1 mM solution). The batch was incubated on ice for a further 50 min.

3. Stop the reaction and check the reaction

The reaction was stopped by addition of dextran ^■ and separation of free nucleotides. The free ATP was separated on a Sephadex G50 column.

Separation of the labeled RII subunit of PKA from free nucleotides via Sephadex G50 columns

Unincorporated nucleotides were separated from the RII subunits by fractionation over Sephadex G 50 columns.

1. Sources of Sephadex G50 material: 20 g were swollen in 400 ml PBS overnight at room temperature. Untreated material was subsequently removed with a Pasteur pipette. The swollen material was aliquotted in 50 ml Falcon tube and stored at 4 ⁰ C. For preservation, sodium azide was added at a final concentration of 0.01%.

2. The material was poured into a 10 ml sterile disposable pipette sealed with a glass ball. To set the column bed, about 50 ml of PBS containing 1 mg / ml BSA (bovine serum albumin) passed through. Until the use of ^'the column was sealed at the top with parafilm.

3. The labeled RII subunits (500 μl) were applied to the column with dextran blue (70 μl of a 20 mg / ml solution) (total volume = 570 μl).

4. After the sample had migrated into the matrix, it was filled up with PBS.

5. Shortly before the dextran blue eluted, fractions were collected (2 fractions each 1.5 ml, the further fractions 1 ml each).

6. To determine the incorporation of ³² P, 1% (5.7 μl) of the sample was used in front of the column (which corresponds to 1% of the radioactivity used) and 3 μl of each fraction. ^'

7 .. The fractions of the first peak containing the probe were pooled. The incorporation rate was calculated in% and the specific activity (cpm / μg protein) was determined.

RII overlay

1. Proteins (40 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (PVDF, polyvinylidene fluoride) by the semi-dry electroblotting method. The membrane-associated proteins were stained with Ponceau S to identify the marker proteins on the membrane. Decolorized with TBS. 2. The membrane was dried in Blotto / BSA for 16 at 4 ⁰ C incubated: 10 mM potassium phosphate buffer, pH 7.4 0.15 M NaCl 8.766 g / l 5% (w / v) skimmed milk powder 50 g / l 0.1% ( w / v) BSA 1 g / l (0.01% antifoam (Sigma)) 0.02% NaN ₃ 0.2 g / l

3. The blotto / BSA was replaced with fresh and ³² P-labeled RII subunits added (10 ⁵ cpm / ml). It was incubated for 4-6 h at room temperature.

4. The membrane was washed 4 x 15 min in Blotto / BSA and 2 x 10 min in 10 mM potassium phosphate buffer, pH 7.4, 0.15 M NaCl.

5.. RII-binding proteins were detected by exposure to a phosphoimage plate.

Results

A peptide library deduced from the wild-type amino acid sequence of the PKA binding domain of AKAP18δ (PEDAELVRLSKRLVENAVLKAVQQY; Henn et al., 2004) was synthesized on a membrane. For this purpose, each amino acid of the wild-type sequence was substituted with the 20 possible amino acids. FIG. 1 shows the detection of the peptides by means of the RII overlay method. As a probe in this case radioactive PKA Rllce and RII / 3 subunits were used simultaneously. In all later experiments, either RIIa or RII / 3 subunits were used as probes. The result shows significant differences in the binding ability of the individual peptides to the R subunits (different signal intensities).

FIG. 2 shows a repetition of the experiment with selected peptides (AKAP18δ-L304T, AKAP18δ-L308D, AKAP18δ-L314E) whose binding ability to RICH or RII / 3 subunits, however, was tested separately in different RII overlay experiments. As controls, the peptides Ht31, Ht31-P, AKAP18δ-RI and AKAP18δ-wt (wild-type sequence) were synthesized on the same membranes and subjected to the RII overlay experiment. For quantification, the signals were densitometrically evaluated and related to the signal obtained for AKAP18δ-wt. The quantification suggests a stronger binding of AKAP18δ-L304T and AKAP18δ-L314E to both RIICÜ and RII / 3 subunits, whereas AKAP18δ-RI and AKAP18δ-L308D are weaker. The well-known peptide Ht31 binds both regulatory. Subunits about 5-fold weaker than the AKAP18δ-wt and about 5-6-fold. weaker than AKAP18δ-L304T and AKAP18δ-L314E. The binding of the Ht31 used herein, regulatory RIIa- RII and / 3-subunits is only slightly stronger than binding of the ^subunits' to Ht31-P, which. The AKAP-PKA interaction does not inhibit (Klussmann et al., 1999, Alto et al., 2003). Thus, the peptides AKAP18δ-wt, AKAP18δ-L304T and and AKAP18δ-L314E are much more efficient inhibitors of AKAP-PKA interaction than Ht.31.

Alto et al. (2003) developed a peptide, AKAP ₁₃ , which binds the interaction between the murine RIIα subunit of PKA with Ξ-fold higher affinity (K _D = 0.45 nM) than the peptide Ht31 (KD = 2.2 nM). In our RII overlay experiments, the peptides AKAPis and Ht31 bind both the human RIIa and RII / 3 subunits of PKA. from the rat, the peptides AKAP18δ-wt and AKAP18δ-L304T and AKAP185-L314E, which we have identified, are strong. This result suggests species differences between the murine and human RIIα subunits, which result in different binding affinities for the same peptides.

Identification of peptides that specifically bind RII / 3 subunits of PKA

To find peptides that bind either RIIa or RII / 3 subunits of PKA and thus specifically inhibit the interaction of AKAP proteins with the type IIa and the type Iljß PKA, were using three-dimensional structural models of the PKA subunits from the wild-type PKA-binding domain derived from AKAPlδδ peptides that could block the AKAP binding pocket. The peptides (1-19) were synthesized in parallel on two membranes and then tested for binding ability to RIIa or RI1 / 3 subunits of PKA in RII overlay experiments (Figure 3A). The quantitative evaluation showed u. a. a clear difference in the binding of the two PKA subunits to the peptide 7, whose sequence is listed next to that of the other peptides in Figure 3B.

Based on the sequence of peptide 7, two peptide libraries were synthesized on membranes and probed for RII overlay experiments with RIIα and RII / 3 subunits, respectively. Figure 4 shows that some peptides bind RIIα but not RIIβ subunits (e.g. Peptides 10/11 and 10/12) and vice versa (for example, peptide 21/4). In addition, some peptides have a stronger binding to Rllce subunits than to RII / 3 subunits. For others it is the other way round. They bind RIIα subunits weaker than they do. RII / 3 subunits. In summary, the results show that we have found the first blockers with the mentioned peptides AlδδRIIαHsl ■ and 2 and A18δRII / 3Rnl, which selectively inhibit the interaction of RIIa and RII / 3 subunits of PKA. identified with AKAP proteins.

Tab. 1 A

No . A ANDAQLVRLSKRLVENAVLKAVQQY no. B ANDAQLVRLSKRLVENAVLKAVQQY No. C ASDAQLVRLSKRLVENAVLKΆVQQY No D ASDAKLVRLSKRLVENAVLKAVQQY No E ARDAKLVRLSKRLVENAVLKAVQQY No F ARDAQLVRLSKRLVENAVLKΆVQQY No. G ANDARLVRLSKRLVENAVLKAVQQY No. H ASDARLVRLSKRLVENAVLKAVQQY No I ASDAKTVRLSKRLVENAVLKAVQQY No J CHANGE CRLVENAVLKAVQQY No. K ANDAKTERLSQRLVENAVLKAVQQY No. L ANDAKTQRLSQRLVENAVLKAVQQY No. M PEDAELVRLSKRLVENAVLKAVQQY No N PEDAELVRLSKRLVENAVLQAVQQY No. O PEDAELVRLSKRLVENAVLNGVQQY ^' No. P PEDAELVRLSKRLVENAVLNGQQQY No ^■ Q PEDAELVRLSKRLVENAVLNGNQQY No. R - PEDAELVRLSKRLVENAVKNGNQQY No. S PEDAELVRLSKRLVENAVKNGAQDY No. T PEDAELVRLSKRLVENAVLKAVQQY (Akapl8δ-wt) No. U PEDAELVRTSKRLVENAVLKAVQQY (AKAP18Ö-L304T) no. V PEDAELVRLSKRDVENAVLKAVQQY (AKAP188-L308D) No. W PEDAELVRLSKRLVENAVEKAVQQY (AKAP18Ö-L314E) No. X • PEDAELVRLSKRLPENAVLKAVQQY (AKAP188-P) No. Y PEDAELVRLSKRLPENAPLKAVQQY (AKAP18δ-PP) No. Z PEDAELVRLDKRLPENAPLKAVQQY (AKAPlδδ-phos) No. AA EPEDAELVRLSKRLVENAVLKAVQQYLEETQ (Akapl8δ-RI) No. BB ANDARLVRLSKRLRENAVLKAVQQY (AlδδRIIαHsl (14/14)) No. CC ANDARLVRLSKRLYENAVLKAVQQY (A18δRIIαHs2 (14/19)) No . DD ANDARLVRLSKRLVENAVLKFVQQY (AlδδRIIßRnl (21/4)) No. EE ANDARLVRLNKRLVENAVLKAVQQY (A18δRIIcxRn2 (10/11)) No. FF ANDARLVRLPKRLVENAVLKAVQQY (A18δRIIαRn3 (10/12)) No. GG ANDARLVRLSKRDVENAVLKAVQQY (A18δRIIαRn4 (13/02)) No. HH YQEQLEEEVAKVIVSMSIAFAQQTE (AKAP450_JL) No. II NLQKIVEEKVAAALVSQIQLEAVQE (AKAP450 2)

Tab. 1

SEQ ID NO. 1 PEDAELVRLSKRLVENAVLKAVQQY (AKAPl8δ-wt) SEQ ID NO: 2 PEDAELVRTSKRLVENAVLKAVQQY. (AKAP18δ-L304T) SEQ ID NO. 3 PEDAELVRLSKRDVENAVLKAVQQY (AKAP18δ-L308D) SEQ ID NO. 4 PEDAELVRLSKRLVENAVEKAVQQY (AKAP18Ö-L314E) SEQ ID NO. 5 PEDAELVRLSKRLPENAVLKAVQQY (AKAP18Ö-P) SEQ ID NO. 6 PEDAELVRLSKRLPENAPLKAVQQY (AKAP18δ-PP) SEQ ID NO. 7 PEDAELVRLDKRLPENAPLKAVQQY (AKAPl8δ-phos) SEQ ID NO. 8 EPEDAELVRLSKRLVENAVLKAVQQYLEETQ (AKAP18δ-RI) SEQ ID NO. 9 NTDEAQEELAWKIAKMIVSDIMQQA SEQ ID NO. 10 VNLDKKAVLAEKIVAEAIEKAEREL SEQ ID NO. 11 NGILELETKSSKLVQNIIQTAVDQF SEQ ID NO. 12 TQDKNYEDELTQVALALVEDVINYA SEQ ID NO. 13 LVDDPLEYQAGLLVQNAIQQAIAEQ SEQ ID NO. 14 QYETLLIETASSLVKNAIQLSIEQL SEQ ID NO. 15 LEKQYQEQLEEEVAKVIVSMSIAFA SEQ ID NO: 16 EEGLDRNEEIKRAAFQIISQVISEA SEQ ID NO: 17 ETSAKDNINIEEAARFLVEKILVNH.. SEQ ID NO. 18 ADRGSPALSSEALVRVLVLDANDNS SEQ ID No. 19 SDRGSPALSSEALVRVLVLDANDNS SEQ ID NO: 20 TDRGFPALSSEALVRVLVLDANDNS SEQ ID NO: 21 FLAGETESLADIVLWGALYPLLQDP SEQ ID NO: 22 SELLKQVSAAASWSQALHDLLQHV SE SEQ ID NO: 23 EKESL ETEATEFLKQILNGVYYLH SEQ ID NO: 24 EKGYYSERDAADAVKQILEAVAYLH SEQ ID NO: 25 WLYLQDQNKAADAVGEILLSLSYLP SEQ ID NO: 26 LKISPVAPDADAVAAQILSLLPLKF SEQ ID NO: 27 SKTEQPAALALDLVNKLVYWVOLYL SEQ ID NO: 28 VLASAYTGRLSMAAADIVNFLTVGS SEQ ID NO: 29 VKLSNLSNLSHDLVQEAIDHAQDLQ SEQ ID NO: 30 APSDPDAVSAEEALKYLLHLVDVNE SEQ ID NO: 31 QMKAKRTKEAVEVLKKALDAISHSD SEQ ID NO: 32 KDKLKPGAAEDDLVLEWIMIGTVS SEQ ID. Nr. 33 EKRVADPTLEKYVLSWLDTINAFF SEQ ID NO. 34 QENLSLIGVANVFLESLFYDVKLQY SEQ ID NO. 35 HQSWYRKQAAMILNELVTGAAGLE SEQ ID NO. 36 QQLQKQLKEAEQILATAVYQAKEKL SEQ ID NO. 37 HSVMDTLAVALRVAEEAIEEAISKA SEQ ID NO. 38 RQVQETLNLEPDVAQHLLAHSHWGA SEQ ID NO. 39 DIPSADRHKSKLIAGKIIPAIATTT. Tab. 2

RIIα Binding RIIβ Binding Peptide K _D [nM] K _D [nM]

AKAP18δwt 0.4 - 1, 5 1 - 6

AKAP185L304T 0.3 - 0.9 35

AKAP186L314E 0.2 - 1, 3 22

AKAP185L308D no bond no binding

AKAP186-P no bond no binding

AKAP186-PP no bond no binding

Ht31 15 35

Ht31-P no bond no binding

AKAPis no bond no binding

AKAPis-P no bond no binding

Table 2. Binding constants for the interaction of rat human RIIa and Rllβ subunits with the indicated peptides derived from the wild-type RII binding domain of AKAPlδδ (AKAP18δwt). The values were obtained by surface plasmon resonance measurements. L, Leucine, T ,. Threonine, D, aspartate, P, proline, IS, in silico. 304, 308 and 314 denote the position of the corresponding amino acids in AKAP188. Other peptides that inhibit AKAP-PKA interactions

In addition to the peptides described in Figures 1-4, more could be identified that act as inhibitors of AKAP-PKA interactions. To demonstrate this property, the peptides listed below were synthesized on a cellulose membrane (SPOT synthesis method) and subjected to a RII overlay (Figure 5). _, All black dots represent peptides that have bound regulatory PKA subunits. The peptides were synthesized in six blocks. The peptides of row A positions 1-17 are positive controls and identical in all blocks. The names of the peptides listed below are derived from their coordinates in blocks 1-6, for example, the peptide is 1.B13. (Sequence: YIALNEDLRSWTAADTAAQISQRKL) in block 1 in row B at position 13.

Figure 5. shows the identification of peptides that inhibit AKAP-PKA interactions. Candidate peptides were synthesized on a membrane and incubated with radioactively labeled PKA regulatory RIID subunits (RII overlay experiment). All black dots represent peptides that have bound regulatory PKA subunits (detected with a phosphoimager). The peptide sequences are listed in the attached list (Table 3):

Tab. 3. Sequence of the peptides

No Sequence Name / ID no. YIALWEDLRSWTAADTAAQISQRKL 1C17 HUMAN 2ISKEHWNPTIVALVYNVLKTLMEMN 2A5A HUMAN 3DAPEFHSRYITTVIQRIFYTVNRSW 2ACA HUMAN 4GSTFQNTYNLKDIAGEAISFASGKI 2ACA HUMAN LSRYNDQASSSRIIERIFSGAVTRG 2ACA HUMAN EASEFHSRYITTVIQRIFYAVNRSW 2ACC HUMAN QSEYSWRKMLSGIAAFLLGLIFLLV 2DOB HUMAN LLLLFWLGWLGMLAGAWI IVRAPR 4F2 HUMAN LYFHLAPKAEVGVIAKALVRLLRSH A3B2 HUMAN VKQNVKMSESQAALPSALKTLQQKL A4E1 HUMAN DPGALGRLGAWALLFFLVTTLLASA AAAT HUMAN 3QEVEGMNILGLWFAIVFGVALRK AAAT HUMAN EPTTGMDPKARRFLWMLILDLIKTG ABC2 HUMAN RLRGI SWKDEARWKWALEKLELTK ABC2 HUMAN LIQSLESNPLTKIAWRAAKPLLMGK ABCR HUMAN AYTTQSLASVAYLINTLANNVLQML ABI2 HUMAN TLSKTKNLRLLILVGRLFMWEEPΞI ABM2 HUMAN RYGAAΞPHTIAAFLGGAAAQEVIKI ABP1 HUMAN NKDDDESPGLYGFLHVIVHSAKGFK ABR HUMAN PTTHAMSPRLRHVLLELLPRLLGSP ACHE HUMAN PGSAQEKSIERRFLNGLFSKLQPRD AD13 HUMAN HFDPTTAFRAPDVARALLRQIQVSR AGRN HUMAN HYKETWKALEALVAKGLVQALGLSN AKA1 HUMAN THAN HUMAN QKLYLDRNLIAAVAPGAFLGLKALR KEIKSYLKRIFQLVRFLFPELPEEG ALS2 HUMAN AISPWKTYQLVYFLDKILQKSPLPP AMPB HUMAN GGAAGDEAREAAAVRALVARLLGPG ANAG HUMAN GARSGHEQWEMLLDRAAPILSKTK ANK3 HUMAN SRTSSPVKSSLFLAPSALKLSTPSS ANK3 HUMAN TSYPWSWARVGPAVELALAQVKARP ANPA HUMAN TIDRETSGNLEQLLLAWKSIRSIP ANX5 HUMAN AVQNR FHGDAQVALLGLASVIKNTP ANX9 HUMAN PFRIYQTTTERPFIQKLFRPVAADG APG5 HUMAN RDRAS YEARERHVAΞRLLMHLEEMQ ARH 1 HUMAN EKLKSRPAHLGVFLRYIFSQADPSP ARHB HUMAN NNTEKTVKKIKAFVEQVANWLYSS ARRS HUMAN ΞINVERDEKLIKVLDKLLLYLRIVH ARS2 HUMAN ΞLLQSETDKWRAVAIALRNLSLDR ARVC HUMAN LVASSQSVREAKAASHVLQTVWSYK ARVC HUMAN EFKTLSEEEIEKVLKNIFNISLQRK ARY1 HUMAN EFKTLTΞEEVEΞVLKNIFKISLGRN ARY2 HUMAN VINLIGQTPISRLVALLVRGLGTEK ASB8 HUMAN JSAAMAAS SVSWLSSLFLKLYRKP AT7A HUMAN PRSVRDTILSRALILKILMSAAIII ATC4 HUMAN EWTGYTAFFVGILVQQIADLI IRKT ATHL HUMAN ADDQGQHPLLKMLLHLLAFSSAATG ATIP HUMAN ΪLGGAWAFVLRD VI YTL IHYINQRP ATM HUMAN STDEYYYALAIWMSIVSIVSSLY ATY3 HUMAN APQRKTTNPLDLAVMRLAALΞQNVE BA2A HUMAN MGALARALLLPLLAQWLLRAAPELA BAE2 HUMAN TTLLRLPQKVPDAWEAVARASL IP BAE2 HUMAN DASYSLILEVQTAIDNVLIQSDVPI BBS7 HUMAN ΞKΞΞVPEGMEEAAVAS WLPARELQ BC13 HUMAN SHΞLRSKILSLQLLLSILQNAGPIF BIG1 HUMAN SHREAWTNLLLLFLTKVLKI SDNRF BIG1 HUMAN HQSNKGYSLASLLAKVAAGKEKS SN BIR6 HUMAN LIQTSSTΞQLRTIIRYLLDTLLSLL BIR6 HUMAN RGNLPTSGNISGFIRRLFLQLMLΞD BIR6 HUMAN VTFHLPHHVLKSIASAIVNELKKIN BIR6 HUMAN GAVYKGSLDERPVAVKVFSFANRQN BMR2 HUMAN LTKISLKNNLDHLLASLLFEΞYISY BP28 HUMAN LVQLVDTLGAEKFLWILLILLFEQY BP28 HUMAN RLTSLKKTLATTLAPRVLLPAIKKT BP28 HUMAN PISGQTYSVEDAVLKGWDPΞFRIR BPA1 HUMAN WAKQHQQRLASALAGLIAKQELLEA BPEA HUMAN HKAELKPLRADYLIARWTVLEKLI BPL1 HUMAN ASTLILTPTSKDVLSNLVMISRGKE BRC2 HUMAN ENREKVNDQAKLIVGIWGLLLAAL C166 HUMAN YRNGKVLHPLEGAWI IFKKEMDPV C166 HUMAN NITDLGAYKLΆEALPSLΆΆSLLRLS C2TA HUMAN DFRKKARQS IQGILEAAFSEΞLTRS C3aR HUMAN SIGLQNFEIAKDFWKVIDRLSRDE CA16 HUMAN VKPSSTRGGVLFAITDAFQKVIYLG CA1 e HUMAN RLGΞQNFHKARRFVEQVARRLTLAR CA26 HUMAN S IGYTNFTLEKNFVINWNRLGAIA CA26 HUMAN IYDERGARQLGLALGPALGLLGDP F CA35 HUMAN ΪS FAWHMNRS I VLLLKVLAQLRDHS CABI HUMAN YKTSTVSADLANLLKRIATIVPRTE CABI HUMAN HSGFIETEELKNFLKDLLΞKANKTV CABV HUMAN TNTQVEGDDEAAFLERLARREERRQ CALD HUMAN FQLQRPPQNLLRLLRKAVERS SLMG CANB HUMAN LYLRQNSMGLFSALRHALAKESLVG CANC HUMAN DKVTQKQFQLKEIVLELVAQVLEHK CAQ2 HUMAN EGFHLLGQPLSHLARRLLDTVWNKG CARF HUMAN ELYLRKRHHEPGVADRLIRLLQETS CARF HUMAN LIRSLYEMQEERLARKAARGLNVGH CARF HUMAN RLLDLATVKANGLAAFLLQHVQELP CARF HUMAN EGFSFNPLKIEVFVQTLLHLAAKSF CB80 HUMAN RITQDAQLKSSKWHKAVLQLNEEG CBG HUMAN VKKAPDAEELDKVARLAAKALASVS CBP4 HUMAN MEQVAHQTIVMQFILELAKSLKVDP CC37 HUMAN FKITRYWNSLSNLVASLLNSVRSIA CCAC HUMAN HIPTPGAALSWQAAIDAARQAKLMG CCAC HUMAN NWLTEVQDTANKALLALFTAEMLLK CCAC HUMAN  95 PNSSKQTVLSWQAAIDAARQAKAAQ CCAD HUMAN 96 RTLLWTFIKSFQALPYVALLIAMIF CCAF HUMAN 97 VRHKYKrFDNLGQALMSLFVLASKDG CCAG HUMAN 98 LRVISRAPGLKLWETLISSLKPIG CCAI HUMAN 99 NYMFTTVFVLEAVLKLVAFGLRRFF CCAI HUMAN 100 VGNLGLLFMLLFFIYAALGVELFGK CCAI HUMAN 101 VHHKYNFDNLGQALMSLFVLASKDG CCAI HUMAN 102 FKITKYWTSLSNLVASLLNSIRSIA CCAS HUMAN 103 HNQPLWLTRLQDIANRVLLSLFTTE CCAS HUMAN 104 VKS KEEAQHVQRVLAQLLRREAALT CD93 HUMAN 105 RVAQDLGLELAELVPRLFRVASKTH CDA1 HUMAN 106 VFSRRGGLGARDLLLWLLLLAAWEV CDA1 HUMAN 107 RIAQDLGLELEELVPRLFRVASKRH CDA2 HUMAN 108 RRGRGAWTRLLSLLLLAAWEVGSGQ CDA2 HUMAN 109 RIAQDLGLELAELVPRLFRVASKRH CDAD HUMAN 1 10 RIAQDLGLELAELVPRLFRVASKGR CDA5 HUMAN 111 RIAQDLGLELAELVPRLFRMASKDR CDA6 HUMAN 1 12 PTSNQQVKPLGLVLRKLLDREETPE CDA9 HUMAN 1 13 RIAQDLGLELAELVQRLFRVAS KRH CDAA HUMAN 1 14 ALLATQAGSAGGAVNKLVPRSVGAG CDAB HUMAN 1 15 RIAQDLGLELAELVQRLFRVASKTH CDAB HUMAN 1 16 VIIGPRGPGSQRLLLSLLLLAAWΞV CDAC HUMAN 1 17 ADRGSPALSSEALVRVLVLDANDNS Cdb2 HUMAN 1 18 HYSVAEETESGSFVANLLKDLGLEI CDB2 HUMAN 1 19 TDRGS PALSSEALVRVLVLDANDNS CDBE HUMAN 120 SDRGSPALSSEALVRVLVLDANDNS CDB9 HUMAN 121TDHGSPALSSEALVRVLVLDANDNS CDBC HUMAN 122 SDHGSPALSSEALVRVWLDANDNS^' CDBD HUMAN 123TDRGFPALSSΞALVRVLVLDANDNS CDBF HUMAN 124 PPQRHPQRSEQVLLLTLLGTLWGAA CDG6 HUMAN 125 TPRFLKEELEVKILENAAPSSRFPL CDG6 HUMAN 126 RGPAGQRRMLFLFLLSLLDQVLSEP CDGF HUMAN 127 SGGGSDEGLASAAARGLVEKVRQLL CDN5 HUMAN 128 TRMAAESRRVLLLAGRLAAQSLDTS CENB HUMAN 129 MGSΞDHLGVIPRAIHDIFQKIKKFP CENE HUMAN 130 KRGPLLTALSAEAVASALHKLHQDL CEP2 HUMAN 131 ΞYHYVQEKASKLAAASLLLALYMKK CGB3 HUMAN 132 YIDENQDRYIKKLAKWVAIQSVSAW CGL1 HUMAN 133 ANPVEIRRGVMLAVDAVIAELKKQS CH60 HUMAN 134 PVAVRLQMSERNILSRLANRAPEPT CHD4 HUMAN 135 ARΞRERLAHSRRAAARAVAAATAAV CIK4 HUMAN 136 LECTISVIPGLECTIVGALIQSVKKLS CIN9 HUMAN 137 LSRFEGMRWVNALLGAIPSIMNVL CIN4 HUMAN 138 LYILTPFNPLRKIAIKILVHSLFSM CIN1 HUMAN 139 LYILTPFNPIRKLAIKILVHSLFNM CIN2 HUMAN 140 LSRFΞGMRWVNAL VGAIPS IMNVL CIN5 HUMAN 141LYILTPLNPVRKIAIKILVHSLFSM CIN3 HUMAN 142 LKTITVIPGLKTTVGALIQSVKKLS C1N4 HUMAN 143LKTISVISGLKTIVGALIQSVKKLA C1N5 HUMAN 144LYVLSPFHPVRRAAVKILVHSLFNM CIN5 HUMAN 145LYILSPFNLIRRIAIKILIHSVFSM CIN8 HUMAN 146EEQLRLRERELTALKGALKEEVASR GING HUMAN 147 NYGKFEKGYL I FWRFLFGL VNQER CIS1 HUMAN 148RGALLAGALAAYAAYLVLGALLVAR CIW6 HUMAN 149AQKVTRQEREEALVRGVFMKWKPK CJ11 HUMAN 150MLRYDVYGGENEVIPEVLRKSHSHF CJ24 HUMAN 151ERQSAEVQGSLALVSRALEAAERAL CK13 HUMAN 152 RSGYIEANELKGFLSDLLKKANRPY CLB2 HUMAN 153VRRKLGEDWIFLVLLGLLMALVSWS CLC1 HUMAN 154AYIVNYFMYVLWALLFAFLAVSLVK CLC5 HUMAN 155LIHSVSDAFSGWLLMLLIGLLSGSL CLC5 HUMAN 156YFPLKTLWRSFFAALVAAFTLRSIN CLC5 HUMAN 157RKKGRESYIETΞLIFALAKTSRVSE CLH2 HUMAN 158FAGSWRSGLAFLAVIKAIDPSLVDM CLMN HUMAN 159GYFGSDVKVAYQLATRLLAHESTQR CLR2 HUMAN 160SPERFLSPLLGLFIQAVAATLATPP CLR2 HUMAN 161LQEQLYVRRAALAARSLLDVLPFDD CLR3 HUMAN 162WQERFLSPLLGRFLEGVAAVLATPA CLR3 HUMAN 163YFSQDVRVTARLLAHLLAFESHQQG CLR3 HUMAN 164LGSVIDISGLQRAVKΞALSAVLPRV CN 2A HUMAN 165SFMΞHIAMPIYKLLQDLFPKAAELY CN2A HUMAN 166WVSFTSLGSLPSALRPLLSGLVGGA CN3B HUMAN 167HVIYQRVDKAVGLAEAALGLARANN CN93 HUMAN 168TDMKDMRLEAEAWNDVLFAVNNMF CNC9 HUMAN 169PKIVGRTKDVKEAVRKLAYQVLAEK CND3 HUMAN 170YGPTNFAPIINHVARFAAQAAHQGT CNE1 HUMAN 171 LAHLRARLKELAALEAAAKHEELVΞ CNG4 HUMAN 172ΞEGGTPEQGVHRALQRLAHLLQADR CNRC HUMAN 173 WKGGSASTWLTAFALRVLGQVNKYV C05 HUMAN 174DVLPNFFYHSNQWRMAALEVYVRR COA1 HUMAN 175RQVQAEVPGSPIFVMRLAKQSRHLE COA1 HUMAN 176 SSQFHMATNSSMFLKQAFEGEYPKL COG5 HUMAN 177VRRLERKYSSIPVIQGIVNΞVRQSM COG8 HUMAN 178TDPDLPPGYVQSLIRRWNNVNIVI C0H1 HUMAN 179GDVKSKTEALKKVIIMILNGEKLPG COPB HUMAN 180TFTLSTIKTLEΞAVGNIVKFLGMHP COPG HUMAN 181LLFGAWAGVLGTALSLLARAELGQP C0X1 HUMAN 182RFIFNRWLENEAVRAAAVSALAKF CPG2 HUMAN 183RREEATRQGELPLVKΞVLLVALGSR CPSA HUMAN 184NATLFTAAEAPFVEILLTNLFKAL CSE1 HUMAN 185 S VNWKHKD AAIYL VTSLASKAQTQK CSE1 HUMAN 186 QRREGGGRN-IGGIVGGIVNPISEAA CSS2 HUMAN 187 IEKESQRKSIDPALSMLIKSIKTKT CT06 HUMAN 188 LDVIYWFRQIIAWLGVIWGVLPLR CT24 HUMAN 189 HRLLSTΞWGLPSIVKSLIGAARTKT CT45 HUMAN 190 NKSGNRSEKEVRAAAL VLQTIWGYK CTD1 HUMAN 191 RHLTQQDPLSEAIVEKLIQSIQKVF CTDB HUMAN 192 RFVKLAVMGLTVALGAAALAWKSA CTEO HUMAN 193 SVKRGNMVRAARALLSAVTRLLILA CTN 1 HUMAN 194 SVKRGTMVRAARALLSAVTRLLILA CTN2 HUMAN 195ARENAGPAIVISFLIAALASVLAGL CTR1 HUMAN 196 VSSSAPSVYSVQALSLLAEVLASLL CU05 HUMAN 197 VYRSDEKEKAVPLISRLLYYVFPYL CU05 HUMAN 198 RGPHGQLSPALPLASSVLMLLMSTL CV03 HUMAN 199 TLRFLHASALLALASGLLAVLLAGL CV03 HUMAN 200 FPNPRRRLRLQDLADRWDASEDEH CYA3 HUMAN 201 LHYYSEREGLQDIVIGIIKTVAQQI CYG1 HUMAN 202WKGAPTTSLISVAVTKIIAKVLEDN D7A1 HUMAN 203 YVASAFTLAVNI lAKKIVLKEQTGS dcor HUMAN 204 LLRILTDALVPYLVGQWAGAQALQ DCUP HUMAN 205 AEKTDEEΞKEDRAAQSLLNKLIRSN DD19 HUMAN 206 DEERRQLIQLRDILKSALQVEQKED DDF2 HUMAN 207 LGHPAAFGRATHAVVRALPESLGQH DDH1 HUMAN 208 ASFQRKWFEVAFVAEΞLVHSEIPAF DEP5 HUMAN 209 KVAFTGST EVGKLIKEAAGKSNLKR DHA1 HUMAN 210 KIAFTGSTΞVGKLIQΞAAGRSNLKR DHA2 HUMAN 211 EAIKFINRQEKPLALYAFSNSRQW. DHA8 HUMAN 212 PRALLAALWALEAAGTAALRIGAFN DHP1 HUMAN 213 RLVSSPPSGVPGLALLALLALLALR DIAC HUMAN 214 RWLKGDVSLKDI IDPAFRASWIAQ DIAC HUMAN 215 FQLPSRQP ALSSFLGHLAAQVQAAL D1S1 HUMAN 216 RSLTSEREGLEGLLSKLLVLSSRNV DIS1 HUMAN 217 LIGPKVRITLMKFLPSVFMDAMRDN DJCD HUMAN 218 QRPRAPRSALWLLAPPLLRWAPPLL DLG4 HUMAN 219 RQRLLGRSWSVPVIRHLFAPLKEYF DM3A HUMAN 220 KRKLEDLSSEWKAVNRLLQELRAKQ DMD HUMAN 221 LPARVPRPGLSEALSLLLFAWLSR DMK HUMAN 222 QRΞLQEALGARAALEALLGRLQAER DMN HUMAN 223 SARLRMVETLSNLLRSWALSPPDL DNL1 HUMAN 224 ELAΞHLNASLAFFLSDLLSLVDRGF D0C6 HUMAN 225 SPFRQQHFLAGLLLTELALALEPEA D0C6 HUMAN 226 LRAHGTHPAISTLARSAIFSVTYPS D0C6 HUMAN 227 IYEPPRYMSVNQAAQQLLEIVQNQR DPH5 HUMAN 228 HIYLYHHAQAHKALFGIFIPSQRRA. DPOE HUMAN 229 FNWRQAHMQARFVILRVLLEAGEGL DPP3 HUMAN 230 GVILGKWAILAILLGIALLPSVLLT DSC3 HUMAN 231PNTELNVSRLEAVLSTIFYQLNKRM DTNA HUMAN 232 RLDEΞHRLIARYAARLAAESSSSQP DTNA HUMAN 233 LTSVLGILASSTVLFMLFRPLFRWQ DUFF HUMAN 234 PQELYESSHIESAINVAIPGIMLRR DUS6 HUMAN 235 SRELYΞSARIGGALSVALPALLLRR DUS9 HUMAN 236 DYYKKQVAQLKTLITMLIGQLSKGD DYH9 HUMAN 237 RDFVEEKLGSKYWGRALDFATSFE DYH9 HUMAN 238 IGVKFLINEATTLADLLALRLHRVE DYHB HUMAN 239 EGKKKQTNYLRTLINELVKGILPRS DYHC HUMAN 240 JPSGSGKSMAWRVLLKALERLEGVE DYHC HUMAN 241KLVAEDIPLLFSLLSDVFPGVQYHR DYHC HUMAN 242 LLSATELDKIRQALVAIFTHLRKIR DYHC HUMAN 243 WRRFRWAIILFIILFILLLFLAIFI DYSF HUMAN 244 PVRREVTDKEQEFAARAAKQLΞYQQ E4L3 HUMAN 245 FPGDILMRMLKMLILPLIISSLITG EAA2 HUMAN 246 KLMVDFFNILNΞIVMKLVIMIMWYS EAA2 HUMAN 247 FPGEILMRMLKLIILPLIISSMITG EAA3 HUMAN 248 PFMSAVSGRAYPAAITILETAQKIA EDD HUMAN 249 RNTFAERLSAVEAIANAI SWS SNG EDD HUMAN 250 NAAQTPRIPSRLLAILLFLLAMLLT EFA5 HUMAN 251GLKΞLPMRNLQEILHGAVRFSNNPA EGFR HUMAN 252 MVETQQLMRVYGALMWALGKWGTP EHD2 HUMAN 253 HGIVSWDTFSVAFIKKIAS FVNKSA ELM1 HUMAN 254 HGIVSWDMVSIT FIKQIAGYVSQPM ELM2 HUMAN 255 VMNQQLQTKAMALLTALLQGASPVE ELM3 HUMAN 256 ESRVQQQEDEITVLKAALADVLRRL EML4 HUMAN 257 5QFGVGFYSAFLVADKVIVTSKHNN ENPL HUMAN 258 ELSSQLPERLSLVIGS ILGALAFLL EPB6 HUMAN 259 MYRTHTRRALQTVAQLILELIEKQE EPPL HUMAN 260 SGRAAALRQWSAVTAL VEAAERQP EPPL HUMAN 261 RLQALRLEREEYVLLKALALANSDS ERR1 HUMAN 262 ΪESAGGΞSVSVLVLSPLAKNLFHRA EST1 HUMAN 263 TPQKNNYNS IAAILIGVLLTSMLVA EV2B HUMAN 264 ALRPAPALLAPAVLLGAALGLGLGL EVC HUMAN 265 NILVTTTQLIPALAKVLLYGLGIVF EYA1 HUMAN 266 NVLVTTTQLIPALAKVLLYGLGSVF eya2 HUMAN 267 PADEKLQΞKAWGAWPLVGKLKKFY HUMAN F49B 268 FACA VEQRKKLSSLLEFAQYLLAHSMFSR HUMAN 269 NRLGIESPRSEKLARELLKELRTQV FACC HUMAN 270 QQRAQTMVQVKAVLGHLLAMSRSSS FACC HUMAN 271 SGQSKLNSWIQGVLSHILSALRFDK FACC HUMAN 272 ETRRGAYLNALKIAKLLLTAIGYGH FAFX HUMAN 273 SQAYDNLSLSDHLLRAVLNLLRREV FAFX HUMAN 274 WWPVLPKGELEVLLEAAIDLSKKG FAFX HUMAN 275 WWPVLPKGELEVLLEAAIDLSVKG Fafy HUMAN 276 RSNDKVYENVTGL VKAVIEMSSKIQ FAK1 HUMAN 277ARTSSAEYNVNNLVSPVLFQΞALWH FAS HUMAN 278 FRYMAQGKHIGKWVQVLAEEPAVL FAS HUMAN 279 AVTIHPVTGSISVLNPAFLGLSRKL FAT2 HUMAN 280 PGPAPLRLLEWRVAAGAAVRIGSVL FCP1 HUMAN 281 MΞEWDRYPRIGDILQKLAPFLKMYG FGD1 HUMAN 282 SGTKKSDFHSQMAVHKLAKSIPLRR FGR1 HUMAN 283 PSHSLLRLPLLQLLLLWQAVGRGL FK10 HUMAN 284 DVFIWLGRKSPRLVRAAALKLGQEL FLIH HUMAN 285 HRLLQQL VLSGNLIKΞAVRRLQRAV FRT2 HUMAN 286 LPHGSGLGTSSILAGTALAALQRAA AUC HUMAN 287 PESTARMQGAGKALHELLLSAQRQG G45G HUMAN 288 PKVDKWSRFLFPLAFGLFNIVYWVY GAAT HUMAN 289 RLFTNLKDTSSKVIQSVANYAKGDL GAK HUMAN 290 ESLRΞVQLEELEAARDL VSKEGFRR GAL1 HUMAN 291 RPFLPYFNVSQQFATFVLKGSFSEI GALC HUMAN 292 NHGMWQTISVEELARNLVIKVNRDA GAS6 HUMAN 293 AGEDPKVTRAKFFIRDLFLRISTAT GBAF HUMAN 294 PSVFGSNPKAHIAAKTVFHLAHRHG GBF1 HUMAN 295 HGRLIFITVLFSIIIWWWISMLLR GC5D HUMAN 296 NVLKDKMEKLKRLLQVAARKSQVTL GCC1 HUMAN 297 TINKFDKNFSAHLLDLLARLSIYST GCP2 HUMAN 298 I INNDTTITLAIWDKLAPRLSQLK GCP5 HUMAN 299 GLLTEKAAPVMNVI HSIFSLVLKFR GCP6 HUMAN 300 IAKQELIAHAREAASRVLSALSDRQ GCP6 HUMAN 301 SYESMSEPPIAHLLRPVLPRAFAFP GCP6 HUMAN 302 JSAVE IVGLSKSAVRWLLELSKKNI GDE HUMAN 303 LRLETAPNI SKDVIRQLLPKAPPLR GDF8 HUMAN 304 VLGDIHTTLLSAVIPNAFRLVKRKP GDL1 HUMAN 305 KDHAGVMGESNRLLSALIRHSKSKD GDS1 HUMAN 306 FNQLTQSASEQGLAKAVASVARLVI GEM4 HUMAN 307GRQKLΆRFNAREFATLIIDILSEAK GIT1 HUMAN 308 KPVYYALEGSVAIAGAVIRWLRDNL GLPK HUMAN 309 VGILSRRLQEALAAKEAADAELGQL GOA3 HUMAN 310 FLRRYPIARVFVI IYMALLHLWVMI GOA5 HUMAN 311 VLGLFWLLFASWLILLLSWVGHVK GPBA HUMAN 312 QASWVRPGVLWDVALVAVAALGLAL GPIX HUMAN 313 FRLVSRRDYASEAIKGAWGIDLGT GR75 GR78 HUMAN 314 EDFKAKKKELEEIVQPIISKLYGSA HUMAN 315 5QPKRYKGFSIDVLDALAKALGFKY GRD1 HUMAN 316 FQGKKNMTLAGRLAGPLFQTLIVAW GRIP HUMAN 317 QKGHKSQREELDAILFIFEKILQLL GRIP HUMAN 318VKTQMQHGLISIAARTVITHLVNHL GRIP HUMAN 319 VPTWDTIRDEEDVLDELLQYLGVTS GRIP HUMAN 320 HEHIERRRKLYLAALPLAFEALIPN GRLF HUMAN 321 GVWSEKGQVEVFALRRLLQWEEPQ GRWD HUMAN 322 MRPEDRMFHIRAVILRALSLAFLLS HA2Q HUMAN 323 SHTRGPEQQVKAILSELLQRENRVL HAPI HUMAN 324 AMSKSRNPRLQTAAQELLEDLRTLE HBP2 HUMAN 325 ALGHKRNSGVPAFLTPLLRNI I ISL HD HUMAN 326ENEDKWKRLSRQIADIILPMLAKQQ HD HUMAN 327FGDAALYQSLPTLARALAQYLVWS HD HUMAN 328 ΪLLKLQERVLNNWIHLLGDΞDPRV HD HUMAN 329NLSSRETSSLESFVRRVANIARTNA HED1 HUMAN 330 5LPRPPMLLALLLATLLAAMLALLT HEXB HUMAN 331 DQRKMLLVGSRKAAΞQVIQDALNQL HIP1 HUMAN 332 JIALAYGSLLLMALLPIFFGALRSV HM13 HUMAN 333PNWKLKVSNLKMVLRSLVEYSQDVL H0K2 HUMAN 3343DQLAQLNTVFQALPTAAWGATLRA HPS6 HUMAN 335LLSSGRPKAVLQAVGQLVQKEQWDR HPS6 HUMAN 336QKHAQQQKWNKLIQFLISLVQSNR HSF1 HUMAN 337AKHAQQQQVIRKIVQFIVTLVQNNQ HSF2 HUMAN 338LNAARRYLGIEDLAGKVFVTSGLGG HUTU HUMAN 339FEKMISGMYLGEIVRHILLHLTSLG HXK3 HUMAN 340IPANLSQNLEAAAATQVAVSVPKRR I4G1 HUMAN 341 IFHKNVFHYLMAFLRELLKNSAKNH I5P2 HUMAN 342FΞQWAHSEDLQSLLLRVANAVSVKG ICE9 HUMAN 343KNSEATLPIAVRFAKTLLANSSPFN IF HUMAN 344SGKVSADNTVGRFLMSLVNQVPKIV IF35 HUM AN 345NPKIWNVHSVLNVLHSLVDKSNINR IF3I HUMAN 346LHGVMEYDLSLRFLENALAVSTKYH IF3X HUMAN 347ENGMYGKRKLLELIGHAVAHLKKAD IFT2 HUMAN 348YRRKDΞPDKAIELLKKALEYIPNNA IFT2 HUMAN 349APSDPDAVSAEEALKYLLHLVDVNE IKAP HUMAN 350QLVKLLGASELPIVTPALRAIGNIV IMA2 HUMAN 351 SSNVENQLQATQAARKLLSREKQPP IMA2 HUMAN 352PLLSHQEVKVQTAALRAVGNIVTGT IMA4 HUMAN 353MRKEEPSNNVKLAATNALLNSLEFT IMB1 HUMAN 354PPEHTSKFYAKGALQYLVPILTQTL IMB1 HUMAN 355PYYDLFMPSLKHIVENAVQKELRLL IMB3 HUMAN 356TAAEEARQMAAVLLRRLLSSAFDEV IMB3 HUMAN 357LIAYRSRKRLETFLSLLVQNLAPAE INPP HUMAN 358FVEPHKNMΞVMGFLHGIFERLKQFL IP11 HUMAN 359LPVPQGPNPWWLQQVFQLIQKVL IP13 HUMAN 360NRERQKLMREQNILKQIFKLLQAPF IP3R HUMAN 361NRERQKLMRΞQNILAQVFGILKAPF IP3S HUMAN 362 EKRVADPTLEKYVLSWLDTINAFF IP3T HUMAN 363 NRERQKLMREQNILKQVFGILKAPF IP3T HUMAN 364 ETIQGLGAASAQFVSRLLPVLLSTA IP04 HUMAN 365 HPAQEHFPKLLGLLFPLLARERHDR IPO4 HUMAN 366 LLRNPSSPRAKΞLAVSALGAIATAA IPO4 HUMAN 367 LMASPTRKPEPQVLAALLHALPLKB IPO4 HUMAN 368 SKALLKNRLLPPLLHTLFPIVAAEP IPO4 HUMAN 369 YMQAVNRERERQWMAVLEALTGVL IPO4 HUMAN 370 DQYRQKEYVAPRVLQQAFNYLNQGV IPO8 HUMAN 371 ILADLNLSVSPFLLGRALWAASRFT IPO9 HUMAN 372 LGFENL VFSIFEFVHALLENSKFKS IPO9 HUMAN 373 PERWTNIPLLVKILKLIINELSNVM IPO9 HUMAN 374 RTSEFTAAFVGRLVSTLISKAGREL IPO9 HUMAN 375 LYNYASNQRΞΞYLLLRLFKTALQEE IQG1 HUMAN 376 NRGARGQNALRQILAPWKE IMDDK IQG1 HUMAN 377 YQDLLQLQYEGVAVMKLFDRAKVNV IQG1 HUMAN 378 LYNYASNQREEYLLLKLFKTALEEE IQG2 HUMAN 379 NRGARGQNTLRQLLAPWKEI IDDK IQG2 HUMAN 380 WSPRKLPSSASTFLSPAFPGSQTHS IRA1 HUMAN 381 WLGGGWPDAIVLAEΞALDKAQEVL IRBP HUMAN 382 GKPLERKLILVQVIPWARMIYEMF IRF6 HUMAN 383 WRYMLLIFSLAFLASWLLFGIIFWV IRKC HUMAN 384 WRWMMLVFSASFWHWLVFAVLWYV IRKD HUMAN 385 ALVIFEMPHLRDVALPALGAVLRGA IRR HUMAN 386 VGWIIAISLLVGILIFLLLAVLLWK ITA9 HUMAN 387 SNSIYPWSEVQTFLRRLVGKLFIDP ITAG HUMAN 388 PIWIIVGSTLGGLLLLALLVLALWK Itah HUMAN 389 QGFTYTATAI Q NWHRLFHAS YGAR ITAX HUMAN 390 ARPRPRPLWVTVLALGALAGVGVGG ITB3 HUMAN 391 LWLLSVMGAILLIGLAALLIWKLL ITB3 HUMAN 392 MAGPRPSPWARLLLAALISVSLSGT^■ ITB4 HUMAN 393 AARQRQEIAAARAADALLKAVAASS^' JPH4 HUMAN 394 LSTLRYADRAKRIVNHAWNEDPNA K13A HUMAN 395 SNINKSLTTLGLVISSLADQAAGKG K13A HUMAN 396 LSTLRYADRAKHIVNNAWNEDPNA K13B HUMAN 397 QENLSLIGVANVFLESLFYDVKLQY K13B HUMAN 398STSFRGGMGSGGLATGIAGGLAGMG K1CR HUMAN 399 ΪFDVNIVEEELGIISRAVKHLFKSI K21A HUMAN 400 HQSWYRKQAAMILNΞLVTGAAGLE K406 HUMAN 401 SLVHRLTRDAPLAVLRAFKVLRTLG K406 HUMAN 402 YLSVKQPVKLQEAARSVFLHLMKVD K406 HUMAN 403 KNVMEFLAPLKPVAIRIVRNAHGNK K682 HUMAN 404 5MRRGNMGHLTRIANAWQNLERGP K685 HUMAN 405 RQDVLHWLNEEKVIQRLVELIHPSQ K685 HUMAN 406 EELVSIPWKVLKWAKVIRALLRIL K830 HUMAN 407 GFIATPFIKLFQLIYYLWAILKNI K830 HUMAN 408 PARTQHPERARDALRVLLHLVEKSL KA43 HUMAN 409 RQDWNWLNΞEKIVQRLIEQIHPSK KB15 HUMAN 410 ALHLATEMEELGL VTHL VTKLRANV KBF2 HUMAN 411 NSQWRQDMSISLAALELLSGLAKVK KC19 HUMAN 412EKGFYTERDASRLIFQVLDAVKYLH KCC1 HUMAN 413 EKGYYSΞRDAADAVKQILΞAVAYLH KCC4 HUMAN 414 LQEFNARRKLKAAVKAWASSRLGS KCC4 HUMAN 415 YALKRSFKELGLLLMYLAVGIFVFS KCF1 HUMAN 416 LLRLASTPDLRRFARSALNL VDLVA KCV2 HUMAN 417 STYFDMNLFLDIILKTVLENSGKRR KE34 HUMAN 418 RGGWRQYWSSSFLVDLLAVAAPW KE72 HUMAN 419 SALESTEEKLHDAASKLLNTVEETT kF11 HUMAN 420 QENEPTVGVIPRVIQLLFKEIDKKS KF4A HUMAN 421 ELSNHQKKRATEILNLLLKDLGEIG KF5C HUMAN 422 EATAFGLGKEDAVLKVAVKMLKSTA KFMS HUMAN 423 KDKLKPGAAEDDLVLEWIMIGTVS KFP3 HUMAN 424 MEGKLHDPQLMGIIPRIARDIFNHI CHIN HUMAN 425 SSAIIDHIFASKAWNAAIPAYHLR KIST HUMAN 426 YLΞVGLSGLSSKAAKD VLGFLRWR KNC1 HUMAN 427 LEKLGYMDLASRLVTRVFKLLQNQI LCAP HUMAN 428 ΞAPAYHLILEGILILWIIRLLFSKT LCB1 HUMAN 429 KTPSGIKLTINKLLDMAAQIAEGMA LCK HUMAN 430 ARGYVQDPFAALLVPGAARRAPLIH LCM2 HUMAN 431 DSLYFRLKTAGRLARAAVWEVDFPD LCM2 HUMAN 432 PLPSLRFLEELRLAGNALTYIPKGA LGR5 HUMAN 433 FWKAFWNITEMEVLSSLANMASATV LIPS HUMAN 434VTITLDLRQVFQV AYVIIKAANAPR LMA1 HUMAN 435 ERTNTRAKSLGΞFIKELARD AEAVN LMA2 HUMAN 436 ETQKE IAEDELVAAEALLKKVKKLF. LMA2 HUMAN 437 QSQAHQQRGLFPAVLNLASNALITT. LMA2 HUMAN 438 NSARDAVRNLTEWPQLLDQLRTVE LMA4 HUMAN 439 VKLSNLSNLSHDLVQEAIDHAQDLQ LMA4 HUMAN 440 GQPLPWELRLGLLLSVLAATLAQAP LMB2 HUMAN 441ALDEKVRERLRMALERVAVLEEELE LPA3 HUMAN 442 DNTKSQLAMSANFLGSVLTLLQKQH LPC4 HUMAN 443 LLGGIKVKLLRGLLPNLVDNLVNRV LPC4 HUMAN 444 KGNKSSYHRLSELVEHVFPLLSKEQ LPN2 HUMAN 445 KNHKSTYERLGEWΞLLFPPVARGP LPN3 HUMAN 446 TVLDQQQTPSRLAVTRVIQALAMKG LPRC HUMAN 447 PGPLPSLPLEPSLLSGWQALRGRL LR10 HUMAN 448 SKTEQPAALALDLVNKLVYWVDLYL LR1 B HUMAN 449 AAKYRDHVTATQLIQKI INILTDKH LRBA HUMAN 450 VSNMS I TERLEHALEKAAPLLRE IF LRBA HUMAN 451 LSLLLVTSVTLLVARVFQKAVDQS LYII HUMAN 452 DHLSQSKVIETQLAKPLFDALLRVA LYST HUMAN 453 SQAELVQKGSELVALRVALREARAT LZT2 HUMAN 454 SRHHHGSSIAGGLVKGALSVAASAY M 172 HUMAN 4555MAAGLYSELFTLLVSLVNRALKSS M18A HUMAN 456AWFSYIATLLYWHAVFSLIRWKS MAL HUMAN 457 PPLNTIRDVSLKIAEKIVKDAYQEK MAOX HUMAN 458TFNDDIQGTASVAVAGLLAALRITK MAOX HUMAN 459KRKTTAAGGESALAPSVFKQAKDKV MAP2 HUMAN 460KGKIKVIKKEGKAAEAVAAAVGTGA MAPB HUMAN 461FHPAVRNSSEVKFAVQAFAALNSNN MC3A HUMAN 462RRLGGLASQEPGAIIELFNSVLQFL MC3A HUMAN 463PPAAARAGGSPTAVRSILTKERRPE MCDL HUMAN 464 RFSWDMAALGGVLGALLLLALLGL MCDL HUMAN 465RGTVARGAGAGVWKDAAAPSQPLR MCDL HUMAN 466AAFMKKYIHVAKIIKPVLTQESATY MCM3 HUMAN 467DLRRKNEKRANRLLNNAFEELVAFQ MCM3 HUMAN 468PFPSWRFPGLLLAAMVLLLYSFSDA MCP HUMAN 469 DVSTLHVQKIISAISELLERLKSYG MDN1 HUMAN 470 LATHRSTAKLLSVLAQVFTELAQKG MDN1 HUMAN 471 SLRNFYSHSLSGAVSNVFKILQPNT MDN1 HUMAN 472VTSIAKAPAVQDLLTRLLQALHIDG MDN1 HUMAN 473 QQLQKQLKEAEQILATAVYQAKEKL MED4 HUMAN 474RLRHWWAIALTTAVTSAFLLAKVIL MENT HUMAN 475MNMAKTSQTVATFLDELAQKLKPLG MEPD HUMAN 476KDYTKVDRSAAYAARWVAKSLVKGG METK HUMAN 477KDYTKVDRSAAYAARWVAKSLVKAG METL HUMAN 478VSPLKHFVLAKKAITAIFDQLLEFV MFN1 HUMAN 479 JYFPMIFRKARΞFIEILFGISLTEV MGC3 HUMA N 480RKPRFMSAWAQVIIASILISVQLTL mgr1 HUMAN 481 LKTAADRQAEDQVLRKLVDLVNQRD MIL1 HUMAN 482YLMVMIGMFSFIIVAILVSTVKSKR MIR1 HUMAN 483SSQPLTLEHVRYFLYQLLRGLKYMH MK07 HUMAN 484GARLALDEHVQFLVYQLLRGLKYIH MK11 HUMAN 485VKKPAGPSISKPAAKPAAAGAPPAK MLEY HUMAN 486RGERLHMFRVGGLVFHAIGQLLPHQ MLL2 HUMAN 487WYVLVIISDLMT11GSILKMEIKAK MLN2 HUMAN 488 SGKPRRKSNLPIFLPRVAGKLGKRP MLPH HUMAN 489 KQKHFNERΞASRWRDVAAALDFLH MNK1 HUMAN 490 KHLERRDAESLKLLKEAIWEEKQGT M0C3 HUMAN 491 VYRKMPEHVLGRIAVAWKGLTYLW MPK5 HUMAN 492DLIFLRGIMESPIVRSLAKVIMVLW MPP2 HUMAN 493STATNPQNGLSQILRLVLQELSLFY MPP4 HUMAN 494ARRRLWGFSESLLIRGAAGRSLYFG mppb HUMAN 495YYAKAFSKDLPRAVEILADIIQNST MPPB HUMAN 496HSVMDTLAVALRVAEEAIEEAISKA MRIP HUMAN 497AVSSAHRAASLEAVSYAIDTLKAKV MS2L HUMAN 498 DECORRILMGLPSFLETVARKELENL MSH5 HUMAN 499SPSLQEKLKSFKAALIALYLLVFAV MSRE HUMAN 500 SVRPLEFTKVKTFVSRI IDTLDIGP mTn3 HUMAN 501 SVRPQNFELVKRFVNQIVDFLDVSP MTN4 HUMAN 502 EEPYRRRNQILSVLAGLAAMVGYAL MTX1 HUMAN 503 VQLRRGLVGSRPWTRWI KAQGLV MU5B HUMAN 504 LVDKGTEDKWDWRNLVFHLKKGY MX1 HUMAN 505 KITINSHTTAGΞWEKLIRGLAMED MY10 HUMAN 506 AGKQGLGPPSTPIAVHAAVKSKSLG MYCD HUMAN 507 VSSTVGAAAVSALAGGALNGVFGRR MYCT HUMAN 508 GKTVNTKRVIQYFATIAVTGΞKKKE MYH4 HUMAN 509 VTKGQTVQQVYNAVGALAKAVYDKM MYH1 HUMAN 510 VTKGQTVEQVSNAVGALAKAVYEKM MYH2 HUMAN 511VTKGQTVQQVYNAVGALAKAIYEKM MYH4 HUMAN 512 GKTVNTKRVIQYFATIAVTGEKKKD MYH8 HUMAN 513 VTKGQTVQQVYNAVGALAKAVYEKM MYH8 HUMAN 514 ΞQLKRNSQRAAEALQSVLDAEIRSR MyHD HUMAN 515 QAAVQLALRAGQI IRKALTEEKRVS Myo2 HUMAN 516 IVWRRFKWVIIGLLFLLILLLFVAV MYOF HUMAN 517 PLDDLDREDEVRLLKYLFTLIRAGM N 107 HUMAN 518 IVLKNHHSRLSDLVNTAILIALNKR N133 HUMAN 519 HΞAQLSEKISLQAIQQL VRKS YQAL N155 HUMAN 520 QGMSRVASVSQNAIVSAAGNIARTI N155 HUMAN 521 YRRAAE KWEVAE WLΞVFYKLLRDY N205 HUMAN 522 LFTFQKHVFSPIFIIGAFVAIFLGR NAH7 HUMAN 523TSGRRWREISASLLYQALPSSPDHE NAL1 HUMAN 524AEKQPPFTL IRSLLRKVLLPΞSFLI NAL5 HUMAN 525 AENMS I TAKLΞRALEKVAPLLRE I F- NBEA HUMAN 526 STVKIQNPMILKWATLLKNSTPSA NBEA HUMAN 527KGGVGKSTFSAHLAHGLAΞDENTQI NBP1 HUMAN 528 FIQEFPGSPAFAALTSIAQKILDAT NBP2 HUMAN 529 WRGPKKNALIKQFVSDVAWGΞLDYL NBP2 HUMAN 530 SARDLQNLMSWRFIMDLVSSLSRTY NEP HUMAN 531 SKLPKDQQDAKHILEHVFFQWEFK NGD5 HUMAN 532 EPEPITASGSLKALRKLLTASVEVP NIBA HUMAN 533 GRVLYREDTSPAVLGLAARYVRAGF NID 2 HUMAN 534 RTKRLHWSRLLFLLGMLi IGSTYQH^■ NKX1 HUMAN 535 YKFGSRHSAESQILKHLLKNLFKIF NNMT HUMAN 536 IVDNGVAPPARRLLRLWRFAPQVE NPHN HUMAN 537 GPVSARVIKALQWDHAFGMLMEGL NPP3 HUMAN 538 SPESDAPGPVYAAASLAVSWVLRSV NPR1 HUMAN 539SPPALPLASSFTALLQAAYESQALR NPR1 HUMAN 540TMPHLSMQQVLLAAKQVLLYLRSTV NPR1 HUMAN 541 TQDMRLTFTLALFIAKAALQILKPE NPR1 HUMAN 542 VLQQRLGELERQLLRKVAELΞDEKS NPX2 HUMAN 543 ISPLIQKSAANWLFDIFVNILTHN NRDC HUMAN 544 VQGNVTSTESMDFLKYWDKLNFKP NRDC HUMAN 545 GFKLLWILLLATLVGLLLQRLAARL NRM2 HUMAN 546 VYVRDLGHVAL YWMWS VAYLGF NRM2 HUMAN 547 RKPGNVLKTLEPILITI IAMSALGV NRP1 HUMAN 548 DYVPIGPRFSNLVLQALLVLLKKAP NSF HUMAN 549 YMIHHGDWFSGKAVGLLVLHLSGMV NSMA HUMAN 550 ILWSGWASNSNYALIGALRAVAQTI NU1M HUMAN 551 IRFLKGMGYSTHAAQQVLHAASGNL NUB1 HUMAN 552 KQIQRTKRGLEILAKRAAETWDPE NUB1 HUMAN 553 ΞLLNMYVGESERAVRQVFQRAKNSA NVL HUMAN 554 RLSLVAGAYVAGLISALVRTVSAFT O9Q1 HUMAN 555 WRRLPGAGLARGFLHPAATVEDAAQ ODBB HUMAN 556 IVFNAHIKGVETIANDWSLATKAR 0DP2 HUMAN 557 DFLNNPFKQENVLARMVASRITNYP OFD 1 HUMAN 558 NLRRDVYIRIASLLKTLLKTEEWVL 0FU2 HUMAN 559 AAETLLSSLLGLLLLGLLLPASLTG. 0S9 HUMAN 560 KRΞNPQLKQIEGLVKELLΞREGLTA. OS9 HUMAN 561 KRVAYARVPSKDLLFSIVEEETGKD OTOF HUMAN 562 TVPVFFNQAERRAVLQAARMAGLKV OXRP HUMAN 563 VGGATRVPRVQΞVLLKAVGKEΞLGK OXRP HUMAN 564 DQKAYKEGKLQKALΞDAFLAIDAKL P2CG HUMAN 565 TKYKMGGD IANRVLRSLVΞASSSGV P2G4 HUMAN 566 NRPS IPYAFSKFLLPIWRYLADQN P4R1 HUMAN 567 HRSPQLLLELDNVISVLFQNSKERG P52K HUMAN 568 HRHMRTIREVRTLVTRVITDVYYVD P531 HUMAN 569 AEQFAPPDIAPPLLIKLVEAIEKKG P85A HUMAN 570 RQVQETLNLEPDVAQHLLAHSHWGA PARC HUMAN 571 VAMGEMΞADVQALVRRAARQLAESG PARC HUMAN 572 VΞLLTNQVGEKMVWQALRLLYLLM PARC HUMAN 573 HPPYLVSKELMSLVSGLLQPVPERR PASK HUMAN 574 STMSPLGSGAFGFVWTAVDKEKNKE PASK HUMAN 575 TSRRRNVTFSQQVANILLNGVKYES PAST HUMAN 576 LFTLDEQSGLLTVAWPLARRANSW PC16 HUMAN 577 KVTDHGKPTLSAVAKLI IRSVSGSL PC17 HUMAN 578 SHINAATGTSASLVYRLVSKAGDAP PCH9 HUMAN 579NGETLVFEESNWFIINVIKLVWRYG • PCL1 HUMAN 580 KADVNLSHSERGALQDALRRLLGLF PCN2 HUMAN 581 NQRKAAHSAELEAVLLALARIRRAL PCN2 HUMAN 582 ISVQLKKTSEVDLAKPLVKFIQQTY PD6I HUMAN 583 NRS IAQMREATTLANGVLASLNLPA PD6I HUMAN 584 PAKTMQGSEWNVLKSLLSNLDΞVK PD6I HUM AN 585 VTEFNSQTSAKIFAARILNHLLLFV PDA2 HUMAN 586 RLALFPGVALLLAAARLAAASDVLΞ PDA3 HUMAN 587 YQGGRTGEAIVDAALSALRQLVKDR PDA6 HUMAN 588 FSEMLAASFS IAWAYAIAVSVGKV PEND HUMAN 589 RHKIPVPIPIEVIVTIIATAISYGA PEND HUMAN 590 ELLSKYIGASBQAVRDIFIRAQAAK PEX1 HUMAN 591 ΪPGPPQLLVSRALLRLLALGSGAWV PEX6 HUMAN 592 EFFTHLDKRSLPALTNI IKILRHDI PH4H HUMAN 593 FESIGKFGLALAVAGGWNSALYNV PHB HUMAN 594 MDRSSKRRQVKPLAASLLEALDYDS PHFE HUMAN 595 TVTWGNYGKSYSVALYLVRQLTSSE pia4 HUMAN 596 TTAKRRLKQSVHLARRVLQLΞKQNS PIBF HUMAN 597 RETAΞPFLFVDΞFLTYLFSRENS IW P1G2 HUMAN 598 LVSTLVPLGLVLAVGAVAVGVARAR pIgR HUMAN 599 EVARGKRAALFFAAVAIVLGLPLWW PIGS HUMAN 600 FTSFDQVAQLSSAARGLAASLLFLL PKD1 HUMAN 601 GAWARWLLVALTAATALVRLAQLGA PKD1 HUMAN 602 LHAAVTLRLEFPAAGRALAALSVRP PKD1 HUMAN 603 RMVASQAYNLTSALMRILMRSRVLN PKD1 HUMAN 604 ESSTNREKYLKSVLRELVTYLLFLI PKD2 HUMAN 605 LLHSRNEGTATYAAAVLFRI SEDKN PLAK HUMAN 606 RQFRTGKVTVΞKVIKILITIVEEVE PLE1 HUMAN 607 RQFRTGRITVEKI IKI I ITWEEQE PLE1 HUMAN 608 JPGPRFLLLLPLLLPPAASASDRPR PL03 HUMAN 609 DILKPGGGTSGGLLGGLLGKVTSVI PLUN HUMAN 610 VLRGLDITLVHDIVNMLIHGLQFVI PLUN HUMAN 611 LGQVPLIVGILLVLMAWLASLIYR PM17 HUMAN 612 PDKRQILLQEΞKLLLAVLKTSLIGM PMS2 HUMAN 613 SLNPSLMAPSQFAAGGALLSLNPGT PO21 HUMAN 614 ITLG YTQADVGLILGVLFGKVFSQK P05L HUMAN 615 ITLGYTQADVGLILGVLFGKVFSQT P05M HUMAN 616 LFSKYTNSKIPYFLLFLIFLITVYH PP3A HUMAN 617 DFVDRGFYSVETFLLLLALKVRYPD PP4C HUMAN 618 LLLARAASLSLGFLFLLFFWLDRSV PPAP HUMAN 619 LVQIIKKTESDAALHPLLQEIYRDM PPAR HUMAN 620 QTWLSALRPSGPALSGLLSLEAEΞN PPCS HUMAN 621 SMEGVTFLQAKQIALHALSLVGEKQ PP04 HUMAN 622 WGTTTTTPSPSAIKAAAKSAALQV PRCC HUMAN 623 NDLTRNRFFENPALWELLFHS IHDA PRES HUMAN 624 PPSGIHLSASRTLAPTLLYSSPPSH PS11 HUMAN 625 KRLFNVDRHVGMAVAGLLADARSLA PSA3 HUMAN 626 RSNFGYNIPLKHLADRVAMYVHAYT PSA3 HUMAN 627 VLYEDEGFRSRQFAALVASKVFYHL PSD1 HUMAN 628 HLLRYGEPTLRRAVPLALALISVSN PSD2 HUMAN 629 SYGHMWS QNATNL VS SLLTLLKQLE PTN5 HUMAN 630 KSLLDPKVAARLAVAEALTNLVFAL PUR4 HUMAN 631AQVGLGVGTSLLALGVIIIVLMYRR PXB1 HUMAN 632 TRLLSMKGTLQKFVDDLFQVILSTS PXB1 HUMAN 633 LWPLPGRDLLLVARGLAGKLSAGV PXB3 HUMAN 634 SELSARQMHLARFLRMLLRLADEFG Ra51 HUMAN 63! IDLVSKLLYSRGLLIDLLIKSNVSR RAE1 HUMAN 636IDLVSKLLYSQGLLIDLLIKSDVSR RAE2 HUMAN 637AIFTGHSAVVEDVAWHLLHiESLFGS RBB7 HUMAN 638EHPAIRTLSARAAAAFVLANENWIA RBP6 HUMAN 639 MARGGRGRRLGLALGLLLALVLAPR RCN 1 HUMAN 640 FEΞYLRALDVNVALRKIANLLKPDK RET1 HUMAN 641MEDYLQALNISLAVRKIALLLKPDK RET5 HUMAN 642 HSYVSVKAKVS S IAQE I LKWAEKI RGE5 HUMAN 643RLLWRLPAPVLWLRYLFTFLNHLA RHG4 HUMAN 644PKPWPKTNVKALVPNLLRAIEAGI RHG5 HUMAN 645YPRKFNETQIKQALRGVLESVKHNL RHG5 HUMAN 646KKNFESLSEAFSVASAAAVLSHNRY RIB2 HUMAN 647VSTTVAKAMAREAAQRVAESSRLEK RIP2 HUMAN 648DPVLRKKNGATPFILAAIAGSVKLL_, RN5A HUMAN 649AHKATNKSSETLALLEILKHIAITE RP1 HUMAN 650LNYVASQPKLAPFVIQALIQVIAKI RP17 HUMAN 651 YGDNHFDNVLQAFVKMLLSVSHSDL RP17 HUMAN 652WVSVLLKKTEKAFLAHLASAVAELR RPL1 HUMAN 653FMKLVGMPYLHEVLKPVISRVFEEK RSG4 HUMAN 654QHADPQTSRSLLLLAKAVQSIGNLG RSG4 HUMAN 655INLKRTWΞKLLLAARAIVAIENPAD RSSA HUMAN 656SQGMVGQLAARRAAGWLEMIREGK RUV2 HUMAN 657EQGKRNFSKAMSVAKQVFNSLTΞYI RYR1 HUMAN 658IDEASWMKRLAVFAQPIVSRARPEL RYR1 HUMAN 659NYLSRNFYTLRFLALFLAFAINFIL RYR1 HUMAN 660QAGKGEALRIRAILRSLVPLΞDLVG RYR1 HUMAN 661 RVRRLRRLTAREAATAVAALLWAAV RYR1 HUMAN 662TAAAGATARWAAAGRALRGLSYRS RYR1 HUMAN 663VEKSPHEQΞIKFFAKILLPLINQYF RYR1 HUMAN 664ARNFYNMRMLALFVAFAINFILLFY RYR2 HUMAN 665 DTPSIEKRFAYSFLQQLIRYVDEAH RYR2 HUMAN 666EQGQRNFSKAIQVAKQVFNTLTEYI RYR2 HUMAN 667GEHFPYΞQEIKFFAKWLPLIDQYF RYR2 HUMAN 668EESGMAWKΞILNLLYKLLAALIRGN RYR3 HUMAN 669HYLARNFYNLRFLALFVAFAINFIL RYR3 HUMAN 670QTGKGEAIRIRSILRSLVPTEDLVG RYR3 HUMAN 671 TEKSPRDQEIKFFAKVLLPLVDQYF RYR3 HUMAN 672TLYQQARLHERGAAEMVLQMISASK RYR3 HUMAN 673FLSGQGLAGIFAALAMLLSM ASGVD S292 HUMAN 6745NSLAYYNMANGAVIHLALKERGGR S3A1 HUMAN 675LQLLSGHPPASEAVASVLSFLYDKK S3T2 HUMAN 676QISLEGYEKALEFATLAARLSTVTG S3T2 HUMAN 677 HTSTLAAMKLMTALVNVALNLS INM SA2 HUMAN 678ΆPPVAAGVGAVLAΆGALLGLVAGAL SBN1 HUMAN 679 DMVKSKWGLALAAWTVLSSLLMSV SCAP HUMAN 680 GMTWSHGLSVSKVLHKAFVEVTEEG SCC2 HUMAN 681 AGKSGGSAGEITPLEALARSESKRD SEN6 HUMAN 682 ILQKYIERI ITRFAPMLVPYIWQMQ SGT1 HUMAN 683 DAQLDYHRQA.VQILDELAE KLKRRM SH31 HUMAN 684 FKKDPPLAAVTTAVQELLRLAQAHP SKIW HUMAN 685 RGLGVHHSGILPILKEIVEMLFSRG SKIW HUMAN 686 RRIDLSKENQIAE IAPDAFQGLRSLN SLT1 HUMAN 687 SSAGPVRPELWLLLWAAAWRLGASA SLT1 HUMAN 688VTVLFALVLSGALIILVASPLRALR SM4A HUMAN 689 PGGMNRKTQETAVAMHVAAMS I QMR SMF1 HUMAN 690 TSTWLDDVEERLFVATALLPEETET SNE1 HUMAN 691 IMSQLARHTSPSVISDLFTDIKKGH SNE2 HUMAN 692 QHVDQRRQGLΞDFLRKVLQNALLLS SNXA HUMAN 693 PDIPEWRKDIGNVIKRALVKVTSVP S0R3 HUMAN 694 LQHRHRLPDLQAILRRILNEEETSP SP90 HUMAN 695 HNNRRLQAESESAATRLLLASKQLG SPA1 HUMAN 696 RAAPRGPGAELQAAGSLVWGVRAAP SPA1 HUMAN 697 RNVFFSPMSISSALAMVFMGAKGST SPB8 HUMAN 698 QLAKQKAQEAEKLLMNVISKLLPTN SPC2 HUMAN 699 SLLDKHSQIINKFVNSVINTLKSTV SPC2 HUMAN 700 IQSLTMYPRLGGFVMNILSRLIMKQ SPK HUMAN 701 VHPAISSINLTTALGSLANIARQRP SPK HUMAN 702 PFYDPEGGS ITQVARWIERIARKG SP01 HUMAN 703 PLKLSRTPALLALALPLAAALAFSD SP01 HUMAN 704 MVGPAPR RRLRPLAALALVLALAPG SPUF HUMAN 705 KTGSFKIRGALNAVRSLVPDALERK SRR HUMAN 706 KHGPGRWWLAAVLIGLLLVLLGIG ST14 HUMAN 707 SNRGLTKENLVFLAQKLFNNSSSHL ST5b HUMAN 708DASKALLGRLTTLIELLLPKLEEWK STA2 HUMAN 709 KGVDLRNAQVTELLQRLLHRAFWE STA2 HUMAN 710 PAAGLGPGHARHVLRSLVNQSVQDG STRC HUMAN 711 PDNTGRGYVLRRILRRAVRYAHEKL SYA HUMAN 712 RRPIMSMHTATHILNFALRSVLGEA SYA HUMAN 713 FLAGETESLADIVLWGALYPLLQDP SYM HUMAN 714 YHQLLEKVRIRDALRSILTISRHGN SYM HUMAN 715 FLKGVLVFLEQALIQYALRTLGSRG SYS HUMAN 716 WEVGVYAAGALALLGIAAVSLWKLW SYTC HUMAN 717 WLYLQDQNKAADAVGEILLSLSYLP SYTC HUMAN 718 TRPWLLDPKILKFWFIVAVLLPVR T10d HUMAN 719 TWKDRFPGYLMNFASILFMIALTFS T16b HUMAN 720 EWKLHPHΞLNMLLSKVLI YLRSAN HUMAN T172 T172 721 YALAVRQDVINTLLPKVLTRIIEGL HUMAN 722 MADPDVLTEVPAALKRLAKYVIRGF T2EA HUMAN 723 FSQGKMYGYVDTLLTMLAMLLKVAM T3C3 HUMAN 724 KLQEIMMHVIWAALAFAAIQLLGML T4S8 HUMAN 725 DTBPAKQTSLDAVAQAWDRVKRIH TAB1 HUMAN 726 LEFAIMRIBALKLARQIALASRSHQ TAC2 HUMAN 727 RANTHIRD FLQVFIYRLFWKSKDRP TAF1 HUMAN 728 DIBRPTYTMLMRLISQIVSSITASL TBA8 HUMAN 729 QTIIAGWREATKAAREALLSSAVDH TCPB HUMAN 730 LDTYLGKYWAIKLATNAAVTVLRVD TCPQ HUMAN 731 KTFGAFS IHPAMMAAAQAALQS SWGm TDBP HUMAN 732 QKLYIPRSTATAALGAAARLATSRS TDR5 HUMAN 733 EEEEKVSQPEVGAAIKIIRQLMEKF TE2I HUMAN 734 LSKKLIYFQLHRALKMIVDPVEPHG TF1 B HUMAN 735 GSRGTTAGSSGDALGKALASIYSPD TFE2 HUMAN 736 LPSIPAQPISADIASRLLRKLKGPV TFR2 HUMAN 737 KTLHKVLQGRLPAVAQAVAQLAGQLL TFR2 HUMAN 738 QRDAWGPGAAKSAVGTAILLΞLVRT TFR2 HUMAN 739 EAFSHFTKIITPAITRWDFAKKLP THB2 HUMAN 740 ΞGQSQQFSVSEKTLLKEAIRAIFPSR THYG HUMAN 741 PASLGKWKKEPELAAFVFKTAWLV TIAM HUMAN 742 RLPSSWALFSPLLAGLALLGVGPVP TIP HUMAN 743 AIKKKLVQRLEHAAKQAAASATQTI TLN 1 HUMAN 744 GQLLRGVGAAATAVTQALKTELLQHV TLN1 HUMAN 745 QQLAAFSKRVAGAVTELIQAAEAMK TLN2 HUMAN 746 SELLKQVSAAASWSQALHDLLQHV TLN2 HUMAN 747 KVLKrLAYNKIKTKIADEAFYGLDKrLQ TLR5 HUMAN 748 GREKFKSRGVGELARLALVISELEG TM26 HUMAN 749 QGH QFLRERΞEHLLΞQLAKLEQELT TM26 HUMAN 750 LKISPVAPDADAVAAQILSLLPLKF TMS3 HUMAN 751 RKARGYLRLVPLFVLLALLVLASAG TMS6 HUMAN 752 YAHPQQKVAVYRALQAALAΞSGGSP TRAD HUMAN 753 YVNYVLSEKSSTFLMKAAAKWESK TRF1 HUMAN 754 AAPAPGAPLLPLLLPALAARLLPPA TRFM HUMAN 755 THIKAPEQQVKNILNELFQRENRVL TRIO HUMAN 756 PHMRKNIKGIHTLLQNLAKASPVYL TRKB HUMAN 757 QHALRNRRLLRKVIKAAALEEHNAA TSC1 HUMAN 758 KSDRLISLQSASWYDLLTIALGRR TT7B HUMAN 759 QMKAKRTKEAVEVLKKALDAISHSD TTC6 HUMAN 760 LARQINHPΞLHMVLSNLAAVLMHRΞ TTCJ HUMAN 761 VAPHGRGPGLLPLLAALAWFSRPAA TTCJ HUMAN 762 HSRTSWVPWLGVLTALVTAAALAL TYO3 HUMAN 763 PLPLPPPPRLGLLLAALASLLLPES TYO3 HUMAN 764 WSWLLGAAMVGAVLTALLAGLVSLL TYRO HUMAN 765 LLHDDRGPVLEALVARAIRNIEMTQ U520 HUMAN 766 RGGGQI IPTARRWYSAFLMATPRL U5S1 HUMAN 767 LFYQDKLKSLHQLLEVLLALLDKDV UB24 UB25 HUMAN 768 YGSGPKRFPLVDVLQYALEFASSKP HUMAN 769 NRKFVDPSAALDLLKGAFRSSEEQQ UB28 HUMAN 770 KIEKVFSKLLYPIVRGAALSVLKYM_, UB35 HUMAN 771 RFLNLLMNDAIFLLDEAIQYLSKIK UB4A HUMAN 772 DI PS ADRHKS KL IAGKI I PAIATTT UBA1 HUMAN 773 GIPPVNRAQSKRIVGQIIPAIATTT Ubal HUMAN 774 PVDKVAAMREFRVLHTALHSSSSYR ULSB HUMAN 775 AARFAKTKEEVΞAΆKΆAALLAKQAE UXD2 HUMAN 776 HLPBKQDTFAEKLVTQI IKNLQVKI V13A HUMAN 777MLNRQDPFTVHMAARIIAKLAAWGK VATH HUMAN 778 ALIEGKNKTVSTLVIQAANVSALYK RPH2 HUMAN 779 ΞVITDNLPGSIRAWNIFL VAKALL Viaa HUMAN 780 VRWLQESRRSRKLILFIVFLALLLD VMT2 HUMAN 781 FVIATTRQRLFQFIGRAAEGAΞAQG VP18 HUMAN 782 WIΞMGSRLDARQLIPAL VNYSQGGE VP18 HUMAN 783 MRDVNKKFSVRYFLNLVLVDEEDRR VP26 HUMAN 784NSFKMKMSVILGIIHMLFGVSLSLF VPP1 HUMAN 785 NSYKMKMSVILGIVQMVFGVILSLF VPP4 HUMAN 786 MQRΞGGPSQIGDALGFAVRYLTSEM VWF HUMAN 787 KDTPSGISKVRKILFTLAKQSKALG WD10 HUMAN 788 LVGLKNGQILKIFVDNLFAIVLLKQ WD10 HUMAN 789 SFEQGGSEFVPVWSRLVLRSMSRR. WD10 HUMAN 790 IPADPEAGGIGRWNGAFMVLKGHR WD22 HUMAN 791 RQNKDΞRYSIKDLLNHAFFQEETGV WNK1 HUMAN 792 RKTSKSKLKAGKLLNPLVRQLKWA WNK2 HUMAN 793 RTDKNERFTIQDLLAHAFFREΞRGV WNK4 HUMAN 794 TAFGADTEGSQWIIGYLLQKVISNL XP04 HUMAN 795 LGLNDETMVLSVFIGKIITNLKYWG XP07 HUMAN 796 LNYLATRPKLATFVTQALIQLYARI XP07 HUMAN 797 AMFVEYRKQLKLLLDRLAQVSPELL XPOT HUMAN 798 QNWQTTRFMEVΞVAIRLL YMLAEAL XPOT HUMAN 799 FPAATSGRMVEAFARRALWDAGLNY XPP2 HUMAN 800 LLKASHVRDAVAVIRYLVWLEKNVP XPP2 HUMAN 801 SILTQPHLYSPVLISQLVQMASQLR Y310 HUMAN 802 ISIGTIYFRAHKLVLAAASLLFKTL Y478 HUMAN RDVLRVQGVSLTALRLLLADAYSGR 803 Y711 Y779 HUMAN 804QLEGLENATARNLLGKLINILLAVM HUMAN 805 PPLIVDRERLKKLLDLLVDKSNNLA YC40 HUMAN 806EDTKEKRTIIHQAIKSLFPGLETKT YI97 HUMAN 807 DQKTNLPEYLQTLLNTLAPLLLFRA Z294 HUMAN 808 RSREHGTLWSLIIAKLILSRSISSD • Z294 HUMAN 809VLASAYTGRLSMAAADIVNFLTVGS- Z297 HUMAN 810 ΪRNKMDPPRSSIFLQEVITTVYGYK ZAN HUMAN 811 VDLAYSNYHVKQFLEALLRNSAAPS ZBT8 HUMAN 812 LKLRSLRVNVGQLLAMI VPDLDLQQ ZCW3 HUMAN 813ELQKQAELMEFEIALKALSVLRYIT ZM10 HUMAN 8 14 DALHMLTDLSAIILTLLALWLSSKS ZNT4 HUMAN  8151MD C; EENYSAAS, KΆVRQVLHQLKRLG ZW10 HUMAN

captions

FIG. 1 Identification of peptides that inhibit the interaction of AKAP proteins with PKA. A library of proteins derived from. derived from the PKA binding domain of AKAP18δ was synthesized on a membrane. The membrane was incubated with radioactively labeled RIIa regulatory and RIIβ subunits of PKA (RII overlay experiment). Each black dot represents a peptide to which the RII subunits have bound ^(■ detected using a phosphoimager). The amino acid sequences of the peptides can be read off with the given abbreviations (single-letter coding). Vertical: sequence of the wild type PKA binding domain of AKAP185; horizontal: the 20th amino acids used for the substitution of the wild-type sequence.

Figure 2 Identification of peptides derived from AKAPlδδ which inhibit the interaction of AKAP proteins with the regulatory RIICK and RII / 3 subunits of PKA. A. Peptides derived from the PKA binding domain of AKAP185 were synthesized on two membranes. The membranes were incubated with radioactively labeled RIICK (top row) or RII / γ (lower row) subunits of PKA (RII overlay experiment). Each black dot represents a peptide to which the RII subunits have bound (detected with a phosphoimager). For quantification, the signals became densitometrically and related to the signal obtained for AKΑP18δ-wt. B: The amino acid sequences of the peptides (single letter coding) given in A.

FIG. 3A shows peptides derived from AKAPlδδ which bind the RIIa and RII / 3 subunits of the PKA to different degrees. A. Peptides 1-19 derived from the PKA binding domain of AKAPlδδ were synthesized on two membranes. Membranes were incubated with radioactively labeled RIIa regulatory (upper panel) or RIIjS (lower panel) subunits of PKA (RII overlay experiment). Each black dot represents a peptide to which the RII subunits have bound (detected with a phosphoimager). For quantification, the signals were densitometrically evaluated and related to the signal obtained for AKAP18δ-wt. The peptide 7 is highlighted by red writing due to the large difference in the binding to the two RII subunits. B: The amino acid sequences of the peptides (single-letter coding) designated A in 1-19.

Figure 4 Different peptides, derived from AKAPlδδ, bind RIIa and RII / 3 subunits of PKA to different degrees. Two libraries of peptides derived from peptide 7 of Figure 3 were synthesized on two membranes. The membranes were incubated with radioactively labeled RIIa regulatory (left side) or RIIjS subunits of PKA (right side) (RII overlay experiment). Each black dot represents one Peptide to which the RII subunits have bound (detected with a phosphoimager). The amino acid sequences ^"of peptides may be using the specified code (Einbuchstabenkodierung) off. Vertical: sequence of peptide 7; horizontal: the 20 amino acids used to substitute the wild-type sequence. Horizontal and vertical rows are additionally labeled by Arabic numerals. These coordinates facilitate the assignment, for example, 10/11 means row 10 peptide 11. The peptides listed below are labeled with AlδδRIIαHsl and 2, respectively, corresponding to their binding to RIIa, or A18δRII / αRnI, corresponding to their binding to RUβ. ^'

Figure 5: Identification of peptides that inhibit AKAP-PKA interactions. Candidate peptides were synthesized on a membrane and probed with. radioactively labeled RIID regulatory subunits of PKA incubated (RII overlay experiment). All black dots represent peptides that have bound regulatory PKA subunits (detected with a phosphoimager).

Figure 6. Influence of hydrogen bonding on binding between peptides and RII (alpha) subunits of PKA. (A, B) Comparative schematic representation of the interaction between RII (alpha) and the peptides AKAP18 (delta) -wt or AKAP18 (delta) -L314E and between RII (alpha), Ht31 or AKAi ₃ . Rllalpha is shown as a rectangle and through selected amino acids, the peptides are represented by their amino acid sequence. Amino acids as a partner of a hydrogen bond are connected by a broken line. Amino acids of peptides that are in positions for hydrophobic Molecular contacts are highlighted in green (Pos. Of the amino acids of AKAP18 (delta) -wt compared to the protein are indicated). (C, D) To investigate the influence of amino acids on bond strength, alanine-substituted peptides were synthesized on membranes, screened for RII (alpha) binding by RII overlay, and quantified densitometrically. The peptides, starting from AKAP18 (delta) -L314E, were substituted in all possible combinations for the amino acids that can form hydrogen bonds (see A). The quantification for all peptides sorted by affinity is shown in C. The quantification for all single substitutions (as indicated), as well as representative "spots" from an RII overlay (above) are shown in D. references

Alto, NM, Soderling, SH, Hoshi, N., Langeberg, LK, Fayos, R., Jennings, PA, Scott, JD Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. Proc. Natl. Acad. Be. USA 100, 4445-4450, 2003.

Bregman, D.B., Bhattacharyya, N., Rubin, C.S. High affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase H-B. J. Biol. Chem. 264, 4648-4656, 1989.

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Frank, R. Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedroή 48, 9217-9232, 1992. ^{■ '•} . ^{■ '}

Fraser, ID, Tavalin, SJ, Lester, LB, Langeberg, L. ^" K., ^" Westphal, AM, Dean, RA, Marrion, NV, Scott, JD A novel lipid-anchored A-kinase anchoring protein facilitating cAMP-responsive membrane events, EMBO J. 17, 2261-2272, 1998.

Henn, V., Edemir, B., Stefan, E., Wiesner, B., Lorenz, D., Theilig, F., Schmitt, R., Vossebein, L., Tamma, G., Beyermann, M., Krause, E., Herberg. FW, Valenti, G., Bachmann, S., Rosenthal, W., Klussmann, E. Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells , J. Biol. Chem. JBC published March 22, 2004 as doi: 10.1074 / jbc.M312835200

Hulme J.T., Lin, T.W., Westenbroek, R.E., Scheuer, T., Catterall, W.A. Beta-adrenergic regulation requires direct anchoring of PKA to cardiac CaVl.2 channels via a leucine zipper interaction with A kinase anchoring protein 15. Proc. Natl. Acad. Be. USA 100, 13093-13098, 2003.

Klussmann, E., Marie, K., Wiesner, B., Beyermann, M., Rosenthal, W. Protein Kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells. J. Biol. Chem. 274, 4934-4938, 1999.

Klussmann, E. Protein kinase A. Online pharmacology reference database. Elsevier Science Inc., Amsterdam, The Netherlands. In print.

Kramer, A., Schneider-Mergener, J. Synthesis and Screening of peptide libraries on continuous cellulose membrane supports. Meth. Mol. Biol. 87, 25-39, 1998.

Tasken, K, Aandahl, E.M. Localized effects of cAMP mediated by distinct pathways of protein kinase A. Physiol. Rev. 84, 137-167, 2004.

Claims

claims

1. Protein kinase A / protein kinase A anchor protein decoupler, characterized in that the decouplers are derived either (i) from an AKAP185 or (ii) from a protein other than AKAP18b and according to (i) at least 8 H-bridging amino acids or according to (ii) have the general formula (1) XXXXXXXXX [AVLISΞ] XX [AVLIF] [AVLI] XX [AVLI] [AVLIF] xx [AVLISE] XXXX (1) where X can be any of the 20 biogenic amino acids.

2. An isolated nucleic acid molecule selected from the group comprising

a) a nucleic acid molecule comprising a nucleotide sequence encoding at least one amino acid sequence according to SEQ. ID Nos. 1 - 39,

b) a nucleic acid molecule which hybridizes with a nucleotide sequence according to a) under stringent conditions,

c) a nucleic acid molecule comprising a nucleotide sequence which has sufficient homology to be functionally analogous to a nucleotide sequence according to a) or b),

d) a nucleic acid molecule which is degenerate as a result of the genetic code to a nucleotide sequence according to a) - c) and / or

d) a nucleic acid molecule according to a nucleotide sequence according to a) - d), which is modified by deletions, additions, substitutions, translocations, inversions and / or insertions and functionally analogous to a nucleotide sequence according ■ ä) ^'to d).

3. Nucleic acid molecule according to claim 2, characterized in that the nucleotide sequence indicated under c) is at least 60%, preferably 70%, preferably 80%, very particularly preferably 90% homologous to one of the nucleotide sequence indicated under a).

4. Nucleic acid molecule according to one of claims 2 or 3, characterized in that it is a genomic DNA, a cDNA and / or an RNA.

5. Vector comprising, a nucleic acid molecule according to any one of claims 2 to 4.

6. host cell comprising the vector according to claim 5.

7. An organism comprising a nucleic acid molecule according to one of claims 2 to 4, a vector according to claim 5 and / or ^' a host cell according to claim 6.

8. An organism according to claim 7, characterized in that the organism is a transgenic mouse or rat, wherein the mouse or rat ^■ preferably by the presence of the nucleic acid molecule according to one of claims 2 to 4, the vector according to claim 5 and / or the Host cell developed according to claim 6 diabetes insipidus.

9. Polypeptide encoded by a nucleic acid molecule according to one of claims 2 to 4.

10. Polypeptide according to claim 9, characterized in that a) the polypeptide comprises an amino acid sequence according to SEQ ID 1 to 39, b) the polypeptide according to a) by deletions, additions, substitutions, translocations, inversions and / or insertions modified and functionally analogous to a polypeptide according to a) and / or c) the polypeptide comprises a polypeptide having sufficient homology to be functionally analogous to a polypeptide according to a) or b).

11. Recognition molecule ^' directed against. ^• a nucleic acid molecule according to any one of claims 2 to 4, a vector according to claim 5, a host cell according to claim 6 and / or a polypeptide according to claim 1, 9 or 10 degrees.

12. Recognition molecule according to claim 11, characterized in that it is an antibody, an antibody fragment and / or an antisense construct, in particular an RNA interference molecule.

13. A pharmaceutical composition, characterized in that it comprises a nucleic acid molecule according to any one of claims 2 to 4, a vector according to claim 5, a host cell according to claim 6, a polypeptide according to claim 1, 9 or 10 and / or a recognition molecule according to any one of claims 11 or 12, optionally with a pharmaceutically acceptable carrier.

14. A pharmaceutical composition according to claim 13, characterized in that it is a Aquaretika.

15. Kit, characterized in that it comprises a nucleic acid molecule according to any one of claims 2 to 4, a vector according to claim 5, a host cell according to claim 6, a polypeptide according to claim 1, 9 or 10, a .Mognkennungsmolekül according to any one of claims 11 or 12 and / or the pharmaceutical composition according to claim 13 or 14.

16. A method ^'for modification, in particular an inhibition of AKAP.-PKA interaction comprising the steps of a) providing a nucleic acid molecule according to any one of claims 2 to 4, a vector according to claim 5, a host cell according to claim 6 and / or a polypeptide according Claim 1, 9 or 10 and b) contacting at least one product according to a) with a cell, a cell culture, a tissue and / or a target organism.

17. The method according to claim 16, characterized in that the modification takes place at a regulatory RII subunit of the ^' PKA.

18. The method according to claim 17, characterized in that the RII subunits RIIa and / or Rllß subunits.

19 .. Use of a nucleic acid molecule according to one of _. Claims 2 to 4, a vector according to claim 5, a host cell according to claim 6, a. An organism according to claim 7 or 8, a polypeptide according to claim 1, 9 or 10, a recognition molecule according to claim 11 or 12, a pharmaceutical composition according to claim 13 or 14 and / or a kit according to claim 15 for modification, in particular inhibition, an AKAP-PKA interaction.

20. Use according to the preceding claim, characterized in ^'that the interaction in a cell, cell culture, tissue, and / or a target organism is performed.

21. Use according to one of the preceding claims, characterized in that the organism according to claim 7 or 8 is used as a model for the tissue and / or cell-specific AKAP-PKA Inter¬ action, in particular as a model for diabetes insipidus.

22. Use according to one of the preceding claims, characterized in that the vasopressin-induced ^' redistribution of AQPII is modified by the modification, in particular prevented.

23. Use according to one of the preceding claims, characterized in that the polypeptide according to claim 1, 9 or 10 and / or the pharmaceutical composition according to claim 13 or 14 are used as water loss-causing agents, in particular 'as aquaretics.

24. Use according to one of the preceding claims, characterized in that the interaction of the RIIa or RII / 3 subunits of the PKA with AKAP is modified, in particular inhibited.

25. Use according to the preceding claim, characterized in that the subunits are of human or murine origin.