EP1576162A1 - Chimeric protein for the screening of agonists and antagonists of cell signalling pathways that are dependent on g-protein-coupled receptors - Google Patents

Abstract

The invention relates to a chimeric protein which is derived from a high-threshold calcium channel and which is characterised in that it consists of at least one β subunit or a fragment of same comprising at least the BID domain, which is fused at the NH2 or COOH end thereof with the I-II loop of an α1 subunit or a fragment of same comprising at least the AID domain. The invention also relates to the applications of said protein in the study of cell signalling pathways that are dependent on G-protein-coupled receptors (GPCR) and the identification of compounds that modulate the activity of G proteins.

Description

CHIMERIC PROTEIN FOR SCREENING AGONISTS AND ANTAGONISTS OF CELL SIGNALING PATHWAYS DEPENDENT ON G PROTEIN COUPLED RECEPTORS

The present invention relates to a chimeric recombinant protein derived from the ai and β subunits of high-threshold calcium channels, as well as to its applications for the study of cell signaling pathways dependent on receptors coupled to G proteins (GPCR). and the identification of compounds modulating the activity of G proteins.

The class of GPCR includes more than a thousand identified members, coded by genes representing 2 to 5% of the coding potential of the vertebrate genome (El Far and Betz, Biochem. J., 2002, 365, 329-336); there are 27 genes coding for Gα subunits, 5 for Gβ subunits, and 14 for Gγ subunits (Albert and Robillard, Cell, 2002, 14, 407-418).

Many biological processes such as synaptic regulation, response to hormones and pheromones, cell guidance (chemoattraction or chemorepulsion) or vision involve receptors coupled to G proteins. Indeed, GPCRs are capable of ensuring recognition and transduction of messages as varied as those of amino acids (glutamic acid ...), peptides (angiotensin, neurotensin, somatostatin ...), proteins (thyrotropin (TSH), follicle stimulating hormone (FSH) ...) , amines (acetylcholine, adrenaline, serotonin ...), lipids (prostaglandins, leukotrienes ...), nucleotides and nucleosides (adenosine or ATP). Ions (Ca ^"1" ), odorous and gustatory molecules, photons and pheromones are also part of the extracellular signals recognized by GPCRs (for review, see Gether, Endocrine reviews, 2000, 21, 90-113 and Albert and Robillard, supra).

The extracellular signal is transduced inside the cell via the heterotrimeric G proteins binding the guanylic nucleotides (GDP and GTP), composed of subunits called Gα, Gβ, Gγ; recognition of the extracellular signal by the RCPG results in the activation of the G proteins, which results in the dissociation of the heterotrimer into Gα and Gβγ, and the binding of the Gα subunit to GTP. Several intracellular effectors can be modulated directly or indirectly by the activation of the different Gα and Gβγ subunits, G-proteins. The effectors controlled by the Gα subunits can be enzymes (phospholipases A2 and C, adenylyl- and guanylylcyclases, c-jun kinase, tyrosine- phosphatase (SH-PTP2) ...), the activation of which will influence the rate of second messengers produced or released (phosphoinositides and diacyls-glycerols, Ca ⁺⁺ , cAMP, cGMP ...), channels (with potassium, calcium, sodium or chlorine conductances), ion exchangers (sodium / proton) or more recently kinases (Btk tyrosine kinases (Bruton's tyrosine kinase), MAP kinases (Mitogen-Activated protein kinase), (Albert and Robillard, supra).

Gβγ can also modulate the activity of effectors at least as numerous as those controlled by Gα, namely: channels (with sodium conductivity, calcium dependent on voltage (N and P / Q) or potassium with incoming rectification (GIRK: G protein inward rectifyer K + channel) ...), the so-called "classic" enzymes (phospholipases A2 and C, adenylyl-cyclase I, II, IV, tyrosine phosphatase (SH-PTP1) ...), as well as a number important kinases (phosphoinositide 3-kinase, β-adrenergic receptor kinases, c-jun kinase, MAP kinases, tyrosine kinases Btk and T-cell-specific kinase (Tsk), (for review see Albert and Robillard, supra).

Thus, the study of these signaling pathways and the search for drugs acting on these signaling pathways are of significant therapeutic interest for the search for new drugs.

It emerges from the above that the activation of G proteins and their dissociation into Gα and Gβγ subunits is the crossroads of a large number of cellular signaling and regulatory pathways. Consequently, the analysis of the activation of these G proteins makes it possible to study the cellular signaling pathways, which are dependent on receptors coupled to the G proteins, and to screen for agonists and antagonists of these signaling pathways.

To analyze the activation of G proteins, Janetopoulos et al. (Science, 2001, 291, 2408-2411) have described a technique for monitoring, in vivo in real time, the Gα-Gβ interaction. This technique, developed in the amoeba Dictyostelium discoideum, is based on the cotransfection of two constructs coding for two fluorescent chimeric proteins: Gβ-YFP and Gα ₂ -CFP. The interaction between the two chimeras induce a fluorescence transfer process (FRET) which allows real-time monitoring of their interaction in the living cell. The two chimeras constructed by Janetopoulos et al. are capable of forming a complex which interacts functionally with the cAMP receptor and can be activated by GTPγS. This fluorescence technique is adaptable to high throughput screening approaches. However, due to competition with endogenous Gα or Gβγ subunits suppressing the FRET process, this approach can only work in cells genetically deleted from their endogenous equivalent G protein. In addition, in vertebrates, the different isoforms of G proteins are involved in the response to the activation of different receptors of RCPG type. Consequently, the approach of Janetopoulos et al. involves the construction of a chimera and a cell line specifically deleted for each protein G isotype studied.

It appears from the above that there is no ubiquitous tool, simple to implement, for evaluating the activation of G proteins in eukaryotic cells.

The calcium channels comprise channels of the low-threshold type, activating by weak depolarizations and channels of the high-threshold type, activating by strong depolarizations. The high-threshold type channels represent a heteromultimeric complex αια ₂ δβ and γ, in which the membrane αj subunit, constituting the channel proper, is associated with an intracellular regulatory subunit β (or Ca _v β), by through its interaction domain (AID domain for alpha interaction domain), with a conserved motif: QQ-E - L-GY-- WI - E (one-letter code; - representing any amino acid ; Pragnell et al., Nature, 1994, 368, 67-70; Figure 1) in which residues Y392, W395 and 1396 are essential for the binding of the β subunit (De Waard et al., FEBS, 1996, 380, 272-276). The β regulatory subunit binds to the AID domain via its BID domain (betα interaction domain; DeWaard et al., J. Biol. Chem., 1995, 270, 12056-12064) which is included in a domain GK-like (Hanlon et al., FEBS, 1999, 445, 366-370). Seven subunits have been identified: αi _A (Ca _v α ₂ .ι), aie (Ca _v a ₂ .2) _> aiE (Ca _v a _{2. 3} ), respectively forming the P / Q type neural channels and N and the R-type channels, regulated by proteins G (channels sensitive to G proteins) and αis (Ca _v αι.ι), αι _c (Ca _v αι _.2 ), αjd (Ca _v αι _.3 ) and αif (Ca _v αι. ₄ ), forming L-type channels, insensitive to G proteins, including the cardiovascular (αι _c > and skeletal (αιs) channels, (Lory et al., m / s, 2001, 10, 979-988). In the central nervous system, the high-threshold calcium channels of type N and P / Q are directly involved in triggering the functioning of the synapse: their opening under the effect of an action potential induces calcium entry into the presynaptic termination. This signal triggers the secretion of neuromediators such as glutamate in the synaptic cleft and, thus, the propagation of nerve impulses in postsynaptic dendrite. The N and P / Q channels are regulated by receptors linked to trimeric G proteins (GPCR) such as metabotropic class III glutamate receptors (for review: El Far and Betz., Supra) or noradrenergic, muscarinic, GABAergic (GABA 5 γ-aminobutyric acid), serotonergic, dopaminergic, and opioid receptors (for review: Hille, Trends NeuroSci., 1994, 17, 531-536) . It has been shown that the Gβγ subcomplex is directly responsible for an inhibition of the activity of P / Q channels which results from a direct fixation of Gβγ on the intra-cytoplasmic loop connecting the membrane domains I and II (loop I-II) of the αj subunit (De Waard et al., Nature, 1997, 385, 446-450). In fact, this loop has several interaction sites with Gβγ, overlapping the binding domain with the regulatory subunit Ca _v β (AID domain; Figure 1), including a consensus motif QQ - RL-GY included in the domain AID, is essential for the binding of Gβγ (Figure 1; De Waard et al., Nature 1997, 385, 446-450; Zamponi et al., Nature, 1997, 385, 442-446). In addition, the Ca _v β regulatory subunit seems to counteract the functional effect of G proteins (Bourinet et al, PNAS, 1996, 93, 1486-1491). Thus, it would seem that this antagonism implies a physical competition between the Ca _v β subunit and the Gβγ protein at the level of the AID region of the loop I-II (Dolphin et al., J. Physiol., 1998, 506, 3-11).

The inventors have constructed chimeric proteins by NH ₂ and / or COOH terminal fusion: (i) of the loop I-II of the subunit ai of high-threshold calcium channels sensitive or insensitive to the G proteins (respectively, αi _A or Ca _v α .ι constituting a P / Q type neural channel and αj _c or Ca _v αι. ₂ constituting a cardiovascular type L channel) or a fragment thereof; said loop corresponds to positions 367 to 487 with reference to the sequence of the Ca _v α ₂ .ι subunit, and comprises the domain for binding to a β subunit of a calcium channel (or AID domain) and the sites of binding to a Gβ subunit of a G protein (FIG. 1), and

(ii) a β subunit of a high-threshold calcium channel, capable of binding to said fragment of the ai subunit. They have shown that all of the chimeric proteins obtained - comprising the loop I-II of an α] subunit derived from a calcium channel sensitive or insensitive to the G proteins or a fragment thereof including at least the domain AID, - has surprising properties of intramolecular interaction between the binding domains of the ai and β subunits of the calcium channel, which prevent the binding of the chimera to the AID domain of an αj subunit. They confirmed that this masking of the binding domain of the β subunit was indeed due to its intramolecular interaction with the AID domain, because the deletion of the AID domain of the chimera reestablishes this fixation. They also showed that the binding of the chimera comprising the loop I-II of a "G protein-sensitive" subunit to the interaction domain of the subunit is restored by the addition of Gβγ. This result was confirmed by the ex vivo demonstration of the interaction of a labeled recombinant β subunit with a fluorophore of Cy3 type with a fluorescent chimera of the αi _A subunit (GFP-αiA), by measurement of fluorescence transfer (FRET) in confocal microscopy. These properties have enabled them to demonstrate that, unexpectedly, the regulation of P / Q channels involves a displacement, by the Gβγ complex, of the interaction between the regulatory subunit β and the α subunit of the calcium channel and not by its inhibition as previously suggested.

More precisely, the inventors have shown that the chimeric protein derived from an ai subunit sensitive to G proteins exists in two "closed" or "open" forms, respectively in the absence or in the presence of Gβ subunit capable of binding to said fragment of the αj subunit, either in the form of Gβ monomer, either in the form of a Gβγ heterodimer. In the absence of Gβ (or Gβγ), the chimeric protein is capable of folding, thus allowing the domains of interaction of the ai and β subunits of the calcium channel to associate by a stable intramolecular bond (closed form). In the presence of Gβ (or Gβγ), the intramolecular bond is destroyed and the domains of interaction of the ai and β subunits of the calcium channel dissociate (open form), thus allowing each of the domains to interact respectively with Gβ (interaction domain of the ai subunit: AID domain) and / or an ai subunit of a calcium channel (interaction domain of the β subunit: BID domain). Similarly, the chimeric protein derived from an αj subunit insensitive to G proteins is capable of folding, thus allowing the interaction domains of the ai and β subunits of the calcium channel to associate by a stable intramolecular bond. (closed form). Consequently, in the presence of antagonists of this bond, other than Gβ or Gβγ, the intramolecular bond can also be destroyed and the interaction domains of the ai and β subunits of the calcium channel dissociate (open form), thus allowing each of the domains to interact respectively with said antagonist other than Gβ or Gβγ (interaction domain of the αj subunit: AID domain) and / or an a subunit of a calcium channel (interaction domain of the β subunit: BID domain). Consequently, due to the modification of their structure in the presence of free Gβ or Gβγ subunits or other antagonists of the interaction between the ai and β subunits (passage from the closed form to the open form), chimeric proteins derived from the ai and β subunits of high-threshold calcium channels represent tools that are simple to implement, sensitive, specific and useful for the following applications:

- the chimeric proteins derived from a subunit ai sensitive to G proteins (for example: α ^, αi _B and α ^) make it possible to determine the variations in cell concentration in free Gβγ subunits, ex vivo, in time and therefore to measure the activation of G proteins in cells: such chimeric proteins represent ubiquitous biosensors for the activation of G proteins perfectly suited to the study of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins and to the screening of agonists / antagonists of these signaling pathways capable of increasing or decreasing the concentration of free Gβγ subunits in cells and therefore of modulating the activity of these regulatory and cellular signaling dependent on receptors coupled to G proteins. - chimeric proteins derived from a subunit ai sensitive or resistant to G proteins (for example: αi _A , αm, αjβ, α _lc , αi, αis and αif) represent simple, sensitive and specific tools, perfectly suited to screening for antagonists of the interaction between the ai and β subunits, capable of modulating the activity of all high-threshold calcium channels. - the chimeric proteins derived from an ai subunit and from a β subunit of a high-threshold calcium channel as defined above, are also useful for the systematic pharmaco-toxicological control of new drugs in phase I and the search for natural orphan receptor agonists. In fact, the cloning of the human genome has made it possible to identify around 350 GPCR receptors. Of these, only 200 have an identified ligand. The others, called orphan receptors, potentially constitute key targets for the identification of new signaling and cellular regulation pathways. The search for agonists and antagonists of these receptors is therefore of major interest both in terms of basic research and in terms of therapy. Consequently, the subject of the present invention is a chimeric protein derived from a high threshold calcium channel, characterized in that it comprises at least one β subunit or a fragment thereof including at least the BID domain, fused (e) at its NH ₂ or COOH end with the loop I-II of an αj subunit or a fragment thereof including at least the AID domain. In accordance with the invention, the AID and BID domains are as defined above; the loop I-II of the ai subunit comprises the AID domain for binding to the β subunit and the sites for binding to the Gβ subunit of a G protein, including a consensus binding site which is included in this AID domain. These different areas are illustrated in Figure 1. The invention encompasses chimeric proteins derived from the ai and β subunits of vertebrates, in particular from human or non-human mammals and from their orthologs in invertebrates.

Chimeric proteins in accordance with the invention are represented in particular by:

a β subunit fused at its NH ₂ or COOH end with the loop I-II of an ai subunit _, and

- The GK-like domain of a β subunit including the BID domain (Hanlon et al., cited above), fused at its NH ₂ or COOH end with the loop I-II of an α i subunit _.

According to the invention, loop I-II or its fragment is either fused directly to the NH ₂ or COOH end of the β subunit or of its fragment, or the two sequences are separated by a spacer peptide, the size and amino acid sequence are such that the AID and BID domains of the chimeric protein containing said spacer are capable of interacting to form an intramolecular bond which is displaced in the presence of an antagonist (change from closed to open form) ; such a spacer peptide is in particular represented by a polyglycine sequence.

According to an advantageous embodiment of said chimeric protein, it comes from a high-threshold calcium channel sensitive to G proteins.

According to an advantageous arrangement of this embodiment, said chimeric protein comprises a fragment of a subunit ai selected from ctiA, "and αi £.

According to another advantageous embodiment of said chimeric protein, said β subunit is selected from the group consisting of βi, β ₂ , β ₃ and β ₄ .

The invention also includes chimeric proteins constituted by sequences functionally equivalent to the sequences as defined above, that is to say of which the β subunit and the loop I-II of the ai subunit or their fragments as defined above are capable of forming an intramolecular bond via their interaction domains; said bond possibly being destroyed in the presence of free Gβ or Gβγ subunits or other antagonists of the interaction between the αj and β subunits ("open form"). Among these sequences, there may be mentioned for example the sequences derived from the preceding sequences by: - mutation (substitution and / or deletion, and / or addition) of one or more amino acids of the sequences as defined above,

modification of at least one peptide bond -CO-NH- of the peptide chain of the chimeric protein as defined above, in particular by replacement with a bond different from the bond -CO-NH- (methylene amino, carba, ketomethylene, thioamide ....) or by the introduction of a retro or retro-inverso type bond, and / or,

- substitution of at least one amino acid of the peptide chain of the chimeric protein as defined above, by a non-proteinogenic amino acid residue. By non-proteinogenic amino acid residue is meant any amino acid which does not form part of a natural protein or peptide, in particular any amino acid whose carbon carrying the side chain R, namely the group - CHR-, located between -CO- and -NH- in the natural peptide chain, is replaced by a motif which does not form part of the constitution of a natural protein or peptide.

The subject of the present invention is in particular a variant chimeric protein derived from a chimeric protein as defined above, characterized in that it has a mutation of at least one amino acid in the sequences of said β subunit and / or loop I-II of a subunit αj. According to another advantageous embodiment of said chimeric protein, said variant has a mutation which modifies the affinity of the β subunit for the loop I-II of the ai subunit and / or vice versa; such mutations make it possible to obtain a chimeric protein more or less sensitive to the concentration of free Gβ or Gβγ subunit. Among these mutations, mention may be made of mutations in the AID domain of the loop I-II of the α _ls subunit as described in Pragnell et al., Cited above and Waard et al. FEBS, 1996, 380, 272-276, namely: Q383A, Q384A, E386D, E386S, L389H, G391R, Y392S, Y392F, W395A, I396A and E400A.

According to another advantageous embodiment of said chimeric protein or its variant, it is coupled, preferably covalently, to at least one suitable marker allowing the detection and / or the purification and / or the immobilization of said protein, for example: an antigenic epitope, a label of the polyhistidine type, a luminescent compound (fluorophore such as GFP or one of its variants: CFP, YFP and BFP), radioactive, or enzymatic.

In accordance with the invention, said coupling is carried out by any suitable means, in particular by a peptide bond via the COOH and / or NH _{2 functions which are} terminal of the peptide chain, or else by another covalent bond, such as for example : an ester, ether, thioether, thioester bond, via reactive functions of the side chain of an amino acid of the peptide chain. According to an advantageous arrangement of this embodiment, said chimeric protein comprises a fluorophore acceptor or donor respectively at its NH ₂ and or COOH end.

The acceptor fluorophores, for example CFP or BFP, can be coupled either to the NH or COOH end of the chimeric protein, the donor fluorophores, for example GFP or YFP are fused to the opposite end of said chimeric protein. Such chimeric proteins are useful for the ex vivo study in real time of the activation of G proteins and the screening of molecules capable of modulating this activation by measurement of fluorescence transfer (FRET).

Indeed, labeling with a luminescent compound has the advantage of obtaining a localized signal which does not require the presence of other reagents as is the case for enzymatic labeling. This type of marking also allows the use of phenomena such as energy transfer which can be carried out by different mechanisms: energy transfer by resonance, transfer of radiative energy (the acceptor absorbs the light emitted by the donor ) and electron transfer. This energy transfer, between a luminescent "donor" compound (D) and a luminescent or not "acceptor" compound (A), which is dependent on the distance between A and D, has been used for carrying out numerous assays. . We choose D and A, which are coupled at each end of the chimeric protein so that the energy transfer takes place only when the intramolecular intercation between the BID and AID domains takes place (closed form). This phenomenon results in a reduction or extinction of the luminescence of D and an emission of luminescence of A if the latter is luminescent, when D is excited. During these assays, the variation in the luminescence of A is measured, or the variation in the luminescence of D; the nature of A and D being variable. For example, to measure the variation in luminescence of A, two fluorescent proteins can be used as donor and acceptor, or else a rare earth complex (europium, terbium) with a chelate, cryptate or macrocycle as donor and a fluorescent protein as acceptor . The measurement of the variation in luminescence of D is based on the ability of a compound (A) to decrease or suppress the luminescence of another compound (D) when these are sufficiently close ("Quench"). The range of molecules A that can be used is therefore more extensive and thus includes non-luminescent compounds such as heavy metals, heavy atoms, chemical molecules such as for example methyl red, nanoparticles such as those sold under the name Nanogold ® by the company Nanoprobes (USA), or even the molecules sold under the names DABCYL® (Eurogentec, Belgium), QSY Dyes (Molecular Probes Inc., USA), ElleQuencher® (Oswell / Eurogentec) or Black Hole Quenchers® ( Biosearch Technologies Inc., USA).

The present invention also relates to a peptide, characterized in that it comprises a fragment of at least 7 amino acids of the sequence of the chimeric protein as defined above, located at the junction of the β subunit and of the loop I-II of the ai subunit or of their fragments as defined above; such peptides make it possible in particular to produce antibodies specific for the chimeric protein according to the invention.

The present invention also relates to antibodies, characterized in that they are directed against a chimeric protein or a peptide as defined above. According to the invention, said antibodies are either monoclonal antibodies or polyclonal antibodies. These antibodies can be obtained by conventional methods, known per se, comprising in particular the immunization of an animal with a protein or a peptide in accordance with the invention, in order to make it produce antibodies directed against said protein or said peptide. Such antibodies are useful in particular for immobilizing the chimeric protein on a solid support, purifying it or even detecting it.

The present invention also relates to a nucleic acid molecule, characterized in that it is selected from the group consisting of the sequences coding for a chimeric protein or a peptide as defined above and the sequences complementary to the preceding, sense or antisense.

The invention also relates to probes and primers, characterized in that they comprise a sequence of approximately 10 to 30 nucleotides corresponding to that located at the junction of the β subunit and of the I-II loop of the subunit ai or their fragments as defined above; these probes and these primers make it possible to specifically detect / amplify said nucleic acid molecules encoding the chimeric protein according to the invention.

The subject of the invention is also other primers making it possible to specifically amplify the β subunit and / or the loop I-II of the ai subunit or their fragments as defined above, characterized in that '' they are selected from the group consisting of the sequences SEQ ID NO: 1, 2, 4, 6, 7, 8 and 9.

The nucleic acid molecules according to the invention are obtained by conventional methods, known in themselves, by following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson lnc, Lïbrary ofCongress, USA).

The sequences coding for a chimeric protein according to the invention can be obtained by amplification of a nucleic sequence by PCR or RT-PCR using an appropriate primer pair or else by screening of genomic DNA libraries by hybridization with a homologous probe. The derivative nucleic acid molecules, coding for a variant of the chimeric protein according to the invention, are obtained by conventional methods, making it possible to introduce mutations into a nucleic acid sequence, known in themselves, following the aforementioned standard protocols. For example, the sequence coding for a variant of the chimeric protein according to the invention can be obtained by site-directed mutagenesis according to the method of Kunkel et al., (PNAS, 1985, 82, 488-492). The present invention also relates to a recombinant eukaryotic or prokaryotic vector, characterized in that it comprises an insert consisting of the nucleic acid molecules coding for a chimeric protein as defined above.

Preferably, said recombinant vector is an expression vector in which said nucleic acid molecule or one of its fragments is placed under the control of regulatory elements for appropriate transcription and translation. In addition, said vector may comprise sequences fused in phase with the 5 'and / or 3' end of said insert, useful for the immobilization, and / or the detection and / or the purification of the protein expressed from said vector. . Many vectors into which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell, are known in themselves; the choice of an appropriate vector depends on the envisaged use for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or else integration into the chromosomal material of the 'host), as well as the nature of the host cell. For example, viral or non-viral vectors such as plasmids can be used.

These vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods, which are known per se.

According to one embodiment of said recombinant vector, it is a eukaryotic expression vector having a sequence selected from the group consisting of the sequences SEQ ID NO: 5 and SEQ ID NO: 10; the plasmid SEQ ID NO: 5 contains the loop I-II of the Ca _v α ₂ .ι rabbit subunit fused to the C-terminal end of the Ca _v β ₃ subunit of rat, under the control of the promoter CMN and the plasmid SEQ ID ΝO: 10 contains an insert consisting of 5 'to 3' by the phase fusion of the following fragments: the sequence GAP-43, the AD ,c coding for EGFP (fluorescence donor), the GK-like domain of the rat Ca _v β ₃ subunit, the loop I-II of the rabbit Ca _v α _2. ι subunit and the cDNA coding for the CFP (fluorescence acceptor).

The present invention also relates to cells modified by a chimeric protein, a nucleic acid molecule or else a recombinant vector as defined above.

According to an advantageous embodiment of the invention, said cells are eukaryotic cells.

According to an advantageous arrangement of this embodiment, said cells express at least one receptor capable of binding to G proteins (RCPG); said cells are either cells constitutively expressing at least one RCPG, or modified cells which express a recombinant RCPG.

Modified cells in accordance with the invention can be obtained by any means, known in themselves, making it possible to introduce a nucleic acid molecule or a protein into a host cell. For example, in the case of animal cells, viral vectors such as adenoviruses, retroviruses, lentiviruses and ANAs, in which the sequence of interest has been inserted, can be used, among others. it is also possible to associate said nucleotide (isolated or inserted into a plasmid vector) or peptide sequence with a substance enabling it to cross the membrane of host cells, for example a preparation of liposomes, lipids or cationic polymers, or else the inject directly into the host cell.

The subject of the present invention is animals and in particular non-human transgenic mammals, characterized in that all or part of their cells are transformed by a nucleic acid molecule according to the invention. These are, for example, animals into which a sequence coding for the chimeric protein according to the invention has been introduced under the control of the control of regulatory elements for appropriate transcription and translation. Such transgenic animals are useful, in particular for the secondary screening stages: i) to evaluate the cellular or even tissue targeting of an active molecule on GPCRs or calcium channels, identified during a primary screening, ii) to study bioavailability of such a molecule, and iii) for research, as a first approach, for possible side effects of such a molecule.

The present invention also relates to the use of a product selected from the group consisting of chimeric proteins, nucleic acid molecules, recombinant vectors, modified cells and transgenic non-human mammals as defined above. above, for the study of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins.

The present invention also relates to the use of a product selected from the group consisting of chimeric proteins, nucleic acid molecules, recombinant vectors, modified cells and transgenic non-human mammals as defined above. above, for the screening of agonists and / or antagonists of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins. The present invention also relates to the use of a product selected from the group consisting of chimeric proteins, nucleic acid molecules, recombinant vectors, modified cells and transgenic non-human mammals as defined above, for the screening of antagonists of the interaction between the ai and β subunits high-threshold calcium channels; such antagonists are useful for modulating the activity of all of the high-threshold calcium channels and therefore represent drugs capable of being used in the treatment of diseases linked to a dysfunction of calcium homeostasis and of pathologies where modulation calcium intake can compensate for a cellular deficit, in particular epilepsies, ataxias, migraines, muscular hypo- and hyper-calcemias, diabetes, and cardiovascular diseases.

According to an advantageous embodiment of the invention, the study of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins is carried out by a method comprising at least the following steps: ai) the culture of modified cells expressing a chimeric protein derived from a calcium channel sensitive to G proteins and a receptor coupled to G protein, as defined above, bi) transducing a signal via said G protein-coupled receptor by any suitable means, and ci) determining, by any suitable means, the proportion of said chimeric protein expressed in said cells which is linked to a Gβγ subunit.

Such a determination makes it possible to evaluate the variations in the cell concentration in free Gβγ subunits and therefore to measure the activation of the G proteins in the cells.

According to an advantageous embodiment of the invention, the screening of agonists / antagonists of the cellular signaling and regulatory pathways dependent on receptors coupled to G proteins is carried out by a method comprising at least the following steps: a ₂ ) the culture of modified cells expressing a chimeric protein originating from a calcium channel sensitive to G proteins and a receptor coupled to G proteins, as defined above, b ₂ ) transduction of a signal via said coupled receptor to protein G, by any suitable means, c ₂ ) the comparative determination, by any appropriate means, of the proportion of said chimeric protein expressed in cells which is linked to a Gβγ subunit, before and after bringing said contacts into contact cells in b ₂ ) with a molecule to be tested, and d ₂ ) the identification of agonist / antagonist molecules of the signaling and regulatory pathways areas dependent on receptors coupled to G proteins, corresponding to those capable of respectively increasing and decreasing the cell concentration in free Gβγ subunits.

Advantageously, said cells modified in ai ₎ or in a ₂ ) express a chimeric protein as defined above coupled at its ends

NH ₂ and COOH, respectively to a donor fluorophore and a fluorescence acceptor fluorophore and said determination in ci) or in c ₂ ) is carried out by the fluorescence transfer technique (FRET). According to an advantageous embodiment of the invention, the screening of antagonists of the interaction between the αj and β subunits of the high-threshold calcium channels is carried out by a method comprising at least the following steps: a) setting in contact with a molecule to be tested with a chimeric protein derived from a calcium channel sensitive or insensitive to G proteins as defined above and with a peptide comprising the AID domain of an ai subunit insensitive to G proteins, b ₃ ) measuring, by any appropriate means, the binding of said chimeric protein to said peptide, and c ₃ ) identifying the antagonists of the interaction between the subunits ai and β corresponding to those with which a binding of said chimeric protein to said peptide.

According to an advantageous embodiment of said method, said peptide comprising the AID domain is immobilized on a solid support, and said chimeric protein is coupled to a marker allowing the measurement of said bond in b), as defined above, including a fluorophore.

The subject of the invention is also a kit for implementing the methods as defined above, characterized in that it includes at least one of the following products: a chimeric protein, an antibody, a recombinant vector, a modified cell or a transgenic non-human mammal, as defined above.

The chimeric protein according to the invention has the following advantages:

- it constitutes a ubiquitous biosensor of endogenous free Gβγ subunits, suitable for the real-time study of cellular signaling and regulation pathways dependent on receptors coupled to protein G, and for systematic screening (high-throughput screening) of molecules capable of modulating them, potentially usable as a drug for the treatment of diseases in which a dysfunction of these pathways is observed, in particular pathologies of the immune system (for a review see Lombardi et al, Crit. Rev. Immunology, 2002, 22, 141 - 163; Onuffer and Horuk, Trends in Pharmacol, 2002, 23, 459-467) and neuropsychiatric and cardiovascular pathologies (Seifert and Wenzel-Seifert, Naumyn- Schmeideberg's Arch. Pharmacol. , 2002, 366, 381-416). In addition, its use is simple to the extent that it makes it possible to partially overcome the problems of stoichiometry since its use only involves two molecules (Ca _v β / Ca _v α-Gβγ) instead of three partners (Ca _v α / Ca _v β / Gβγ) for the methods of the prior art.

- it is suitable for systematic screening (high-throughput screening) of molecules capable of modulating the activity of high-threshold calcium channels, potentially usable as a drug for the treatment of diseases in which there is a dysfunction of calcium homeostasis and pathologies where the modulation of calcium entry can compensate for a cell deficit as defined above.

In addition to the foregoing arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of use of the chimeric protein which is the subject of the present invention as well as to annexed drawings, in which:

- Figure 1 illustrates the overlap, in the loop I-II of the Ca _v α .ι _j subunit of the binding domains to the β subunit (AID domain) and to the Gβγ complex. The AID domain is represented by a black box (positions 383 to 400). The binding sites for the Gβ (Gβγ) subunit are represented by hatched boxes; the site in the central position (QQ - RL-GY) which is essential for the binding of the Gβ subunit (Gβγ) is included in the AID domain.

FIGS. 2 and 3 illustrate the displacement of the interaction Ca _v α .ι- Ca _v β by the Gβγ complex of the G proteins:

- Figure 2a illustrates the linkage of the β ₃ subunit (1 to 3 pM) with the AIDι domain. ₂ of the GST-AIDι fusion protein. ₂ (1 μM),

- Figure 2b shows that the fusion of the β ₃ subunit with the loop I-II of the α ₂ .ι subunit (Chimera Ca _v β ₃ - I-II ₂ .ι) prevents its binding with the domain AIDj.2 of the GST-AIDι _.2 fusion protein,

FIG. 2c shows that the deletion of the 18 amino acids from the AID ₂ .ι domain (Chimera Ca _v β ₃ - I-II ₂ .ιΔAID) restores the bond of the β ₃ subunit with the AIDι _.2 domain of the GST-AIDι fusion protein _. , - Figure 3 shows that the addition of Gβγ complex displaces the intramolecular interaction between the Ca _v β subunit and the loop I-II of the α _2. ι subunit of the chimera Ca _v β ₃ - I- II _2.1 , thus allowing the β ₃ subunit to bind with the domain AIDj. ₂ of the fusion protein GST-AID1.2; the concentration of Gβγ, capable of displacing 50% of the bond between the Ca _v β subunit and the AID domain _. ι (IC ₅₀ ) is l60nM,

FIGS. 4 to 7 illustrate the FRET analysis of the disassembly of the P / Q calcium channel, induced by the Gβγ complex:

- Figure 4a illustrates the labeling with Cy3 of the purified His-Ca _v β ₃ subunit. CB: Coomassie blue staining of an SDS-PAGE gel illustrating the purity of the protein. FS = recording of the fluorescence of an uncolored gel showing the covalent labeling of the protein,

- Figure 4b illustrates the effect of the Ca _v β subunit coupled to a fluorochrome (Cy3-Ca _v β ₃ ) on the current-voltage relationship of Ca _v α ₂ .ι channels expressed in xenopus oocytes, by comparison with the unlabeled Ca _v β ₃ subunit (injection of cRNA),

- Figure 5a illustrates the observation by confocal microscopy of two distinct regions, xenopic oocytes containing Ca _v α _2. j and Cy3-Ca _v β ₃ . T = transmission, F = fluorescence, - Figure 5b illustrates the emission spectrum of the fluorescence of

GFP-Ca _v α _2.b Cy3-Ca _v β ₃ and (GFP-Ca _v α _2. ι + Cy3-Ca _v β ₃ ),

- Figure 6 illustrates the kinetics of decrease in the fluorescence transfer induced by the injection of 100 nM of Gβγ. Upper panel: variations in the fluorescence emission spectrum and lower panel: variations in the ratio of fluorescence intensities (R _f ) at 585 irai and 525 nm,

- Figure 7 illustrates the R _f values of non-injected oocytes (-), injected with Gβγ (100 nM) or with heat-inactivated Gβγ (HI-Gβγ).

- Figure 8 (a to c) illustrates the sequence of the plasmid pcDNA3Cavβ3- I-II2.1 (SEQ ID NO: 5) containing the loop I-II of the subunit Ca _v α _2. ι rabbit fused to l C-terminal end of Ca _v β ₃ subunit of rat, under the control of the CMN promoter. FIG. 9 (a to c) illustrates the sequence of the plasmid pCHIC (SEQ ID NO: 10) derived from the vector pEYFPmemb. (CLONTECH), containing an insert consisting of 5 'in 3' by the phase fusion of the following fragments: the GAP-43 sequence, the cDNA coding for EGFP (fluorescence donor), the GK-like domain of the rat Ca _v β ₃ subunit, the loop I-II of the Ca _v α subunit ₂ .ι rabbit and cDNA coding for the CFP (fluorescence acceptor).

It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation. EXAMPLE 1: CONSTRUCTION OF A RECOMBINANT CHEMICAL PROTEIN Cavβ3-I-II ₂ .ι 1). Materials and methods

The PCR amplification and the cloning of the recombinant DNA are carried out by the conventional techniques known to those skilled in the art, following standard protocols such as those described for example in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000 , Wiley and son Inc, Library of Congress, USA).

An expression plasmid containing a cDNA encoding a chimeric protein according to the invention, constituted by the C-terminal fusion of the rat β3 subunit with the intracellular loop I-II of the rabbit ai subunit and has been constructed as follows:

The cDNA of the rat Ca _v β ₃ subunit (corresponding to positions 98 to 1545 of the GENBANK M88755 sequence) is amplified by PCR using the following sense and antisense primers: - 5'-TTTGGTACCATGGATGACGACTCCTACGTGCCCGGGTTTGAGGACTCGGAGGCGGGTT- 3 '(SEQ ID NO: 1), and - δ'-GCGGAATTCGTAGCTGTCCTTAGGCCAAGGCCGGTTACGCTGCCAGTT-S', (SEQ ID NO: 2).

The fragment thus obtained was cloned between the Kpn I and EcoR I sites of the expression plasmid (pcDNA3, IN VITROGEN) to give the recombinant plasmid pcDNA3-Ca _v β3.

The cDNA fragment corresponding to loop I-II of the Ca _v α ₂ .ι rabbit subunit (positions 1383 to 1754 of the GENBANK X57477 sequence), the sequence is illustrated in FIG. 1, was amplified by PCR using the following sense and antisense primers:

- 5'-GGGGMTTCGCCAAAGAAAGGGAGCGGGTGGAGAAC-3 '(SEQ ID NO: 3; De Waard et al., Cited above and Bichet et al., Neuron, 2001, 25, 177-190), and - S'-TTTGAATTCTTACTGAGTTTTGACCATGCGACGGATGTAGAC ID NO: 4).

The fragment obtained was cloned at the EcoR I site of the plasmid pcDNA3-Cavβ3 to give the recombinant plasmid pcDNA3-Ca _v β3-I-II ₂ .ι.

A control plasmid containing a cDNA encoding a chimeric protein constituted by the C-terminal fusion of the rat β3 subunit with the intracellular loop I-II of the rabbit Cavα ₂ .ι subunit deleted from the AID domain has been constructed in the same way ; the recombinant plasmid thus obtained is called pcDNA3-Cavβ3-I-II ₂ .ιΔAID.

2) Results

The recombinant plasmid pcDNA3-Ca _v β3-I-II _2. ι has the sequence SEQ ID NO: 5. The peptide sequence deduced from the nucleotic sequence obtained by automatic sequencing of the cloned insert in the plasmid pcDNA3-

Ca _v β3-I-II _2. ι presents the expected sequence for a chimeric protein according to the invention. Similarly, the peptide sequence deduced from the nucleotic sequence obtained by automatic sequencing of the cloned insert in the plasmid pcDNA3- Ca _v β3-I-II ₂ .ιΔAID corresponds to that expected for a chimeric protein deleted from the AID domain.

EXAMPLE 2: IN VITRO HIGHLIGHTING OF THE MOVEMENT OF

THE INTERACTION Ca _v α ₂ .ι-Ca _v β THROUGH THE PROTEIN Gβγ COMPLEX

G. 1) Materials and methods

The expression of the recombinant DNA and the analysis of the recombinant proteins are carried out by the conventional techniques known to those skilled in the art, following standard protocols such as those described for example in

Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library ofCongress, USA) and in Current protocols in Immunology (John E.

Coligan, 2000, Wiley and son Inc, Library ofCongress, USA). a) Expression of the recombinant chimeric proteins and of the GST-AIDv fusion?

The chimeric proteins Ca _v β3-I-II ₂ .ι and Ca _v β3-I-II _2. ιΔAID, and the subunit Ca _v β ₃ , are transcribed and translated in vitro in the presence of [ ³⁵ S] -methionine , from the plasmids as described in Example 1, using the TNT system PROMEGA kit, following the manufacturer's instructions.

The GST-AIDι fusion protein. ₂ described in Pragnell et al., Supra, is produced and purified as described by the previous authors. The GST protein produced and purified under the same conditions is used as a control. b) In Vitro Analysis of the Regulation of the Cayα ^ -Cayβ Interaction by the Gβγ Complex of the G Proteins

The in vitro analysis of the regulation of the interaction Ca _v α _2. ι-Ca _v β by the Gβγ complex of the G proteins, is carried out according to the protocols as described in De Waard et al., Nature, 1997, 385, 446-450. More precisely, the β ₃ subunit and the labeled chimeric proteins ([ ³⁵ S] Ca _v β3, [ ³⁵ S] Ca _v β3-I-II _2. ι and [ ³⁵ S] Ca _v β3-I- II ₂ .ιΔAID) are incubated in the absence or in the presence of the GST-AIDj _.2 fusion protein or of the GST protein, and optionally in the presence of increasing amounts of Gβγ (10 to 900 nM, CALBIOCHEM).

The incubation product is separated by polyacrylamide gel electrophoresis (SDS-PAGE) and the gel is autoradiographed. D Results

The results illustrated in Figures 2 and 3 are as follows:

- Figure 2a shows that the β ₃ subunit (1 to 3 pM) binds with the AID _1.2 domain of the GST-AID fusion protein]. (LμM)

- Figure 2b shows that the fusion of the β ₃ subunit with the loop I-II of the α ₂ .ι subunit (Chimera Ca _v β ₃ - I-II _2.1 ) prevents its binding with the domain

AIDι, from the GST-AIDι fusion protein. ₂ ,

FIG. 2c shows that the deletion of the 18 amino acids from the AID ₂ domain. ₁ (Chimera Ca _v β ₃ - I-II2.1ΔAID) restore the binding of the β ₃ subunit with the domain AIDι _.2 of the fusion protein GST-AIDι _.2 , - Figure 3 shows that the addition of Gβγ complex displaces the intramolecular interaction between the Ca _v β subunit and the I-II loop of the subunit α ₂ .ι of the chimera Ca _v β ₃ - I-II _2.1 , thus allowing the β ₃ subunit to bind with the AID _1.2 domain of the fusion protein GST-AIDι. ₂ ; the IC ₅₀ concentration of Gβγ, capable of displacing 50% of the bond between the Ca _v β subunit and AID ₂ domain _. ι, after 30 min of incubation at 30 ° C, is 160nM; this value is 2 to 3 times higher than that relating to the affinity of Gβγ for the loop I-II ₂ .ι, previously reported (De Waard et al., Nature, 1997, 385, 446-450).

EXAMPLE 3: EX VIVO HIGHLIGHTING OF THE DISPLACEMENT OF THE INTERACTION Ca _v α ₂ . Ca _v β THROUGH THE Gβγ COMPLEX OF PROTEINS G. 1) Materials and methods a) Cy3 labeling of the purified recombinant His-Cayβ ₃ protein.

The purified His-Ca _v β ₃ recombinant protein (Geib et al., Biochem J., 2002, 364, 285-292; Fathallah et al., Eur. J. Neurosci., 2002, 16, 219-228) is coupled to the monoreactive Cy3 maleimide following the manufacturer's instructions (Amersham Pharmacia Biotech). b) Injection of xenopic oocytes and electrophysiological recordings.

The preparation, the injection of xenopic oocytes and the electrophysiological recordings are carried out as described in Geib et al., Cited above. The effects of the Gβγ complexes on the current-voltage relationship and the inactivation of the steady state are analyzed 30 min after the injection. c) Measurement of fluorescence transfer (FRET).

The oocytes are analyzed by confocal microscopy (TCS-SP2 microscope, LEICA, in "XYλ" mode), 4 to 7 days after the injection.

The fluorescence emission is recorded using an argon laser with an excitation at 488 nm and a dicliroic mirror (488/543/633). Fluorescence is measured through 14 filters (10 nm thick) to reconstruct the emission spectrum. For each measurement, two different regions are analyzed to ensure the reproducibility of the measurement. FRET levels are estimated by the ratio (585/525) between fluorescence at 585 nm (emission peak of the Cy3 acceptor) and fluorescence at 525 nm (emission peak of the GFP donor). 2) Results

- The Ca _v β ₃ subunit coupled to Cy3 (Figure 4a) is as active as the Ca _v β ₃ subunit on the regulation of Ca _v α ₂ ι channels expressed in xenopic oocytes (Figure 4b). - The injection of the protein Cy3-Ca _v β ₃ or GFP-Ca _v α2.ι or of the cDNA coding for said protein, alone or in combination, results in the emission of a signal of high fluorescence at the plasma membrane (Figure 5 a).

Analysis of the fluorescence emission between 500 and 640 nm, after excitation at 488 nm (Figure 5b), shows that GFP-Ca _v α _2. ι produces a high signal with a maximum at 525 nm while Cy3-Ca _v β3 alone is not very excited and produces a weak signal with a maximum at 585 nm. When the two proteins are in the oocytes, the signal emitted at 525 nm decreases drastically while that at 585 nm increases significantly. These changes are easily quantifiable by determining the ratio of the fluorescence signals at 585 nm and 525 nm (R _f = 0.34 ± 0.03 for GFP-Ca _v α _2. ι (n = 3), R _f = 1 , 9 ± 0.10 for Cy3-Ca _v β ₃ (n = 3) and R _f = 3.9 ± 0.22 for GFP-Ca _v α ₂ .ι + Cy3-Ca _v β ₃ (n = 7) Also large changes resulting from a significant transfer of fluorescence, demonstrate the proximity of the fluorochromes GFP-Cavα _2. ι and Cy3-Cavβ3 - The injection of Gβγ in oocytes containing both GFP-

Cavα2 _. ι and Cy3-Cavβ ₃ induces a rapid disappearance of the fluorescence transfer (Figure 6). By comparison, the injection of Gβγ has no effect in cells containing only GFP-Cavα ₂ .ι or Cy3-Cavβ ₃ .

The final report of the fluorescence signals (0.82 ± 0.06, n = 7) is of the order of that observed in oocytes containing only GFP-Cavα ₂ or Cy3-Cavβ ₃ indicating that the dissociation of the Cy3 channel -Cavβ ₃ is important (Figure 7). By comparison, the injection of heat-inactivated Gβγ has no effect (R _f = 3.74 ± 0.4, n = 3).

These results demonstrate that Gβγ is also capable of displacing the Cavβ ₃ subunit ex vivo from its binding site to the Cavα .ι channel. EXAMPLE 4 CONSTRUCTION OF A BIOSENSOR FOR MEASURING THE ACTIVITY OF G PROTEINS BY THE FREIGHT TECHNIQUE

A chimeric protein containing a fluorescence donor fluorophore (EGFP) at its NH end and a fluorescence acceptor fluorophore (CFP) at its COOH end is constructed from the vector pEYFPmemb (CLONTECH). This vector has the advantage of having:

- A GAP-43 sequence which allows the chimera to be anchored to the plasma membrane via its NH end. Anchoring to the membrane has the advantage on the one hand of maintaining the protein on the membrane and on the other hand of increasing the probability of encounter between the chimeric protein and its ligand Gβγ which is itself anchored to the membrane by a palmitoylation type bond, and

- an EYFP sequence downstream of GAP-43. Construction is carried out in two stages:

- the ^1st cloning step: The DNA fragment coding for the GK-like domain of the β subunit (Hanlon et al, FEBS, 1999, 445, 366-370) fused to the I-II loop of the sub -unit ai is amplified by PCR from the plasmid pcDNA3-Ca _v β3-I-II _2. ι (example 1) then it is cloned in 3 ′ of the EYFP gene.

More specifically, the PCR amplification is carried out using the following sense and antisense primers: BsiWI Pvu 1 - 5'- AGCCGTACGCGATCGCATCTCTAGCCAAGCAGAAGCAAA - 3 '(SEQ ID NO: 6)

Hpa I Spe I

5'- CCCGTTAACCCCACTAGTCTGAGTTTTGACCATGCGACGGAT-3 '(SEQ ID NO: 7) The PCR product obtained is cloned between the BsiW I and Hpa I sites of the plasmid pEYFPmemb to give the plasmid pEYFmemChimBéta3I-II.

- 2 ^-th cloning step:

The cDNA coding for the ECP is amplified by PCR and then cloned into the preceding plasmid, 3 'to the insert β 3 -I-II. More specifically, the ECFP is amplified by PCR from the pECFP vector (Clontech), using the following sense and antisense primers: Spe l

- 5'- GGGACTAGTATGGTGAGCAAGGGCGAGGAGCTG- 3 '(SEQ ID NO: 8)

Hpa l

- 5'- CCCGTTAACTGCCGAGAGTGATCCCGGCGGCGGT-3 '(SEQ ID NO: 9)

The PCR product obtained is cloned between the Spe I and Hpa I sites of the plasmid pEYFmemChimBéta3I-II to give the plasmid pCHIC corresponding to the sequence SEQ ID NO: 10.

Claims

1 °) Chimeric protein derived from a high threshold calcium channel, characterized in that it comprises at least one β subunit or a fragment thereof including at least the BID domain, fused at its NH end ₂ or COOH with loop I-II of an ai subunit or a fragment thereof including at least the AID domain.

2 °) chimeric protein according to claim 1, characterized in that it consists of a β subunit fused at its NH ₂ or COOH end with the loop I-II of an ai subunit. 3 °) chimeric protein according to claim 1, characterized in that it consists of the GK-like domain of a β subunit fused at its NH ₂ or COOH end with the loop I-II of a sub- unit αj.

4 °) Protein according to any one of claims 1 to 3, characterized in that the β subunit or its fragment and the loop I-II or its fragment are separated by a spacer peptide.

5 °) chimeric protein according to any one of claims 1 to 4, characterized in that it comes from a high-threshold calcium channel sensitive to G proteins.

6 °) chimeric protein according to claim 5, characterized in that it comprises the loop I-II of a subunit ai selected from αi _A, ie and αi _E or a fragment thereof.

7 °) chimeric protein according to any one of claims 1 to 6, characterized in that it comprises a β subunit selected from the group consisting of βi, β ₂ , β ₃ and β ₄ or a fragment thereof. this. 8 °) variant chimeric protein derived from a chimeric protein according to any one of claims 1 to 7, characterized in that it has a mutation of at least one amino acid in the sequences of the said β subunit and / or of the loop I-II of a subunit _\ . 9 °) variant chimeric protein according to claim 8, characterized in that said mutation modifies the affinity of the β subunit for the fragment of the loop I-II of the α subunit and / or vice versa.

10 °) chimeric protein variant according to claim 8 or claim 9, characterized in that said mutations are selected from the following mutations of the AID domain of the loop I-II of the subunit ai: Q383A, Q384A, E386D, E386S , L389H, G391R, Y392S, Y392F, W395A, I396A and E400A.

11 °) chimeric protein according to any one of claims 1 to 10, characterized in that it is coupled, preferably covalently, to at least one suitable marker allowing the detection and / or the purification and / or the immobilization of said protein.

12 °) chimeric protein according to claim 11, characterized in that it comprises an acceptor or donor fluorophore respectively at its NH ₂ and / or COOH end. 13 °) chimeric protein according to claim 12, characterized in that the acceptor fluorophore is the fluorescent protein CFP or BFP and the donor fluorophore is the fluorescent protein GFP or YFP.

14 °) Peptide, characterized in that it comprises a fragment of at least 7 amino acids of the sequence of the chimeric protein according to any one of claims 1 to 13 located at the junction of the β subunit and the loop I-II of the ai subunit of a calcium channel or of their fragments as defined in claim 1.

15 °) Antibodies, characterized in that they are directed against a protein according to any one of claims 1 to 13 or a peptide according to claim 14.

16 °) Nucleic acid molecule, characterized in that it is selected from the group consisting of the sequences coding for a chimeric protein according to any one of claims 1 to 13 or a peptide according to claim 14, and the sequences complementary to the previous ones, sense or antisense. 17 °) Probes and primers, characterized in that they comprise a sequence of approximately 10 to 30 nucleotides corresponding to that located at the junction of the β subunit and of the loop I-II of the ai d subunit 'a calcium channel or their fragments as defined in claim 1. 18 °) Primers capable of amplifying the β subunit and / or the loop I-II of the ai subunit of a calcium channel or their fragments as defined in claim 1, characterized in that they are selected from the group consisting of the sequences SEQ ID NO: 1, 2, 4, 6, 7, 8 and 9.

19 °) Recombinant vector, characterized in that it comprises an insert selected from the group consisting of the nucleic acid molecules according to claim 16.

20 °) Recombinant vector according to claim 19, characterized in that it is a eukaryotic expression vector having a sequence selected from the group consisting of the sequences SEQ ID NO: 5 and SEQ ID NO: 10.

21 °) Cells modified by a recombinant vector according to claim 19 or 20, by a nucleic acid molecule according to claim 16 or by a chimeric protein according to any one of claims 1 to 13.

22 °) Modified cells according to claim 21, characterized in that they are eukaryotic cells.

23 °) modified cells according to claim 21 or claim 22, characterized in that they express at least one receptor capable of binding to the G proteins.

24 °) non-human transgenic mammal, characterized in that all or part of its cells are transformed by a nucleic acid molecule according to claim 16.

25 °) Use of a product selected from the group consisting of chimeric proteins according to any one of claims 1 to 13, the nucleic acid molecules according to claim 16, the recombinant vectors according to claim 19 or claim 20 , the modified cells according to any one of claims 21 to 23 and the non-human transgenic mammals according to claim 24, for the study of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins.

26 °) Use of a product selected from the group consisting of chimeric proteins according to any one of claims 1 to 13, the nucleic acid molecules according to claim 16, the recombinant vectors according to claim 19 or the claim 20, the modified cells according to any one of claims 21 to 23 and the non-human transgenic mammals according to claim 24 for the screening of agonists and / or antagonists of receptor-dependent cellular signaling and regulatory pathways coupled to proteins G. 27 °) Use of a product selected from the group consisting of chimeric proteins according to any one of claims 1 to 13, the nucleic acid molecules according to claim 16, the recombinant vectors according to claim 19 or claim 20, the modified cells according to any one of claims 21 to 23 and the mammalian transgendered Non-human nicks according to claim 24 for the screening of antagonists of the interaction between the ai and β subunits of high-threshold calcium channels.

28 °) Method for studying cellular signaling and regulatory pathways dependent on receptors coupled to G proteins, characterized in that it comprises at least the following steps: ai) the culture of modified cells expressing a chimeric protein derived from a calcium channel sensitive to G proteins and a receptor coupled to protein G according to claim 23, bi) transduction of a signal via said receptor coupled to protein G, by any appropriate means, and ci) determination, by any suitable means, of the proportion of said chimeric protein expressed in said cells which is linked to a Gβγ subunit.

29 °) Method for screening agonists / antagonists of cellular signaling and regulatory pathways dependent on receptors coupled to G proteins, characterized in that it comprises at least the following steps: a ₂ ) the culture of modified cells expressing a chimeric protein derived from a calcium channel sensitive to G proteins and a receptor coupled to G proteins according to claim 23, b ₂ ) transduction of a signal via said coupled receptor to G proteins, by any suitable means,

C ₂ ) the comparative determination, by any appropriate means, of the proportion of said chimeric protein expressed in cells which is linked to a Gβγ subunit, before and after contacting said cells in b ₂ ) with a molecule to test, and d ₂ ) the identification of the agonist / antagonist molecules of the cellular signaling and regulatory pathways dependent on receptors coupled to G proteins, corresponding to those capable respectively of increasing and decreasing the cellular concentration in free Gβγ subunits .

30 °) Method according to claim 28 or claim 29, characterized in that said cells modified in ai) or in a ₂ ) express a chimeric protein coupled, at its ends NH ₂ and COOH, respectively to a donor fluorophore and a fluorophore fluorescence acceptor and said determination in ci) or in C ₂ ) is carried out by the fluorescence transfer technique (FRET).

31 °) Method for screening for antagonists of the interaction between the ai and β subunits of high-threshold calcium channels, characterized in that it comprises at least the following steps: a ₃ ) bringing into contact a molecule to be tested with a chimeric protein derived from a calcium channel sensitive or insensitive to G proteins according to any one of claims 1 to 13, and with a peptide comprising the AID domain of an αj subunit insensitive to G proteins , b ₃ ) measuring, by any suitable means, the binding of said chimeric protein to said peptide, and c ₃ ) the identification of the antagonists of the interaction between the αj and β subunits, corresponding to those with which a binding of said chimeric protein to said peptide is observed.

32 °) screening method according to claim 31, characterized in that said peptide comprising the AID domain is immobilized on a solid support and said chimeric protein is a chimeric protein according to any one of claims 11 to 13.

33 °) kit for the implementation of a method according to any one of claims 28 to 32, characterized in that it comprises at least one product selected from the group consisting of chimeric proteins according to any one of claims 1 to 13, the nucleic acid molecules according to claim 16, the recombinant vectors according to claim 19 or claim 20, the modified cells according to any one of claims 21 to 23 and the non-human transgenic mammals according to the claim 24.

1/13

Figure 1

2/13

Figure 2 3/13

T GST 0 10 30 1 10 400 900 Gβγ concentration (nM)

Gβγ concentration (nM)

Figure 3

4/13

tz- ta J

5/13

( ^• nB) Λ-jisiiθjiπ

H PΉ

F585 / F525 ratio

7/13

Gβγ HI Gβγ

Figure 7 8/13

pcDNA3-Ca _v β3-I-II2.1 (SEQ ID NO: 5)

1 ATGGATGACG ACTCCTACGT GCCCGGGTTT GAGGACTCGG AGGCGGGTTC 51 AGCCGACTCC TACACCAGCC GCCCCTCTCT GGACTCAGAC GTTTCCCTGG 101 AGGAGGACCG GGAGAGTGCC CGGCGAGAAG TGGAGAGTCA GGCTCAGCAG 151 CAGCTGGAAA GAGCCAAGCA CAAACCTGTG GCATTTGCTG TGAGGACCAA 201 TGTCAGCTAC TGTGGAGTTC TGGATGAGGA ATGCCCAGTC CAGGGCTCTG 251 GAGTCAACTT CGAGGCCAAA GATTTTCTGC ACATTAAAGA GAAGTACAGC 301 AATGACTGGT GGATCGGGAG GCTAGTGAAA GAAGGTGGCG ATATTGCCTT 351 CATCCCCAGC CCCCAACGCC TGGAGAGCAT CCGGCTCAAA CAGGAACAGA 401 AGGCCAGGAG ATCCGGGAAC CCTTCCAGCC TGAGTGACAT TGGCAACCGA 451 CGTTCCCCTC CTCCATCTCT AGCCAAGCAG AAGCAAAAGC AGGCGGAACA 501 TGTCCCCCCG TATGATGTGG TGCCCTCCAT GCGGCCTGTG GTGCTGGTGG 551 GACCCTCTCT GAAAGGTTAT GAGGTCACAG ACATGATGCA GAAGGCTCTC 601 TTCGACTTCC TTAAACACAG GTTTGATGGC AGGATCTCCA TCACCCGCGT 651 CACGGCTGAC CTCTCACTGG CCAAGCGCTC TGTGCTCAAC AATCCTGGCA 701 AGAGGACCAT CATCGAGCGC TCTTCTGCCC GCTCCAGCAT TGCTGAGGTG 751 CAGAGTGAGA TTGAGCGCAT ATTCGAGCTG GCCAAATCCC TGCAGCTAGT 801 GGTGTTGGAT GCTGACACCA TCAACCACCC AGCACAGCTA GCCAAGACCT 851 CACTGGCCCC CATCATCGTC TTCGTCAAAG TGTCCTCGCC AAAGGTACTG 901 CAGCGACTGA TCCGCTCCAG GGGGAAGTCC CAGATGAAGC ACCTCACTGT 951 ACAGATGATG GCATATGATA AGCTGGTTCA GTGCCCACCT GAGTCATTTG 1001 ATGTGATTCT GGATGAGAAC CAGCTGGACG ACGCCTGTGA GCACCTAGCT 1051 GAATACCTAG AGGTTTACTG GCGCGCTACC CACCACCCAG CACCGGGCCC 1101 CGGGATGCTG GGTCCGCCCA GTGCCATCCC TGGACTTCAG AACCAGCAGC 1151 TGCTGGGGGA GCGAGGTGAG GAGCATTCAC CCCTGGAGCG GGACAGTTTG 1201 ATGCCCTCGG ATGAGGCCAG TGAGAGCTCC CGCCAGGCCT GGACCGGATC 1251 TTCACAGCGC AGCTCCCGCC ATCTGGAGGA GGACTATGCA GATGCCTACC 1301 AGGACCTGTA CCAGCCTCAC CGTCAACACA CCTCGGGGCT ACCCAGTGCT 1351 AACGGGCATG ACCCCCAAGA CCGGCTCCTA GCCCAGGACT CGGAGCATGA 1401 CCACAATGAC CGGAACTGGC AGCGTAACCG GCCTTGGCCT AAGGACAGCT 1451 ACGAATTCGC CAAAGAAAGG GAGCGGGTGG AGAACCGGCG CGCATTCCTG 1501 AAGCTGCGGC GGCAGCAGCA GATTGAACGC GAGCTCAACG GGTACATGGA 1551 GTGGATCTCA AAAGCAGAAG AGGTGATCCT CGCAGAGGAC GAGACCGACG 1601 TGGAGCAGAG ACATCCCTTT GATGGAGCTC TGCGGAGAGC CACTATCAAG 1651 AAGAGCAAGA CGGACCTGCT CCACCCAGAG GAGGC GGAGG ATCAGCTGGC 1701 CGACATCGCC TCCGTGGGGT CTCCCTTTGC CCGAGCCAGC ATTAAAAGTG 1751 CCAAGCTGGA GAACTCGAGT TTTTTCCACA AAAAAGAGAG GAGAATGCGT 1801 TTCTACATCC GTCGCATGGT CAAAACTCAG TAAGAATTCT GCAGATATCC 1851 ATCACACTGG CGGCCGCTCG AGCATGCATC TAGAGGGCCC TATTCTATAG 1901 TGTCACCTAA ATGCTAGAGC TCGCTGATCA GCCTCGACTG TGCCTTCTAG 1951 TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 2001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG 2051 CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA 2101 CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG 2151 TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG 2201 TATCCCCACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 2251 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT 2301 TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA 2351 GCTCTAAATC GGGGCATCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA 2401 CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT 2451 CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC CACGTTCTTT 2501 AATAGTGGAC TCTTGTT CCA AACTGGAACA ACACTCAACC CTATCTCGGT 2551 CTATTCTTTT GATTTATAAG GGATTTTGGG GATTTCGGCC TATTGGTTAA 2601 AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG 2651 TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG CAGGCAGAAG 2701 TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC

Figure 8a 9/13

2751 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 2801 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 2851 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 2901 CAGAGGCCGA GGCCGCCTCT GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 2951 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT 3001 ATCCATTTTC GGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA 3051 TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG 3101 CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 3151 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG 3201 ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG 3251 TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC 3301 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC 3351 TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT 3401 GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 3451 CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC 3501 TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC 3551 GAACTGTTCG CCAGGCTCAA GGCGCGCATG CC CGACGGCG AGGATCTCGT 3601 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC 3651 GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT 3701 CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 3751 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC 3801 AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC 3851 TGGGGTTCGA AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT 3901 TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT 3951 CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT 4001 TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA 4051 AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC 4101 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGTATAC 4151 CGTCGACCTC TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT 4201 GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG 4251 CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA 4301 TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG 4351 CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG 4401 GCGCTCTTCC GCTT CCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC 4451 TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA 4501 GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA 4551 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC 4601 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA 4651 AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 4701 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT 4751 TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT 4801 CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC 4851 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT 4901 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC 4951 AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 5001 GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC 5051 TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC 5101 AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT 5151 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG 5201 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG5251 AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 5301 TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC 5351 AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA 5401 TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG 5451 CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC 5501 CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC

Figure 8b 10/13

5551 AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 5601 CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG 5651 TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT 5701 TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT 5751 GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA 5801 GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT 5851 TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 5901 CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT 5951 GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA 6001 GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT 6051 ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT 6101 CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA 6151 AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 6201 ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT 6251 GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA 6301 GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCGACGGATC 6351 GGGAGATCTC CCGATCCCCT ATGGTCGACT CT CAGTACAA TCTGCTCTGA 6401 TGCCGCATAG TTAAGCCAGT ATCTGCTCCC TGCTTGTGTG TTGGAGGTCG 6451 CTGAGTAGTG CGCGAGCAAA ATTTAAGCTA CAACAAGGCA AGGCTTGACC 6501 GACAATTGCA TGAAGAATCT GCTTAGGGTT AGGCGTTTTG CGCTGCTTCG 6551 CGATGTACGG GCCAGATATA CGCGTTGACA TTGATTATTG ACTAGTTATT 6601 AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA TATGGAGTTC 6651 CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA 6701 CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA 6751 TAGGGACTTT CCATTGACGT CAATGGGTGG ACTATTTACG GTAAACTGCC 6801 CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA 6851 CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT 6901 TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT 6951 ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG GATAGCGGTT 7001 TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT 7051 TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC 7101 GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT 7151 AAGCAGAGCT CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG 7201 AAATTAATAC CTAG CACTAT AGGGAGACCC AAGCTTGGTA CC

Figure 8c 11/13 pCHIC (SEQ ID NO: 10)

1 ATGCTGTGCT GTATGAGAAG AACCAAACAG GTTGAAAAGA ATGATGAGGA 51 CCAAAAGATC ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC 101 CCATCCTGGT CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG 151 TCCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT 201 CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA 251 CCTTCGGCTA CGGCCTGCAG TGCTTCGCCC GCTACCCCGA CCACATGAAG 301 CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG 351 CACCATCTTC TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA 401 AGTTCGAGGG CGACACCCTG GTGAACCGCA TCGAGCTGAA GGGCATCGAC 451 TTCAAGGAGG ACGGCAACAT CCTGGGGCAC AAGCTGGAGT ACAACTACAA 501 CAGCCACAAC GTCTATATCA TGGCCGACAA GCAGAAGAAC GGCATCAAGG 551 TGAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC 601 GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC 651 CGACAACCAC TACCTGAGCT ACCAGTCCGC CCTGAGCAAA GACCCCAACG

701 AGAAGCGCGA TCACATGGTC CTGCTGGAGT TCGTGACCGC CGCCGGGATC 751 ACTCTCGGCA TGGACGAGCT GTACGCGATC GCATCTCTAG CCAAGCAGAA

801 GCAAAAGCAG GCGGAACATG TCCCCCCGTA TGATGTGGTG CCCTCCATGC 851 GGCCTGTGGT GCTGGTGGGA CCCTCTCTGA AAGGTTATGA GGTCACAGAC 901 ATGATGCAGA AGGCTCTCTT CGACTTCCTT AAACACAGGT TTGATCCCTCCGCCGCCGCCCC

1001 TGCTCAACAA TCCTGGCAAG AGGACCATCA TCGAGCGCTC TTCTGCCCGC 1051 TCCAGCATTG CTGAGGTGCA GAGTGAGATT GAGCGCATAT TCGAGCTGGC 1101 CAAATCCCTG CAGCTAGTGG TGTTGGATGC TGACACCATC AACCACCCAG 1151 CACAGCTAGC CAAGACCTCA CTGGCCCCCA TCATCGTCTT CGTCAAAGTG 1201 TCCTCGCCAA AGGTACTGCA GCGACTGATC CGCTCCAGGG GGAAGTCCCA 1251 GATGAAGCAC CTCACTGTAC AGATGATGGC ATATGATAAG CTGGTTCAGT 1301 GCCCACCTGA GTCATTTGAT GTGATTCTGG ATGAGAACCA GCTGGACGAC 1351 GCCTGTGAGC ACCTAGCTGA ATACCTAGAG GTTTACTGGC GCGCTACCCA 1401 CCACCCAGCA CCGGGCCCCG GGATGCTGGG TCCGCCCAGT GCCATCCCTG 1451 GACTTCAGAA CCAGCAGCTG CTGGGGGAGC GAGGTGAGGA GCATTCACCC 1501 CTGGAGCGGG ACAGTTTGAT GCCCTCGGAT GAGGCCAGTG AGAGCTCCCG 1551 CCAGGCCTGG ACCGGATCTT CACAGCGCAG CTCCCGCCAT CTGGAGGAGG 1601 ACTATGCAGA TGCCTACCAG GACCTGTACC AGCCTCACCG TCAACACACC 1651 TCGGGGCTAC CCAGTGCTAA CGGGCATGAC CCCCAAGACC GGCTCCTAGC 1701 CCAGGACTCG GAGCATGACC ACAATGACCG GAACTGGCAG CGTAACCGGC 1751 CTTGGCCTAA GGACAGCTAC GAATTCGCCA AAGAAAGGGA GCGGGTGGAG 1801 AACCGGCGCG CATTCCTGAA GCTGCGGCGG IT GCAGCAGA TTGAACGCGA 1851 GCTCAACGGG TACATGGAGT GGATCTCAAA AGCAGAAGAG GTGATCCTCG 1901 CAGAGGACGA GACCGACGTG GAGCAGAGAC ATCCCTTTGA TGGAGCTCTG 1951 CGGAGAGCCA CTATCAAGAA GAGCAAGACG GACCTGCTCC ACCCAGAGGA 2001 GGCGGAGGAT CAGCTGGCCG ACATCGCCTC CGTGGGGTCT CCCTTTGCCC 2051 GAGCCAGCAT TAAAAGTGCC AAGCTGGAGA ACTCGAGTTT TTTCCACAAA 2101 AAAGAGAGGA GAATGCGTTT CTACATCCGT CGCATGGTCA AAACTCAGAC 2151 TAGTATGGTG AGCAAGGGCG AGGAGCTGTT CACCGGGGTG GTGCCCATCC 2201 TGGTCGAGCT GGACGGCGAC GTAAACGGCC ACAGGTTCAG CGTGTCCGGC 2251 GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA AGTTCATCTG 2301 CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCCTGA 2351 CCTGGGGCGT GCAGTGCTTC AGCCGCTACC CCGACCACAT GAAGCAGCAC 2401 GACTTCTTCA AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT 2451 CTTCTTCAAG GACGACGGCA ACTACAAGAC CCGCGCCGAG GTGAAGTTCG 2501 AGGGCGACAC CCTGGTGAAC CGCATCGAGC TGAAGGGCAT CGACTTCAAG 2551 GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT ATATCAGCCA 2601 CAACGTCTAT ATCACCGCCG ACAAGCAGAA GAACGGCATC AAGGCCAACT 2651 TCAAGATCCG CCW AACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC 2701 TACCAGCAGA ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA

Figure 9a 12/13

2751 CCACTACCTG AGCACCCAGT CCGCCCTGAG CAAAGACCCC AAGGAGAAGC

2801 GCGATCACAT GGTCCTGCTG GAGTTCGTGA CCGCCGCCGG GATCACTCTC

2851 GGCAGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA

2901 GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT

2951 GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA

3001 ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT

3051 AACCAATAGG CCGAAATCGG CAAAATCCCT T TAAATCAA AAGAATAGAC

3101 CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA

3151 AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT

3201 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG

3251 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT

3301 GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA

3351 GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC

3401 CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA

3451 CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA

3501 CATTCAAATA'TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

3551 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA

3601 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA

3651 GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

3701 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

3751 AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC

3801 CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT

3851 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG

3901 AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA

3951 TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC

4001 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA

4051 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG

4101 GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA

4151 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG

4201 CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC

4251 GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA

4301 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG

4351 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT

4401 ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA

4451 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC

4501 CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT

4551 ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT

4601 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG

4651 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT

4701 ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA

4751 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC

4801 CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT

4851 TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC

4901 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA

4951 AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA

5001 AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG 5051 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG 5101 GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT 5151 CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC 5201 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT 5251 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA 5301 AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA 5351 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG 5401 CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC 5451 AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG 5501 ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA

Figure 9b 13/13

5551 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG

5601 TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA

5651 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG

5701 CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC

5751 AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC 5801 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT

5851 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC

5901 TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA

5951 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG

6001 GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA

6051 ACCGTATTAC CGCCATGCAT TAGTTATTAA TAGTAATCAA TTACGGGGTC

6101 ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA

6151 ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA

6201 ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

6251 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT

6301 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC

6351 GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA

6401 GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC

6451 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT

6501 CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

6551 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG

6601 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC

6651 CGTCAGATCC GCTAGCGCTA CCGGACTCAG ATCTCGAGCT CAAGCTTCGA

6701 ATTCTGCAGT CGACGGTACC GCGGGCCCGG GATCCACCGG TCGCCACC

Figure 9c SEQUENCE LISTING

<110> Atomic Energy Commission

National Institute of Health and Medical Research

DE WAARD, Michel DUPUIS, Alain GRUNWALD, Didier SANDOZ, Guillaume

<120> Chimeric protein for the screening of agonists and antagonists of cell signaling pathways dependent on receptors coupled to G proteins

<130> 263PCT87

<160> 10

<170> Patentln version 3.1

<210> 1

<211> 58

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 1 tttggtacca tggatgacga ctcctacgtg cccgggtttg aggactcgga ggcgggtt 58

<210> 2

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> primer

Page 1 <400> 2 gcggaattcg tagctgtcct taggccaagg ccggttacgc tgccagtt 48

<210> 3

<211> 36

<212> DNA

<213> arti fi ci al sequence

<220>

<223> primer

<400> 3 ggggaattcg ccaaagaaag ggagcgggtg gagaac 36

<210> 4

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 4 tttgaattct tactgagttt tgaccatgcg acggatgtag aaacgcattc t 51

<210> 5

<211> 7242

<212> DNA

<213> artificial sequence

<220>

<223> recombinant plasmid

<400> 5 atggatgacg actcctacgt gcccgggttt gaggactcgg aggcgggttc agccgactcc 60 tacaccagcc gcccctctct ggactcagac gtttccctgg aggaggaccg ggagagtgcc 120 cggcgagaag tggagagtca ggctcagcag cagctggaaa gagccaagca caaacctgtg 180 gcatttgctg tgaggaccaa tgtcagctac tgtggagttc tggatgagga atgcccagtc 240 cagggctctg gagtcaactt cgaggccaaa gattttctgc acattaaaga gaagtacagc 300 aatgactggt ggatcgggag gctagtgaaa gaaggtggcg atattgcctt catccccagc 360 ccccaacgcc tggagagcat ccggctcaaa caggaacaga aggccaggag atccgggaac 420

Page 2 ccttccagcc tgagtgacat tggcaaccga cgttcccctc ctccatctct agccaagcag 480 aagcaaaagc aggcggaaca tgtccccccg tatgatgtgg tgccctccat gcggcctgtg 540 gtgctggtgg gaccctctct gaaaggttat gaggtcacag acatgatgca gaaggctctc 600 ttcgacttcc ttaaacacag gtttgatggc aggatctcca tcacccgcgt cacggctgac 660 ctctcactgg ccaagcgctc tgtgctcaac aatcctggca agaggaccat catcgagcgc 720 tcttctgccc gctccagcat tgctgaggtg cagagtgaga ttgagcgcat attcgagctg 780 gccaaatccc tgcagctagt ggtgttggat gctgacacca tcaaccaccc agcacagcta 840 gccaagacct cactggcccc catcatcgtc ttcgtcaaag tgtcctcgcc aaaggtactg 900 cagcgactga tccgctccag ggggaagtcc cagatgaagc acctcactgt acagatgatg 960 gcatatgata agctggttca gtgcccacct gagtcatttg atgtgattct ggatgagaac 1020 cagctggacg acgcctgtga gcacctagct gaatacctag aggtttactg gcgcgctacc 1080 caccacccag caccgggccc cgggatgctg ggtccgccca gtgccatccc tggacttcag 1140 aaccagcagc tgctggggga gcgaggtgag gagcattcac ccctggagcg ggacagtttg 1200 atgccctcgg atgaggccag tgagagctcc cgccaggcct ggaccggatc ttcacagcgc 1260 agctcccgcc ATCT ggagga ggactatgca gatgcctacc aggacctgta ccagcctcac 1320 cgtcaacaca cctcggggct acccagtgct aacgggcatg acccccaaga ccggctccta 1380 gcccaggact cggagcatga ccacaatgac cggaactggc agcgtaaccg gccttggcct 1440 aaggacagct acgaattcgc caaagaaagg gagcgggtgg agaaccggcg cgcattcctg 1500 aagctgcggc ggcagcagca gattgaacgc gagctcaacg ggtacatgga gtggatctca 1560 aaagcagaag aggtgatcct cgcagaggac gagaccgacg tggagcagag acatcccttt 1620 gatggagctc tgcggagagc cactatcaag aagagcaaga cggacctgct ccacccagag 1680 gaggcggagg atcagctggc cgacatcgcc tccgtggggt ctccctttgc ccgagccagc 1740 attaaaagtg ccaagctgga gaactcgagt tttttccaca aaaaagagag gagaatgcgt 1800 ttctacatcc gtcgcatggt caaaactcag taagaattct gcagatatcc atcacactgg 1860 cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc 1920 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1980 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 2040 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 2100 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 2160 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 2220 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 2280 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2340 tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca 2400 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 2460

Page 3 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2520 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg 2580 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 2640 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag 2700 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 2760 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 2820 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 2880 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 2940 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 3000 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 3060 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 3120 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 3180 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 3240 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 3300 tgaagc ggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 3360 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 3420 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 3480 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 3540 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 3600 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 3660 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 3720 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 3780 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 3840 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 3900 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 3960 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 4020 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 4080 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 4140 gtctgtatac c gtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 4200 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4260 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4320 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4380 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4440 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4500 Page 4 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4560 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4620 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4680 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4740 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 4800 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 4860 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 4920 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 4980 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5040 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5100 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5160 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5220 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5280 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5340 gacagt tacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5400 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5460 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5520 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5580 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5640 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5700 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5760 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5820 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5880 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5940 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6000 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6060 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6120 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6180 gcgacacgga has atgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6240 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6300 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc 6360 ccgatcccct atggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 6420 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 6480 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 6540 Page 5 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 6600 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 6660 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 6720 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 6780 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 6840 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgaect 6900 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 6960 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 7020 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 7080 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 7140 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 7200 aaattaatac gactcactat agggagaccc this aagcttggta 7242

<210> 6

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 6 agccgtacgc gategeatet ctagccaagc agaagcaaa 39

<210> 7

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 7 cccgttaacc ccactagtct gagttttgac catgcgacgg at 42

<210> 8

<211> 33

<212> DNA

Page 6 <213> arti fi ci al sequence

<220>

<223> primer

<400> 8 gggactagta tggtgagcaa gggcgaggag ctg 33

<210> 9

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 9 cccgttaact gccgagagtg atcccggcgg cggt 34

<210> 10

<211> 7242

<212> DNA

<213> artificial sequence

<220>

<223> recombinant plasmid

<400> 10 atggatgacg actcctacgt gcccgggttt gaggactcgg aggcgggttc agccgactcc 60 tacaccagcc gcccctctct ggactcagac gtttccctgg aggaggaccg ggagagtgcc 120 cggcgagaag tggagagtca ggctcagcag cagctggaaa gagccaagca caaacctgtg 180 gcatttgctg tgaggaccaa tgtcagctac tgtggagttc tggatgagga atgcccagtc 240 cagggctctg gagtcaactt cgaggccaaa gattttctgc acattaaaga gaagtacagc 300 aatgactggt ggatcgggag gctagtgaaa gaaggtggcg atattgcctt catccccagc 360 ccccaacgcc tggagagcat ccggctcaaa caggaacaga aggccaggag atccgggaac 420 ccttccagcc tgagtgacat tggcaaccga cgttcccctc ctccatctct agccaagcag 480 aagcaaaagc aggcggaaca tgtccccccg tatgatgtgg tgccctccat gcggcctgtg 540 gtgctggtgg gaccctctct gaaaggttat gaggtcacag acatgatgca gaaggctctc 600 ttcgacttcc ttaaacacag gtttgatggc aggatctcca tcacccgcgt cacggctgac 660 ctctcactgg ccaagcgctc tgtgctcaac aatcctggca agaggaccat catcgagcgc 720

Page 7 tcttctgccc gctccagcat tgctgaggtg cagagtgaga ttgagcgcat attcgagctg 780 gccaaatccc tgcagctagt ggtgttggat gctgacacca tcaaccaccc agcacagcta 840 gccaagacct cactggcccc catcatcgtc ttcgtcaaag tgtcctcgcc aaaggtactg 900 cagcgactga tccgctccag ggggaagtcc cagatgaagc acctcactgt acagatgatg 960 gcatatgata agctggttca gtgcccacct gagtcatttg atgtgattct ggatgagaac 1020 cagctggacg acgcctgtga gcacctagct gaatacctag aggtttactg gcgcgctacc 1080 caccacccag caccgggccc cgggatgctg ggtccgccca gtgccatccc tggacttcag 1140 aaccagcagc tgctggggga gcgaggtgag gagcattcac ccctggagcg ggacagtttg 1200 atgccctcgg atgaggccag tgagagctcc cgccaggcct ggaccggatc ttcacagcgc 1260 agctcccgcc atctggagga ggactatgca gatgcctacc aggacctgta ccagcctcac 1320 cgtcaacaca cctcggggct acccagtgct aacgggcatg acccccaaga ccggctccta 1380 gcccaggact cggagcatga ccacaatgac cggaactggc agcgtaaccg gccttggcct 1440 aaggacagct acgaattcgc caaagaaagg gagcgggtgg agaaccggcg cgcattcctg 1500 aagctgcggc ggcagcagca gattgaacgc gagctcaacg ggtacatgga gtggatctca 1560 aaagcagaag aggtgatcct cgcagaggac gagaccgacg tggagcagag acatcccttt 1620 gatggagctc tgcggagagc cactatcaag aagagcaaga cggacctgct ccacccagag 1680 gaggcggagg atcagctggc cgacatcgcc tccgtggggt ctccctttgc ccgagccagc 1740 attaaaagtg ccaagctgga gaactcgagt tttttccaca aaaaagagag gagaatgcgt 1800 ttctacatcc gtcgcatggt caaaactcag taagaattct gcagatatcc atcacactgg 1860 cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc 1920 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1980 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 2040 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 2100 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 2160 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 2220 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 2280 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2340 tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca 2400 cctcgacccc aaaaa acttg attagggtga tggttcacgt agtgggccat cgccctgata 2460 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2520 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg 2580 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 2640 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag 2700 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 2760 Page 8 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 2820 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 2880 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 2940 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 3000 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 3060 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 3120 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 3180 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 3240 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 3300 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 3360 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 3420 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 3480 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 3540 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 3600 cgtgac ccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 3660 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 3720 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 3780 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 3840 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 3900 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 3960 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 4020 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 4080 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 4140 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 4200 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4260 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4320 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4380 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4440 gtcgttcggc t gcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4500 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4560 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4620 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4680 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4740 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 4800 Page 9 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 4860 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 4920 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 4980 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5040 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5100 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5160 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5220 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5280 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5340 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5400 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5460 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5520 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5580 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5640 cgcaac gttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5700 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5760 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5820 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5880 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5940 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6000 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6060 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6120 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6180 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6240 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6300 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc 6360 ccgatcccct atggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 6420 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 6480 caacaaggca has ggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 6540 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 6600 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 6660 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 6720 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 6780 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 6840

Page 10 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgaect 6900 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 6960 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 7020 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 7080 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 7140 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 7200 aaattaatac gactcactat agggagaccc this aagcttggta 7242

11/11 1/13

Figure 1

2/13

Figure 2 3/13

T GST 0 10 30 1 10 400 900 Gβγ concentration (nM)

Gβγ concentration (nM)

Figure 3

4/13

tz- ta J

5/13

( ^• nB) Λ-jisiiθjiπ

H PΉ

F585 / F525 ratio

7/13

Gβγ HI Gβγ

Figure 7 8/13

pcDNA3-Ca _v β3-I-II2.1 (SEQ ID NO: 5)

1 ATGGATGACG ACTCCTACGT GCCCGGGTTT GAGGACTCGG AGGCGGGTTC 51 AGCCGACTCC TACACCAGCC GCCCCTCTCT GGACTCAGAC GTTTCCCTGG 101 AGGAGGACCG GGAGAGTGCC CGGCGAGAAG TGGAGAGTCA GGCTCAGCAG 151 CAGCTGGAAA GAGCCAAGCA CAAACCTGTG GCATTTGCTG TGAGGACCAA 201 TGTCAGCTAC TGTGGAGTTC TGGATGAGGA ATGCCCAGTC CAGGGCTCTG 251 GAGTCAACTT CGAGGCCAAA GATTTTCTGC ACATTAAAGA GAAGTACAGC 301 AATGACTGGT GGATCGGGAG GCTAGTGAAA GAAGGTGGCG ATATTGCCTT 351 CATCCCCAGC CCCCAACGCC TGGAGAGCAT CCGGCTCAAA CAGGAACAGA 401 AGGCCAGGAG ATCCGGGAAC CCTTCCAGCC TGAGTGACAT TGGCAACCGA 451 CGTTCCCCTC CTCCATCTCT AGCCAAGCAG AAGCAAAAGC AGGCGGAACA 501 TGTCCCCCCG TATGATGTGG TGCCCTCCAT GCGGCCTGTG GTGCTGGTGG 551 GACCCTCTCT GAAAGGTTAT GAGGTCACAG ACATGATGCA GAAGGCTCTC 601 TTCGACTTCC TTAAACACAG GTTTGATGGC AGGATCTCCA TCACCCGCGT 651 CACGGCTGAC CTCTCACTGG CCAAGCGCTC TGTGCTCAAC AATCCTGGCA 701 AGAGGACCAT CATCGAGCGC TCTTCTGCCC GCTCCAGCAT TGCTGAGGTG 751 CAGAGTGAGA TTGAGCGCAT ATTCGAGCTG GCCAAATCCC TGCAGCTAGT 801 GGTGTTGGAT GCTGACACCA TCAACCACCC AGCACAGCTA GCCAAGACCT 851 CACTGGCCCC CATCATCGTC TTCGTCAAAG TGTCCTCGCC AAAGGTACTG 901 CAGCGACTGA TCCGCTCCAG GGGGAAGTCC CAGATGAAGC ACCTCACTGT 951 ACAGATGATG GCATATGATA AGCTGGTTCA GTGCCCACCT GAGTCATTTG 1001 ATGTGATTCT GGATGAGAAC CAGCTGGACG ACGCCTGTGA GCACCTAGCT 1051 GAATACCTAG AGGTTTACTG GCGCGCTACC CACCACCCAG CACCGGGCCC 1101 CGGGATGCTG GGTCCGCCCA GTGCCATCCC TGGACTTCAG AACCAGCAGC 1151 TGCTGGGGGA GCGAGGTGAG GAGCATTCAC CCCTGGAGCG GGACAGTTTG 1201 ATGCCCTCGG ATGAGGCCAG TGAGAGCTCC CGCCAGGCCT GGACCGGATC 1251 TTCACAGCGC AGCTCCCGCC ATCTGGAGGA GGACTATGCA GATGCCTACC 1301 AGGACCTGTA CCAGCCTCAC CGTCAACACA CCTCGGGGCT ACCCAGTGCT 1351 AACGGGCATG ACCCCCAAGA CCGGCTCCTA GCCCAGGACT CGGAGCATGA 1401 CCACAATGAC CGGAACTGGC AGCGTAACCG GCCTTGGCCT AAGGACAGCT 1451 ACGAATTCGC CAAAGAAAGG GAGCGGGTGG AGAACCGGCG CGCATTCCTG 1501 AAGCTGCGGC GGCAGCAGCA GATTGAACGC GAGCTCAACG GGTACATGGA 1551 GTGGATCTCA AAAGCAGAAG AGGTGATCCT CGCAGAGGAC GAGACCGACG 1601 TGGAGCAGAG ACATCCCTTT GATGGAGCTC TGCGGAGAGC CACTATCAAG 1651 AAGAGCAAGA CGGACCTGCT CCACCCAGAG GAGGC GGAGG ATCAGCTGGC 1701 CGACATCGCC TCCGTGGGGT CTCCCTTTGC CCGAGCCAGC ATTAAAAGTG 1751 CCAAGCTGGA GAACTCGAGT TTTTTCCACA AAAAAGAGAG GAGAATGCGT 1801 TTCTACATCC GTCGCATGGT CAAAACTCAG TAAGAATTCT GCAGATATCC 1851 ATCACACTGG CGGCCGCTCG AGCATGCATC TAGAGGGCCC TATTCTATAG 1901 TGTCACCTAA ATGCTAGAGC TCGCTGATCA GCCTCGACTG TGCCTTCTAG 1951 TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG 2001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG 2051 CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA 2101 CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG 2151 TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG 2201 TATCCCCACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT 2251 TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT 2301 TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA 2351 GCTCTAAATC GGGGCATCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA 2401 CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT 2451 CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC CACGTTCTTT 2501 AATAGTGGAC TCTTGTT CCA AACTGGAACA ACACTCAACC CTATCTCGGT 2551 CTATTCTTTT GATTTATAAG GGATTTTGGG GATTTCGGCC TATTGGTTAA 2601 AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG 2651 TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG CAGGCAGAAG 2701 TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC

Figure 8a 9/13

2751 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 2801 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 2851 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 2901 CAGAGGCCGA GGCCGCCTCT GCCTCTGAGC TATTCCAGAA GTAGTGAGGA 2951 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT 3001 ATCCATTTTC GGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA 3051 TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG 3101 CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 3151 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG 3201 ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG 3251 TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC 3301 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC 3351 TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT 3401 GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 3451 CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC 3501 TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC 3551 GAACTGTTCG CCAGGCTCAA GGCGCGCATG CC CGACGGCG AGGATCTCGT 3601 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC 3651 GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT 3701 CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 3751 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC 3801 AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC 3851 TGGGGTTCGA AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT 3901 TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT 3951 CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT 4001 TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA 4051 AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC 4101 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGTATAC 4151 CGTCGACCTC TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT 4201 GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG 4251 CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA 4301 TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG 4351 CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG 4401 GCGCTCTTCC GCTT CCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC 4451 TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA 4501 GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA 4551 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC 4601 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA 4651 AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 4701 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT 4751 TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT 4801 CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC 4851 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT 4901 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC 4951 AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 5001 GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC 5051 TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC 5101 AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT 5151 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG 5201 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG5251 AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 5301 TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC 5351 AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA 5401 TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG 5451 CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC 5501 CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC

Figure 8b 10/13

5551 AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 5601 CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG 5651 TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT 5701 TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT 5751 GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA 5801 GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT 5851 TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 5901 CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT 5951 GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA 6001 GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT 6051 ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT 6101 CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA 6151 AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 6201 ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT 6251 GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA 6301 GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCGACGGATC 6351 GGGAGATCTC CCGATCCCCT ATGGTCGACT CT CAGTACAA TCTGCTCTGA 6401 TGCCGCATAG TTAAGCCAGT ATCTGCTCCC TGCTTGTGTG TTGGAGGTCG 6451 CTGAGTAGTG CGCGAGCAAA ATTTAAGCTA CAACAAGGCA AGGCTTGACC 6501 GACAATTGCA TGAAGAATCT GCTTAGGGTT AGGCGTTTTG CGCTGCTTCG 6551 CGATGTACGG GCCAGATATA CGCGTTGACA TTGATTATTG ACTAGTTATT 6601 AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA TATGGAGTTC 6651 CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA 6701 CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA 6751 TAGGGACTTT CCATTGACGT CAATGGGTGG ACTATTTACG GTAAACTGCC 6801 CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA 6851 CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT 6901 TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT 6951 ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG GATAGCGGTT 7001 TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT 7051 TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC 7101 GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT 7151 AAGCAGAGCT CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG 7201 AAATTAATAC CTAG CACTAT AGGGAGACCC AAGCTTGGTA CC

Figure 8c 11/13 pCHIC (SEQ ID NO: 10)

1 ATGCTGTGCT GTATGAGAAG AACCAAACAG GTTGAAAAGA ATGATGAGGA 51 CCAAAAGATC ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC 101 CCATCCTGGT CGAGCTGGAC GGCGACGTAA ACGGCCACAA GTTCAGCGTG 151 TCCGGCGAGG GCGAGGGCGA TGCCACCTAC GGCAAGCTGA CCCTGAAGTT 201 CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTCGTGACCA 251 CCTTCGGCTA CGGCCTGCAG TGCTTCGCCC GCTACCCCGA CCACATGAAG 301 CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG 351 CACCATCTTC TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA 401 AGTTCGAGGG CGACACCCTG GTGAACCGCA TCGAGCTGAA GGGCATCGAC 451 TTCAAGGAGG ACGGCAACAT CCTGGGGCAC AAGCTGGAGT ACAACTACAA 501 CAGCCACAAC GTCTATATCA TGGCCGACAA GCAGAAGAAC GGCATCAAGG 551 TGAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC 601 GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC 651 CGACAACCAC TACCTGAGCT ACCAGTCCGC CCTGAGCAAA GACCCCAACG

701 AGAAGCGCGA TCACATGGTC CTGCTGGAGT TCGTGACCGC CGCCGGGATC 751 ACTCTCGGCA TGGACGAGCT GTACGCGATC GCATCTCTAG CCAAGCAGAA

801 GCAAAAGCAG GCGGAACATG TCCCCCCGTA TGATGTGGTG CCCTCCATGC 851 GGCCTGTGGT GCTGGTGGGA CCCTCTCTGA AAGGTTATGA GGTCACAGAC 901 ATGATGCAGA AGGCTCTCTT CGACTTCCTT AAACACAGGT TTGATCCCTCCGCCGCCGCCCC

1001 TGCTCAACAA TCCTGGCAAG AGGACCATCA TCGAGCGCTC TTCTGCCCGC 1051 TCCAGCATTG CTGAGGTGCA GAGTGAGATT GAGCGCATAT TCGAGCTGGC 1101 CAAATCCCTG CAGCTAGTGG TGTTGGATGC TGACACCATC AACCACCCAG 1151 CACAGCTAGC CAAGACCTCA CTGGCCCCCA TCATCGTCTT CGTCAAAGTG 1201 TCCTCGCCAA AGGTACTGCA GCGACTGATC CGCTCCAGGG GGAAGTCCCA 1251 GATGAAGCAC CTCACTGTAC AGATGATGGC ATATGATAAG CTGGTTCAGT 1301 GCCCACCTGA GTCATTTGAT GTGATTCTGG ATGAGAACCA GCTGGACGAC 1351 GCCTGTGAGC ACCTAGCTGA ATACCTAGAG GTTTACTGGC GCGCTACCCA 1401 CCACCCAGCA CCGGGCCCCG GGATGCTGGG TCCGCCCAGT GCCATCCCTG 1451 GACTTCAGAA CCAGCAGCTG CTGGGGGAGC GAGGTGAGGA GCATTCACCC 1501 CTGGAGCGGG ACAGTTTGAT GCCCTCGGAT GAGGCCAGTG AGAGCTCCCG 1551 CCAGGCCTGG ACCGGATCTT CACAGCGCAG CTCCCGCCAT CTGGAGGAGG 1601 ACTATGCAGA TGCCTACCAG GACCTGTACC AGCCTCACCG TCAACACACC 1651 TCGGGGCTAC CCAGTGCTAA CGGGCATGAC CCCCAAGACC GGCTCCTAGC 1701 CCAGGACTCG GAGCATGACC ACAATGACCG GAACTGGCAG CGTAACCGGC 1751 CTTGGCCTAA GGACAGCTAC GAATTCGCCA AAGAAAGGGA GCGGGTGGAG 1801 AACCGGCGCG CATTCCTGAA GCTGCGGCGG IT GCAGCAGA TTGAACGCGA 1851 GCTCAACGGG TACATGGAGT GGATCTCAAA AGCAGAAGAG GTGATCCTCG 1901 CAGAGGACGA GACCGACGTG GAGCAGAGAC ATCCCTTTGA TGGAGCTCTG 1951 CGGAGAGCCA CTATCAAGAA GAGCAAGACG GACCTGCTCC ACCCAGAGGA 2001 GGCGGAGGAT CAGCTGGCCG ACATCGCCTC CGTGGGGTCT CCCTTTGCCC 2051 GAGCCAGCAT TAAAAGTGCC AAGCTGGAGA ACTCGAGTTT TTTCCACAAA 2101 AAAGAGAGGA GAATGCGTTT CTACATCCGT CGCATGGTCA AAACTCAGAC 2151 TAGTATGGTG AGCAAGGGCG AGGAGCTGTT CACCGGGGTG GTGCCCATCC 2201 TGGTCGAGCT GGACGGCGAC GTAAACGGCC ACAGGTTCAG CGTGTCCGGC 2251 GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA AGTTCATCTG 2301 CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCCTGA 2351 CCTGGGGCGT GCAGTGCTTC AGCCGCTACC CCGACCACAT GAAGCAGCAC 2401 GACTTCTTCA AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT 2451 CTTCTTCAAG GACGACGGCA ACTACAAGAC CCGCGCCGAG GTGAAGTTCG 2501 AGGGCGACAC CCTGGTGAAC CGCATCGAGC TGAAGGGCAT CGACTTCAAG 2551 GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT ATATCAGCCA 2601 CAACGTCTAT ATCACCGCCG ACAAGCAGAA GAACGGCATC AAGGCCAACT 2651 TCAAGATCCG CCW AACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC 2701 TACCAGCAGA ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA

Figure 9a 12/13

2751 CCACTACCTG AGCACCCAGT CCGCCCTGAG CAAAGACCCC AAGGAGAAGC

2801 GCGATCACAT GGTCCTGCTG GAGTTCGTGA CCGCCGCCGG GATCACTCTC

2851 GGCAGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA

2901 GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT

2951 GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA

3001 ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT

3051 AACCAATAGG CCGAAATCGG CAAAATCCCT T TAAATCAA AAGAATAGAC

3101 CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA

3151 AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT

3201 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG

3251 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT

3301 GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA

3351 GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC

3401 CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA

3451 CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA

3501 CATTCAAATA'TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

3551 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA

3601 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA

3651 GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

3701 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

3751 AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC

3801 CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT

3851 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG

3901 AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA

3951 TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC

4001 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA

4051 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG

4101 GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA

4151 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG

4201 CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC

4251 GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA

4301 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG

4351 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT

4401 ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA

4451 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC

4501 CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT

4551 ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT

4601 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG

4651 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT

4701 ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA

4751 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC

4801 CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT

4851 TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC

4901 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA

4951 AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA

5001 AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG 5051 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG 5101 GGGCCAATAC GCCCGGGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT 5151 CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC 5201 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT 5251 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA 5301 AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA 5351 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG 5401 CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC 5451 AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG 5501 ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA

Figure 9b 13/13

5551 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG

5601 TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA

5651 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG

5701 CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC

5751 AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC 5801 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT

5851 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC

5901 TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA

5951 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG

6001 GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA

6051 ACCGTATTAC CGCCATGCAT TAGTTATTAA TAGTAATCAA TTACGGGGTC

6101 ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA

6151 ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA

6201 ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

6251 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT

6301 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC

6351 GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA

6401 GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC

6451 AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT

6501 CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

6551 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG

6601 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC

6651 CGTCAGATCC GCTAGCGCTA CCGGACTCAG ATCTCGAGCT CAAGCTTCGA

6701 ATTCTGCAGT CGACGGTACC GCGGGCCCGG GATCCACCGG TCGCCACC

Figure 9c SEQUENCE LISTING

<110> Atomic Energy Commission

National Institute of Health and Medical Research

DE WAARD, Michel DUPUIS, Alain GRUNWALD, Didier SANDOZ, Guillaume

<120> Chimeric protein for the screening of agonists and antagonists of cell signaling pathways dependent on receptors coupled to G proteins

<130> 263PCT87

<160> 10

<170> Patentln version 3.1

<210> 1

<211> 58

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 1 tttggtacca tggatgacga ctcctacgtg cccgggtttg aggactcgga ggcgggtt 58

<210> 2

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> primer

Page 1 <400> 2 gcggaattcg tagctgtcct taggccaagg ccggttacgc tgccagtt 48

<210> 3

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 3 ggggaattcg ccaaagaaag ggagcgggtg gagaac 36

<210> 4

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 4 tttgaattct tactgagttt tgaccatgcg acggatgtag aaacgcattc t 51

<210> 5

<211> 7242

<212> DNA

<213> artificial sequence

<220>

<223> recombinant plasmid

<400> 5 atggatgacg actcctacgt gcccgggttt gaggactcgg aggcgggttc agccgactcc 60 tacaccagcc gcccctctct ggactcagac gtttccctgg aggaggaccg ggagagtgcc 120 cggcgagaag tggagagtca ggctcagcag cagctggaaa gagccaagca caaacctgtg 180 gcatttgctg tgaggaccaa tgtcagctac tgtggagttc tggatgagga atgcccagtc 240 cagggctctg gagtcaactt cgaggccaaa gattttctgc acattaaaga gaagtacagc 300 aatgactggt ggatcgggag gctagtgaaa gaaggtggcg atattgcctt catccccagc 360 ccccaacgcc tggagagcat ccggctcaaa caggaacaga aggccaggag atccgggaac 420

Page 2 ccttccagcc tgagtgacat tggcaaccga cgttcccctc ctccatctct agccaagcag 480 aagcaaaagc aggcggaaca tgtccccccg tatgatgtgg tgccctccat gcggcctgtg 540 gtgctggtgg gaccctctct gaaaggttat gaggtcacag acatgatgca gaaggctctc 600 ttcgacttcc ttaaacacag gtttgatggc aggatctcca tcacccgcgt cacggctgac 660 ctctcactgg ccaagcgctc tgtgctcaac aatcctggca agaggaccat catcgagcgc 720 tcttctgccc gctccagcat tgctgaggtg cagagtgaga ttgagcgcat attcgagctg 780 gccaaatccc tgcagctagt ggtgttggat gctgacacca tcaaccaccc agcacagcta 840 gccaagacct cactggcccc catcatcgtc ttcgtcaaag tgtcctcgcc aaaggtactg 900 cagcgactga tccgctccag ggggaagtcc cagatgaagc acctcactgt acagatgatg 960 gcatatgata agctggttca gtgcccacct gagtcatttg atgtgattct ggatgagaac 1020 cagctggacg acgcctgtga gcacctagct gaatacctag aggtttactg gcgcgctacc 1080 caccacccag caccgggccc cgggatgctg ggtccgccca gtgccatccc tggacttcag 1140 aaccagcagc tgctggggga gcgaggtgag gagcattcac ccctggagcg ggacagtttg 1200 atgccctcgg atgaggccag tgagagctcc cgccaggcct ggaccggatc ttcacagcgc 1260 agctcccgcc ATCT ggagga ggactatgca gatgcctacc aggacctgta ccagcctcac 1320 cgtcaacaca cctcggggct acccagtgct aacgggcatg acccccaaga ccggctccta 1380 gcccaggact cggagcatga ccacaatgac cggaactggc agcgtaaccg gccttggcct 1440 aaggacagct acgaattcgc caaagaaagg gagcgggtgg agaaccggcg cgcattcctg 1500 aagctgcggc ggcagcagca gattgaacgc gagctcaacg ggtacatgga gtggatctca 1560 aaagcagaag aggtgatcct cgcagaggac gagaccgacg tggagcagag acatcccttt 1620 gatggagctc tgcggagagc cactatcaag aagagcaaga cggacctgct ccacccagag 1680 gaggcggagg atcagctggc cgacatcgcc tccgtggggt ctccctttgc ccgagccagc 1740 attaaaagtg ccaagctgga gaactcgagt tttttccaca aaaaagagag gagaatgcgt 1800 ttctacatcc gtcgcatggt caaaactcag taagaattct gcagatatcc atcacactgg 1860 cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc 1920 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1980 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 2040 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 2100 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 2160 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 2220 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 2280 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2340 tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca 2400 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 2460 Page 3 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2520 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg 2580 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 2640 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag 2700 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 2760 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 2820 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 2880 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 2940 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 3000 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 3060 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 3120 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 3180 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 3240 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 3300 tgaagc ggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 3360 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 3420 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 3480 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 3540 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 3600 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 3660 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 3720 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 3780 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 3840 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 3900 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 3960 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 4020 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 4080 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 4140 gtctgtatac c gtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 4200 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4260 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4320 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4380 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4440 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4500 Page 4 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4560 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4620 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4680 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4740 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 4800 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 4860 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 4920 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 4980 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5040 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5100 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5160 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5220 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5280 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5340 gacagt tacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5400 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5460 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5520 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5580 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5640 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5700 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5760 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5820 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5880 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5940 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6000 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6060 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6120 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6180 gcgacacgga has atgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6240 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6300 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc 6360 ccgatcccct atggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 6420 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 6480 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 6540 Page 5 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 6600 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 6660 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 6720 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 6780 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 6840 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgaect 6900 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 6960 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 7020 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 7080 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 7140 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 7200 aaattaatac gactcactat agggagaccc this aagcttggta 7242

<210> 6

<211> 39

<212> DNA

<213> artifi ci al sequence

<220>

<223> primer

<400> 6 agccgtacgc gategeatet ctagccaagc agaagcaaa 39

<210> 7

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 7 cccgttaacc ccactagtct gagttttgac catgcgacgg at 42

<210> 8 <211> 33 <212> DNA

Page 6 <213> artificial sequence

<220>

<223> primer

<400> 8 gggactagta tggtgagcaa gggcgaggag ctg 33

<210> 9

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 9 cccgttaact gccgagagtg atcccggcgg cggt 34

<210> 10

<211> 7242

<212> DNA

<213> artificial sequence

<220>

<223> recombinant plasmid

<400> 10 atggatgacg actcctacgt gcccgggttt gaggactcgg aggcgggttc agccgactcc 60 tacaccagcc gcccctctct ggactcagac gtttccctgg aggaggaccg ggagagtgcc 120 cggcgagaag tggagagtca ggctcagcag cagctggaaa gagccaagca caaacctgtg 180 gcatttgctg tgaggaccaa tgtcagctac tgtggagttc tggatgagga atgcccagtc 240 cagggctctg gagtcaactt cgaggccaaa gattttctgc acattaaaga gaagtacagc 300 aatgactggt ggatcgggag gctagtgaaa gaaggtggcg atattgcctt catccccagc 360 ccccaacgcc tggagagcat ccggctcaaa caggaacaga aggccaggag atccgggaac 420 ccttccagcc tgagtgacat tggcaaccga cgttcccctc ctccatctct agccaagcag 480 aagcaaaagc aggcggaaca tgtccccccg tatgatgtgg tgccctccat gcggcctgtg 540 gtgctggtgg gaccctctct gaaaggttat gaggtcacag acatgatgca gaaggctctc 600 ttcgacttcc ttaaacacag gtttgatggc aggatctcca tcacccgcgt cacggctgac 660 ctctcactgg ccaagcgctc tgtgctcaac aatcctggca agaggaccat catcgagcgc 720

Page 7 tcttctgccc gctccagcat tgctgaggtg cagagtgaga ttgagcgcat attcgagctg 780 gccaaatccc tgcagctagt ggtgttggat gctgacacca tcaaccaccc agcacagcta 840 gccaagacct cactggcccc catcatcgtc ttcgtcaaag tgtcctcgcc aaaggtactg 900 cagcgactga tccgctccag ggggaagtcc cagatgaagc acctcactgt acagatgatg 960 gcatatgata agctggttca gtgcccacct gagtcatttg atgtgattct ggatgagaac 1020 cagctggacg acgcctgtga gcacctagct gaatacctag aggtttactg gcgcgctacc 1080 caccacccag caccgggccc cgggatgctg ggtccgccca gtgccatccc tggacttcag 1140 aaccagcagc tgctggggga gcgaggtgag gagcattcac ccctggagcg ggacagtttg 1200 atgccctcgg atgaggccag tgagagctcc cgccaggcct ggaccggatc ttcacagcgc 1260 agctcccgcc atctggagga ggactatgca gatgcctacc aggacctgta ccagcctcac 1320 cgtcaacaca cctcggggct acccagtgct aacgggcatg acccccaaga ccggctccta 1380 gcccaggact cggagcatga ccacaatgac cggaactggc agcgtaaccg gccttggcct 1440 aaggacagct acgaattcgc caaagaaagg gagcgggtgg agaaccggcg cgcattcctg 1500 aagctgcggc ggcagcagca gattgaacgc gagctcaacg ggtacatgga gtggatctca 1560 aaagcagaag aggtgatcct cgcagaggac gagaccgacg tggagcagag acatcccttt 1620 gatggagctc tgcggagagc cactatcaag aagagcaaga cggacctgct ccacccagag 1680 gaggcggagg atcagctggc cgacatcgcc tccgtggggt ctccctttgc ccgagccagc 1740 attaaaagtg ccaagctgga gaactcgagt tttttccaca aaaaagagag gagaatgcgt 1800 ttctacatcc gtcgcatggt caaaactcag taagaattct gcagatatcc atcacactgg 1860 cggccgctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc 1920 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1980 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 2040 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 2100 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 2160 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 2220 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 2280 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2340 tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca 2400 cctcgacccc aaaaa acttg attagggtga tggttcacgt agtgggccat cgccctgata 2460 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2520 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg 2580 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 2640 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag 2700 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 2760 Page 8 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 2820 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 2880 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 2940 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 3000 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 3060 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 3120 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 3180 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 3240 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 3300 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 3360 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 3420 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 3480 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 3540 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 3600 cgtgac ccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 3660 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 3720 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 3780 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 3840 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 3900 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 3960 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 4020 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 4080 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 4140 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 4200 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4260 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4320 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4380 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4440 gtcgttcggc t gcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4500 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4560 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4620 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4680 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4740 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 4800 Page 9 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 4860 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 4920 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 4980 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5040 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5100 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5160 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5220 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5280 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5340 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5400 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5460 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5520 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5580 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5640 cgcaac gttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5700 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5760 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5820 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5880 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5940 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6000 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 6060 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 6120 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6180 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6240 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6300 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc 6360 ccgatcccct atggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 6420 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 6480 caacaaggca has ggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 6540 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 6600 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 6660 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 6720 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 6780 actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 6840

Page 10 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgaect 6900 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 6960 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 7020 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 7080 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 7140 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 7200 aaattaatac gactcactat agggagaccc this aagcttggta 7242

Page 11