CN1549858A - Endogenous and non-endogenous versions of human G protein-coupled receptors - Google Patents

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown, and to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity.

Description

Endogenous and non-endogenous versions of human G protein-coupled receptors

The cross reference of related application

The application is the United States serial 09/170 of on October 13rd, 1998 in United States Patent and Trademark Office's application, 496 part continuation application, the corresponding PCT application number PCT/US99/23938 of United States serial 09/170,496 is open on April 20th, 2000, and publication number is WO00/22129.Below presents requires all these by U.S. Express Mail in the appointed day right of priority in the provisional application of United States Patent and Trademark Office's application: the interim sequence number 60/253,404 of the U.S. of application on November 27th, 2000; The interim sequence number 60/255,366 of the U.S. of application on December 12nd, 2000; The interim sequence number 60/270,286 of the U.S. of application on February 20 calendar year 2001; The interim sequence number 60/282,356 of the U.S. of application on April 6 calendar year 2001, this application require the right of priority of the U.S. Provisional Application 60/270,266 of application on February 20 calendar year 2001; The interim sequence number 60/282,032 of the U.S. of application on April 6 calendar year 2001; The interim sequence number 60/282,358 of the U.S. of application on April 6 calendar year 2001; The interim sequence number 60/282,365 of the U.S. of application on April 6 calendar year 2001; The interim sequence number 60/290,917 of the U.S. of application on May 14 calendar year 2001; The interim sequence number 60/309,208 of the U.S. of application on July 31 calendar year 2001; The disclosure of all these provisional application all intactly is attached to herein by reference.

Invention field

The present invention relates to transmembrane receptor, relate to g protein coupled receptor in some embodiments, relate in some preferred embodiments and be subjected to changing to set up or to strengthen the endogenous GPCR of the constitutive activity of described acceptor.In some embodiments, use composing type activatory GPCR directly to identify receptor stimulant or the inverse agonist (inverse agonist) that has the therapeutical agent applicability in the candidate compound.

Background of invention

Though there are many receptor families in the human body, the abundantest at present and maximally related with treatment is g protein coupled receptor (GPCR) family.According to estimates, have an appointment 30 in the human genome, 000-40,000 gene is estimated wherein about 2% coding GPCR.The acceptor (comprising GPCR) of having identified its endogenous ligands is called " known " acceptor, and does not identify that the acceptor of endogenous ligands is called " solitarily " acceptor.

GPCR is the key areas of development pharmaceutical prod: about 20 kinds from 100 kinds of known GPCR develop and in whole prescription drugss about 60% medicine.For example, in 1999, in a prescription drugs of 100 brands, below medicine and GPCR interact (refer in the parenthesis treated disease and/or disorder):

Claritin  (transformation reactions) Prozac  (depression) Vasotec  (hypertension)

Paxil  (depression) Zoloft  (depression) Zyprexa  (psychosis)

Cozaar  (hypertension) Imitrex  (migraine) Zantac  (anti-stream)

Propulsid  (reflux disease) Risperdal  (schizophrenia Serevent  (asthma)

Disease)

Pepcid  (anti-stream) Gaster  (ulcer) Atrovent  (segmental bronchus convulsion

Contraction)

Effexor  (depression) Depakote  (epilepsy) Cardura  (prostate gland fertilizer

Greatly)

Allegra  (transformation reactions) Lupron  (prostate cancer) Zoladex  (prostate cancer)

(sensation lacks BuSpar  (anxiety) Ventolin  (segmental bronchus convulsion to Diprivan 

Lose) contraction)

Hytrin  (hypertension) Wellbutrin  (depression) Zyrtec  (rhinitis)

Plavix  (MI/ apoplexy) Toprol-XL  (hypertension) Tenormin  (angor)

Xalatan  (glaucoma) Singulair (asthma) Diovan  (hypertension)

(prostate gland increases Harnal 

Give birth to)

(Med Ad News 1999 annual datas).

GPCR has an apokoinou construction motif jointly, this motif comprises seven by 22 to 24 sequences that hydrophobic amino acid is formed, these seven sequences form seven α spirals, and each α spiral is all striden film (stride the film number with figure denote, promptly stride film-1 (TM-1), stride film-2 (TM-2) etc.).Described transbilayer helix is being striden film-2 and is being striden film-3, strides film-4 and stride film-5 and stride film-6 and the amino acid chain of striding between the film-7 connects (these amino acid chains are called " born of the same parents outside " and distinguish 1,2 and 3 (EC-1, EC-2 and EC-3)) by cytolemma outside or " outside ".Described transbilayer helix is striding film-1 and striding film-2, stride film-3 and stride film-4 and stride film-5 and the amino acid chain of striding between the film-6 connects (these amino acid chains are called " born of the same parents in " and distinguish 1,2 and 3 (IC-1, IC-2 and IC-3)) by cytolemma the inside or " inboard " also." carboxyl " of described acceptor (" C ") is terminal at intracellular born of the same parents' inside clearance, and " amino " of described acceptor (" N ") is terminal at extracellular extracellular space.

In general,, change, allow in described intracellular region and born of the same parents, between " G-albumen " coupling to take place at the intracellular region occurred conformation when endogenous ligands during in conjunction with described acceptor (being commonly referred to " activation " of described acceptor).It is reported that GPCR and G albumen are " choosing friends indiscriminately ", promptly GPCR can with more than one G protein-interacting.See Kenakin, T., 43 Life Sciences 1095 (1988).Though there is other G albumen, the G albumen of having identified at present is G _q, G _s, G _i, G _zAnd G _oGPCR start signal cascade process (being called " signal transduction ") with the ligand activation of G albumen coupling.Under normal operation, signal transduction finally causes cell activation or cell to suppress.Though without wishing to be bound by theory, think the C-terminal and the described G protein-interacting of IC-3 winding and described acceptor.

Under physiological condition, GPCR is present on the cytolemma, is in two kinds of not balances between isomorphic map: " inactivation " state and " activity " state.The acceptor of inactivated state can not the entering signal transduction pathway with the initial signal transduction that causes biologically.Changing described receptor conformation makes it possible to enter transduction pathway (by G albumen) and produces biologically to active condition.

Can acceptor be stable at active condition by part or compound such as medicine.Nearest discovery provides and promoted and stablized the method for described acceptor in activity conformation except that part or medicine, and described nearest discovery comprises but is not only limited to the aminoacid sequence of modifying described acceptor.These methods are effectively stablized described acceptor in active condition by stimulating the effect of part in conjunction with described acceptor.Stable being called " composing type receptor activation " that obtains by the described mode that is independent of part.

The invention summary

Endogenous form and non-endogenous form and their application of the open people GPCR of this paper.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:2, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:1, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:4, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the non-endogenous composing type activated form of the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:3, described plasmid and the host cell that comprises them.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:6, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:5, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:8, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the non-endogenous composing type activated form of the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:7, described plasmid and the host cell that comprises them.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:10, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:9, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:12, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:11, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:14, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:13, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:16, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:15, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:18, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:17, and the host cell that comprises described plasmid.

Embodiments more of the present invention relate to by the non-endogenous composing type activated form of the amino acid sequences encoded g protein coupled receptor of SEQ.ID.NO.:20, described acceptor and the host cell that comprises them.

Embodiments more of the present invention relate to the plasmid of the cDNA that comprises carrier and SEQ.ID.NO.:19, and the host cell that comprises described plasmid.

The accompanying drawing summary

Fig. 1 illustrates and uses 293 cells measuring inositol monophosphate (IP ₃) during the second messenger of accumulation measures, RUP32, G _q(del)/G _i, and G _q(del)/G _iThe RUP32 of cotransfection and CMV (contrast; Expression vector) activation.

Fig. 2 illustrates endogenous form RUP35 and compares the second messenger IP that causes with RUP36 with contrast (" CMV ") ₃Produce.

Detailed Description Of The Invention

Scientific literature around the acceptor development has been used many terms are named various effects to acceptor part. Be clear and uniformity, definition below in whole this patent document, will using. When other definition of these definition and these terms during contradiction, with the following standard that is defined as:

Activator should refer to such material (such as part, candidate compound): when as described in during the material bind receptor, activating cell internal reaction or increase GTP and be combined with film. In some embodiments, activator is the unknown activating cell internal reaction or conjunctival material of increase GTP when bind receptor before those.

Amino acid abbreviations used herein is stated in Table A:

Table A

Alanine	ALA	A
			Arginine	ARG	R
Asparagine	ASN	N
			Aspartic acid	ASP	D
Cysteine	CYS	C
			Glutamic acid	GLU	E
Glutamine	GLN	Q
			Glycine	GLY	G
Histidine	HIS	H
			Isoleucine	ILE	I
Leucine	LEU	L
			Lysine	LYS	K
Methionine	MET	M
			Phenylalanine	PHE	F
Proline	PRO	P
			Serine	SER	S
Threonine	THR	T
			Tryptophan	TRP	W
Tyrosine	TYR	Y
			Valine	VAL	V

Antagonist should refer to such material (such as part, candidate compound): as described in the same site of material and activator competition bind receptor, therefore but the cell internal reaction that the activity form that does not activate described acceptor is initial can suppress the cell internal reaction of activator. Antagonist does not reduce the basic cell internal reaction when lacking activator. In some embodiments, antagonist is the unknown activating cell internal reaction or conjunctival material of increase GTP when bind receptor before those.

Candidate compound should refer to use the molecule (such as but not limited to compound) that triage techniques screens. Preferred phrase " candidate compound " do not comprise the public known be selected from following compound: inverse agonist, activator or the antagonist (" compound of indirectly identifying ") of the acceptor of determining according to indirect authentication method in the past; Determined at least a mammal, to have the compound of the indirect evaluation of therapeutic efficiency before more preferably not comprising; Determined in human body, to have the compound for the treatment of the indirect evaluation of using before most preferably not comprising.

Composition refers to comprise the material of at least a composition; " Pharmaceutical composition " is an example of composition.

Compound efficacy should refer to compare with receptors bind affinity, the tolerance of the ability (i.e. activation/Inhibitory signal transduction pathway) of compound inhibition or costimulatory receptor function. The illustrative methods of detection compound effect is partly open at the embodiment of this patent document.

Codon should refer to the combination of three nucleotides (or equivalent of nucleotides), described nucleotides generally comprises the nucleosides (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) with the phosphate group coupling, described nucleotides be combined in amino acid of when translation coding.

The acceptor of composing type activation should refer to be subject to the acceptor of composing type receptor activation effect. The acceptor of composing type activation can be endogenous or non-endogenous.

The composing type receptor activation should refer to stablize described acceptor in activated state, and the method for described stable use except following methods finished: acceptor is combined with part or its chemical equivalent.

No matter contact should refer in the system, at least two parts be approached in vitro system or body.

Direct Identification or Direct Identification with regard to phrase " candidate compound ", should pointer to the acceptor screening candidate compound of composing type activation and estimate the effect of described compound, the acceptor of described composing type activation is the orphan receptor of composing type activation preferably, most preferably is the G albumen coupling cell surface orphan receptor of composing type activation. This phrase should be explained in no instance or be interpreted as and comprise or be included in phrase " indirectly identify " or " indirectly identifying ".

The endogenous material that should refer to the natural generation of mammal. Endogenous with regard to regard to term " acceptor ", should refer to by mammal (such as but not limited to the people) or viral natural production. Different therewith, herein non-endogenous should refer to not be by mammal (such as but not limited to the people) or viral natural production. Such as but not limited to this example, be not that composing type has activity when a kind of acceptor is in endogenous form, but through becoming the activated acceptor of composing type after the operation, then described acceptor most preferably is referred to herein as " acceptor of non-endogenous composing type activation ". These two terms may be used to describe " in the body " and " external " system. Such as but not limited to this example, in screening technique, endogenous or non-endogenous acceptor can refer to the in-vitro screening system. As another example but be not limited to this example, when operating mammiferous genome when comprising the acceptor of non-endogenous composing type activation, be feasible by screening system candidate compound in the body.

G protein coupled receptor fusion and gpcr fusion protein are in context disclosed herein, all refer to non-intrinsic protein, described non-intrinsic protein comprises the GPCR of the endogenous composing type activation of merging with the alpha subunit (this subunit is the subunit in conjunction with GTP) of at least a G albumen, best described G albumen or the GPCR of non-endogenous composing type activation, described G albumen preferably with the G albumen same type of the described endogenous lonely GPCR of natural coupling. Such as but not limited to this example, at endogenous state, if G albumen " G_sα " be and the main G albumen of GPCR coupling that the gpcr fusion protein based on specific GPCR is to comprise and G so_sThe non-intrinsic protein of the GPCR that α merges; In some cases, such as hereinafter statement, non-main G albumen and described GPCR can be merged. Described G albumen can directly merge with described composing type activation GPCR, and spacer region is perhaps arranged between the two.

Host cell should refer to add therein the cell of plasmid and/or carrier. In the situation of prokaryotic host cell, plasmid is the autonomous molecular replication of conduct (generally separating subsequently described plasmid to introduce eukaryotic host cell) when host cell copies usually; In the situation of eukaryotic host cell, plasmid integration is advanced the cell DNA of described host cell, when copying with the described eukaryotic host cell of box lunch, described plasmid replication. In some embodiments, described host cell is eukaryotic, is more preferably mammalian cell, most preferably is selected from 293,293T and COS-7 cells.

The conventional method of the finger drug discovery process of indirectly identifying or indirectly identifying, described conventional method relates to identifies that specificity is for the endogenous ligands of endogenous recipient, for described acceptor screening candidate compound determining those interference and/or to compete the compound of described ligand-receptor interaction, and the effect of estimating the described compounds affect at least a second messenger approach relevant with described activated receptor.

Inhibition should refer to compare with the situation that does not have compound existence reduction or the prophylactic response of described compound with regard to term " reaction ".

Inverse agonist should refer to such material (such as part, candidate compound): as described in compound in conjunction with as described in acceptor endogenous form or as described in the composing type activated form of acceptor; and suppress by the initial basic cell internal reaction of the activated form of described acceptor; it is dropped to below viewed arm's length basis activity level in the situation that does not have activator, perhaps reduce the GTP binding film. Preferably compare with the basic reaction in the situation that does not have described inverse agonist, exist described inverse agonist to suppress described basic cell internal reaction at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, most preferably at least 99%.

Known receptor should refer to such endogenous recipient: identified that specificity is for the endogenous ligands of described acceptor.

Part should refer to that specificity is for the molecule of natural receptor.

Sudden change or sudden change with regard to the nucleotide sequence and/or aminoacid sequence of endogenous recipient, specific one that should refer to described endogenous sequence is made changes or a plurality of change, so that described endogenous non-composing type activated receptors shows the composing type activation of described acceptor.With regard to the equivalent of particular sequence, the mutant form subsequently of people's acceptor is considered to the equivalent of first sudden change of described people's acceptor under following situation: (a) level with first sudden change performance of described acceptor is identical substantially for the composing type activation levels of the mutant form subsequently of people's acceptor; (b) sequence (amino acid and/or nucleic acid) the identity percentage between first sudden change of the mutant form subsequently of described acceptor and described acceptor is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99%.In some embodiments, consider disclosed hereinly to be used to obtain some preferred cartridge of composing type activatory and between the endogenous form of described GPCR and non-endogenous form, to comprise that monamino acid and/or codon change that preferred sequence identity percentage should at least 98%.

Non-orphan receptor should refer to specificity at the endogenous natural molecule of identifying part, wherein the signal pathway in the active cells that combines of part and acceptor.

Orphan receptor should refer to such endogenous recipient: do not identify the endogenous ligands of specificity at described acceptor, perhaps described part is unknown.

Medicinal compositions should refer to comprise the composition of at least a active ingredient, so composition is adapted at the specific effective effect of research in the Mammals (such as but not limited to the people).The needs that persons skilled in the art can be understood and estimate according to the technician are suitable for the technology whether the detection of active composition has needed effect.

Plasmid should refer to the combination of carrier and cDNA.In general, plasmid is introduced host cell and become albumen so that duplicate described cDNA and/or express described cDNA.

The second messenger should refer to the cell internal reaction that produces as the receptor activation result.The second messenger can comprise, for example, and inositoltriphosphoric acid (IP3), diacylglycerol (DAG), ring-type AMP (cAMP) and ring-type GMP (cGMP).Can measure second messenger's reaction to determine receptor activation.In addition, can measure second messenger reaction with direct evaluation candidate compound, described candidate compound comprises, for example, and inverse agonist, agonist and antagonist.

The signal that signal to noise ratio should refer to respond activation, strengthens or stimulate and produce, wherein said signal than do not have activation, do not have strengthen or non-stimulated situation under background noise or basal level strong.

Transcribed spacer should refer to be positioned at after gene (for example purpose GPCR) last codon or last amino acid but in purpose G albumen initiator codon or several amino acid of the translation before beginning to distinguish, and several amino acid of wherein said translation place that described purpose G is proteic to begin in the district with meeting frame.Can customize the amino acid whose quantity of described translation according to technician's needs, this quantity is generally from about amino acid, preferred two amino acid, more preferably three amino acid, more preferably four amino acid, more preferably five amino acid, more preferably six amino acid, more preferably seven amino acid, more preferably eight amino acid, more preferably nine amino acid, more preferably ten amino acid, more preferably 11 amino acid even more preferably 12 amino acid.

Stimulation should refer to compare with the situation that does not have compound with regard to term " reaction ", described compound have an intensified response.

Should refer in fact the result results of comparison 40% with interior, preferred 35% with interior, more preferably 30% with interior, more preferably 25% with interior, more preferably 20% with interior, more preferably 15% with interior, more preferably 10% with interior, more preferably 5% with interior, more preferably 2% with interior, most preferably in 1%.For example, under the function of receptors implementations, if according to the method or the similar methods measure known to the skilled of this paper instruction, the signal of being transduceed is then tested acceptor and may be showed the result similar in fact to contrasting acceptor in the signal 40% that control signal produces.

Carrier should refer to add at least a cDNA therein and itself can mix the cyclic DNA of host cell with regard to cDNA.

The order of lower part is in order to state conveniently, is not and should not be construed as disclosure or claim below the restriction.

A. introduce

The tradition research of acceptor is generally carried out according to an a priori assumption (based on history): must at first identify endogenous ligands, further then discovery may influence antagonist and other molecule of described acceptor.Even under the situation of at first known antagonist, research also extends to the searching endogenous ligands immediately.Even after finding the composing type activated receptors, this thoughtcast still continues in acceptor research.What therefore, do not recognize in the past is that the active condition of acceptor is the most useful for agonist and the inverse agonist of finding described acceptor.For those diseases that is caused by the acceptor or the active insufficient acceptor of overactivity, the needs of medicine are the compounds that reduces the receptor active state respectively or strengthen receptor active, and the antagonist pharmaceuticals of endogenous ligands not necessarily.This is because minimizing or the active compound of enhanced activity receptor status not necessarily combine same site with endogenous ligands.Therefore, the method for instruction according to the present invention, the research of any searching treatment compound should be from screening the compound to the active condition that is independent of part.

B. identifier GPCR

The too much information of people's gene group about nucleotide sequence that is positioned at has been identified in the enforcement of the Human Genome Project; In this effort, can obtain genetic sequence information and not understand or be familiar with any specific gene group sequence whether to comprise and maybe may comprise the open reading-frame (ORF) information of translating into human protein.In several method those skilled in the art's in this area of the interior nucleotide sequence of identifier's genome the limit of power.Such as but not limited to this example, various human GPCR disclosed herein is by search GenBank ^TMDatabase is found.Below table B list several endogenous GPCR that we have found that, and with disclosed other GPCR of GPCR homologous.

Table B

The lonely GPCR of disclosed people	The registration number of identifying	Open reading-frame (ORF) (base pair)	Homology GPCR contrast	With the identity percentage of specifying GPCR
					hRUP28	??AC073957	????1,002bp	????hGPR30	????34％
hRUP29	??AC083865	????918bp	????hGPR18	????27％
					hRUP30	??AC055863	????1,125bp	????hBRB1	????27％
hRUP31	??AL356214	????1,086bp	????hGALR-1	????31％
					hRUP32	??AL513524	????1,038bp	????hPNR	????43％
hRUP33	??AL513524	????1,020bp	????GPR57 ????GPR58	????50％ ????51％
					hRUP34	??AL513524	????1,029bp	????hPNR	????45％
hRUP35	??AC021089	????1,062bp	H κ-3 type opioid	????27％
					hRUP36	??AC090099	????969bp	????GPR90	????42％
hRUP37	??AC090099	????969bp	????hMRG	????41％

Acceptor identity is used to obtain the understanding that described acceptor acts in human body.Along with further specifying of this patent file, the technology of these acceptors of sudden change with the non-endogenous composing type activated form of setting up these acceptors will be discussed.

Technology disclosed herein also can be applicable to other people GPCR known in the art, and this is conspicuous for those skilled in that art.

C. screen acceptor

Non-endogenous composing type activated form screening candidate compound at GPCR disclosed herein allows direct the evaluation to act on the candidate compound of cell surface receptor, and does not require the endogenous ligands of using described acceptor.The technology that people use conventional common commercialization to get, the described endogenous form that can determine people GPCR disclosed herein are expressed and/or the position of overexpression in vivo.Expression of receptor is positioned particular organization makes scientist can determine the physiological function of described acceptor.Also might use these technology to determine and described receptor expression and/or the relevant relative disease/disturbance state of overexpression; This patent file discloses such method.In addition, the expression of acceptor in the disease organ can help people to determine the importance of described acceptor clinical correlation.

The GPCR of constitutive expression disclosed herein is based on the distance of the proline residue of the TM6 that is positioned at GPCR apart from supposition; This algorithmic technique is open in the common unsettled and common patent document PCT application number PCT/US99/23938 that transfers the possession of, this patent document is open as WO 00/22129 on April 20th, 2000, and other patent document of listing with this paper is attached to herein by reference.Described algorithmic technique is based on traditional sequence " arrange contrast ", and is based on specific range apart from aforementioned TM6 proline residue (certainly, or the endogenous composing type of described proline residue replace).The amino-acid residue that is positioned at 16 amino-acid residues that begins from this residue (supposing to be positioned at the IC3 district of described acceptor) by sudden change can activate described acceptor by composing type to lysine residue most preferably.Can use other amino-acid residue to reach this purpose in this site mutation, this will go through hereinafter.

D. identify and/or screen disease/disorder

As mentioned below, can identify that described non-endogenous composing type activates inverse agonist and the agonist of GPCR by method of the present invention.Described inverse agonist and agonist are the ideal candidate as lead compound, are used to seek the drug discovery programs of treatment and this receptor diseases associated.Also therefore allow the development medicinal compositions because can directly identify inverse agonist, correspondingly seek disease and the disorder relevant with described GPCR at described GPCR.Expression of receptor is positioned particular organization the ability of distributing described acceptor physiological function is provided for scientist.For example, the existence of GPCR is not only academic utilization at present in scanning diseased tissue sample and the healthy tissues sample, and people can use the endogenous ligands of this method evaluation at specific GPCR.Can carry out tissue scanning to the health tissues and the diseased tissue of broad range.Described tissue scanning provides the potential the first step for special receptor and disease and/or disorder are got in touch.In addition, expressed receptor can help people to determine the importance of described acceptor clinical correlation in the disease organ.

Can use the dna sequence dna of GPCR to make probe/primer.In some preferred embodiments, use described dna sequence dna manufacturing probe to be used for (a) and carry out the dot hybridization analysis, and/or (b) RT-PCR identifies the expression of described acceptor in tissue sample at tissue mRNA.Existence or the described acceptor of acceptor in source tissue or diseased tissue exists with the concentration higher than healthy tissues in diseased tissue and can be used for location and functional cohesion, point out the physiological action/function of described acceptor, and create the treatment therapy, be used for the treatment of the disease that includes but not limited to described function/effect is relevant.Also can be with this technology with the district of receptor mapping in organ.According to the known or supposition effect/function of the particular organization of described receptor mapping, the physiological function of inferring of the described acceptor of can deriving.Such as but not limited to this example, location/expressed proteins is relevant with awakening with the sensorimotor processing in the thalamus zone (sees Goodman; Gilman ' s, The Pharmacological Basis of Therapeutics, the 9th edition, the 465th page (1996)).The albumen of hippocampus perhaps prosperous cell inner expression respectively with learning and memory and peripheroneural myelin form relevant (see Kandel, E. etc., Essentials of Neural Science And BehaviorRespectively the 657th, 680 and 28 page (1995)).

E. screen candidate compound

1.GPCR class screening assay technology

When G protein receptor composing type activates, it in conjunction with G albumen (as G _q, G _s, G _i, G _z, G _o) and stimulate GTP in conjunction with described G albumen.Described G albumen works as the GTP enzyme subsequently, and hydrolysis GTP becomes GDP, and described thus acceptor is inactivation under normal operation.Yet the composing type activated receptors continues to transform GDP and becomes GTP.The analogue of the non-hydrolysable of a kind of GTP [ ³⁵S] GTP γ S can be used to monitor the enhancing combination of the film of expressing the composing type activated receptor.It is reported, [ ³⁵S] GTP γ S be used in that part exists and non-existent situation under monitor G albumen coupling film.In and the example that those skilled in that art use well-known at other, an example of this monitoring is reported in nineteen ninety-five by Traynor and Nahorski.The application of this mensuration system generally is the preliminary screening that is used for candidate compound, because this system can be widely used in all g protein coupled receptors, comprises and the interactional specific G albumen of recipient cell intracellular domain.

2. specificity GPCR screening assay technology

Identify candidate compound in case use " class " g protein coupled receptor to measure (mensuration that is used to be selected to the compound of agonist or inverse agonist), preferably further screen, confirm that described compound has interaction described acceptor site.For example, measure compounds identified not necessarily in conjunction with described acceptor, but may only make described G albumen " uncoupling " from the described cell internal area by described " class ".

A.G _s, G _zAnd G _i

G _sStimulate adenylate cyclase.On the other hand, G _i(and G _zAnd G _o) the inhibition adenylate cyclase.Adenylate cyclase enzyme catalysis ATP is to the conversion of cAMP; Therefore, coupling G _sProteic composing type activation GPCR is relevant with level increase in the cAMP cell.On the other hand, coupling G _I(or G _z, G _o) proteic composing type activation GPCR is relevant with level reduction in the cAMP cell.Generally as seen, " indirect mechanism that transmits of cynapse ", the 8th chapter, From Neuron to Brain(the 3rd edition) Nichols, editors such as J.G., Sinauer Associates, Inc. (1992).Therefore, can utilize the mensuration that detects cAMP to determine whether candidate compound is the inverse agonist (being the level that described compound can reduce cAMP) of for example described acceptor.Can utilize the several different methods of measurement cAMP known in the art; Most preferred method depends on the anti-cAMP antibody of application based on the ELISA pattern.It is that full cell second messenger reports systems measurement that utilizable other type is measured.Gene promoter drives the expression of specific gene encoded protein.Ring-type AMP promotes the combination of the conjugated protein or transcription factor (CREB) of the reactive DNA of cAMP by following mechanism drives genetic expression: ring-type AMP, and the conjugated protein or transcription factor of the reactive DNA of described cAMP is subsequently in conjunction with the promotor (cAMP response element) of specific site and drive described genetic expression.Can make up the report system, described report system has the promotor that comprises a plurality of cAMP response elements before at reporter gene (as beta-galactosidase enzymes or luciferase).Therefore, composing type activation G _sThe link coupled acceptor causes the accumulation of cAMP, and the accumulation of cAMP activating gene subsequently causes the expression of described reporter gene.Use standard biological chemical assay (Chen etc. 1995) to detect report albumen such as beta-galactosidase enzymes or luciferase subsequently.

B.G _oAnd G _q

G _qAnd G _oRelevant with the activation of Phospholipase C, and Phospholipase C hydrolytic phosphatide PIP subsequently ₂, discharge courier in two kinds of cells: diacylglycerol (DAG) and inositol 1,4,5-triphosphoric acid (IP ₃).IP ₃The increase and the G of accumulation _qAnd G _oThe activation of associated receptor is relevant.Generally as seen, " indirect mechanism that transmits of cynapse ", the 8th chapter, From Neuron to Brain(the 3rd edition) Nichols, editor such as J.G. Sinauer Associates, Inc. (1992).Can utilize and detect IP ₃The mensuration of accumulation determines that whether candidate compound is G for example _qAnd G _oThe inverse agonist of associated receptor (is that described compound reduces IP ₃Level).Also can use the AP1 report to measure and check G _qAssociated receptor wherein depends on G _qPhospholipid hydrolase cause comprising the gene activation of AP1 element; Therefore, activatory G _qAssociated receptor will show described expression of gene to be increased, and its inverse agonist will show the reduction of described genetic expression thus, and agonist will show the increase of described expression.Can obtain the commercialization test of this detection.

3.GPCR fusion rotein

Using endogenous composing type activation GPCR or non-endogenous composing type activation GPCR screens candidate compound and causes an interesting screening difficult problem with direct evaluation inverse agonist, because according to definition, acceptor even when lacking, also be activated with its bonded endogenous ligands.Therefore, for distinguish as exist under the candidate compound situation non-endogenous recipient and at the non-endogenous recipient that does not exist under the candidate compound situation, whether the purpose of this differentiation is to understand described compound is inverse agonist or agonist or to the not influence of described acceptor, preferred utilization can strengthen the method for described differentiation.Preferred method is to use gpcr fusion protein.

In general, in case use determination techniques mentioned above (and other technology) to determine non-endogenous GPCR composing type activation, just might determine the main G albumen of the described endogenous GPCR of coupling.The described G albumen of coupling provides the signal pathway that can estimate to described GPCR.In some embodiments, preferably use mammalian expression system to screen, expect that described system will comprise endogenous G albumen therein.Therefore, according to definition, in described system, described non-endogenous composing type activation GPCR will continue to provide signal.In some embodiments, preferably strengthen described signal,, more likely more easily under the screening situation, distinguish the acceptor that contacts with described inverse agonist so that in the presence of the inverse agonist of for example described acceptor.

Described gpcr fusion protein will strengthen and the proteic effect of described non-endogenous GPCR link coupled G.Described gpcr fusion protein is preferred for the screening of endogenous composing type activation GPCR or non-endogenous composing type activation GPCR, because described method strengthens the signal that utilizes in described triage techniques.This is for obtaining effectively " noise " than being important; Screening as candidate compound disclosed herein preferred as described in signal to noise ratio.

Structure is used to express in construction those skilled in the art's in this area the limit of power of gpcr fusion protein.Commercialization expression vector and system provide the whole bag of tricks that is fit to the concrete needs of investigator.The major criterion that makes up described gpcr fusion protein construction includes but not limited to: described endogenous GPCR sequence and described G protein sequence all meet frame (preferably the sequence of described endogenous GPCR is in described G protein sequence upstream), and " termination " codon of described GPCR disappearance or replaced is so that also can express described G albumen when expressing described GPCR.Other embodiment comprises such construction: do not meet frame and/or described " termination " codon at endogenous GPCR sequence described in the described construction and described G protein sequence and do not lack or replaced.Described GPCR can directly connect described G albumen, and transcribed spacer residue (though residue quantity can be easily definite by those skilled in that art, preferably being no more than about 12) is perhaps arranged between the two.From needing consideration easily, preferably use transcribed spacer.The described G albumen of the described non-endogenous GPCR of coupling has obtained identifying before making described gpcr fusion protein construction.Because only there is minority G albumen to obtain identifying, thus preferably obtain comprise described G protein sequence construction (being general G albumen construction (seeing embodiment)) to insert endogenous GPCR sequence therein; This makes the multiple efficient with the endogenous GPCR of not homotactic difference of extensive screening higher.

As indicated above, expection coupling G _i, G _zAnd G _oComposing type activation GPCR suppress the formation of cAMP, this makes mensuration based on these GPCR types become possible (be that the cAMP signal reduces, therefore make directly identify become possibility as inverse agonist (inverse agonist will further reduce this signal)) when activation.As hereinafter open, we determine: for these type receptors, might create not based on the proteic gpcr fusion protein of the endogenous G of described GPCR, so that set up the feasible mensuration based on cyclase.Therefore, for example, endogenous G _iCoupled receptor can with G _sAlbumen merges-" driving " or " ordering about " described endogenous GPCR coupling such as G when described fusion construct is expressed _sRather than " natural " G _iAlbumen is so that can set up mensuration based on cyclase.Therefore, for G _i, G _zAnd G _oCoupled receptor, in some embodiments, when using gpcr fusion protein and described mensuration based on right

During the detection of adenylate cyclase, preferably use G _s(perhaps stimulate adenylate cyclase forms be equal to G albumen) sets up described fusion construct.

G albumen	CAMP produces the effect to GPCR activation (being composing type activation or agonist combination)	IP 3Accumulation is to the effect of GPCR activation (being composing type activation or agonist combination)	CAMP produce to inverse agonist bonded effect	IP 3Accumulation for inverse agonist bonded effect
					G s	Increase	N/A	Reduce	N/A
G i	Reduce	N/A	Increase	N/A
					G z	Reduce	N/A	Increase	N/A
G o	Reduce	Increase	Increase	Reduce
					G q	N/A	Increase	N/A	Reduce

On an equal basis effectively utilize and G _s, G _i, G _zOr G _oThe G that albumen merges _qProteic G albumen fusion construct.In some embodiments, can use G _qAlbumen obtains preferred fusion construct, described G _qSix (a 6) individual amino acid of protein delation G protein alpha subunit (" G α q "), and last five (5) the individual amino acid of the C-terminal of G α q are by the corresponding aminoacid replacement of purpose G Protein G α.For example, fusion construct can have and G _iThe G that albumen merges _q(lacking 6 amino acid) produces " G _q/ G _iFusion construct ".This fusion construct will be ordered about endogenous G _iCoupled receptor is non-endogenous G Protein G with it _qCoupling is measured the cAMP generation so that can measure second messenger's (for example inositoltriphosphoric acid or diacylglycerol) to substitute.

4. target G _iCoupling GPCR and signal toughener G _sThe cotransfection of coupling GPCR (based on the mensuration of cAMP)

Known G _iCoupled receptor suppresses adenylate cyclase, and therefore reduces the level of cAMP output, and this makes the level of estimating cAMP become possibility.Measure cAMP output and reduce (main coupling G when activation _iAcceptor composing type activatory index) effective technology can followingly finish: cotransfection signal toughener and and G _iMain coupling G when the GPCR that connects, described signal toughener for example are activation _sNon-endogenous composing type activated receptor (as disclosed TSHR-A623I hereinafter).Be apparent that, can measure G according to the increase of cAMP output _sThe composing type activation of coupled receptor.G _iThe composing type activation of coupled receptor causes cAMP output to reduce.Therefore, described cotransfection method will advantageously be utilized these " opposition " effects.The non-endogenous composing type activatory G of cotransfection for example _sCoupled receptor (described " signal toughener ") and described endogenous G _iCoupled receptor (described " target acceptor ") provides basic cAMP signal (though promptly described G _iCoupled receptor can reduce the cAMP level, but is somebody's turn to do " increase " for composing type activatory G _sSubstantive the increasing of the cAMP level that coupling signal toughener is set up is relative).By the composing type activated form of described signal toughener of cotransfection and described target acceptor, expection cAMP will be owing to described G then _iThe activity of target increases (promptly reducing cAMP) and further reduces with respect to the basis.

Can use mensuration screening candidate compound then, wherein two " changes " be arranged with respect to using described endogenous recipient/G albumen syzygy based on cAMP: at first, described relatively G _iLink coupled target acceptor will produce " opposition " effect, promptly described G _iThe inverse agonist of coupling target acceptor can increase the cAMP signal of measurement, and described C _iThe agonist of coupling target acceptor will reduce this signal; The second, be apparent that, should use the directly candidate compound of evaluation of this method by independent assessment, to guarantee their the not described signal enhancing of target acceptors (this can finish) before or after screening at described cotransfection acceptor.

F. pharmaceutical chemistry

Generally speaking, but be not in all cases, will produce compound and directly identify that candidate compound combines and carry out that preparation is used for several thousand kinds of compounds of described analysis at random thus by combinatorial chemistry technique.In general, described screening has generation the compound of unique core texture; After this, these compounds are carried out other chemically modified, further strengthen their medicinal property around preferred core texture.Described technology is that those skilled in that art are known, and will not describe in detail in this patent file.

G. medicinal compositions

Can use the well-known technology of those skilled in that art to be mixed with medicinal compositions with selecting the candidate compound that is used to further develop.Suitable pharmaceutically acceptable carrier is that those skilled in that art know; For example, see Remington ' s PharmaceuticalSciences, the 16th edition, 1980, Mack Publishing Co., (editor such as Osol).

H. other application

Though the advantageous applications of the non-endogenous form of GPCR disclosed herein is the candidate compound of directly identifying as inverse agonist or agonist (preferably as medicine), the GPCR of these forms has other application.For example, can utilize in the vitro system that adds GPCR and the body system further to illustrate and understand the effect of these acceptors in human body situation (normal or disease), and understand composing type activatory effect when it is applied to signal cascade.In some embodiments, preferred described endogenous recipient is " orphan receptor ", does not promptly identify the endogenous ligands at described acceptor.Therefore, in some embodiments, can use the non-endogenous GPCR of described modification to understand endogenous recipient, therefore identify endogenous ligands then in the intravital effect of people.Also can use described endogenous ligands further to illustrate known receptor and the approach by described acceptor transduction signal.After particularly studying this patent file, other application of described open acceptor will become obvious for those skilled in that art.

Embodiment

The invention provides the following examples for illustrating, rather than restriction the present invention.Though herein disclosed is concrete nucleotide sequence and aminoacid sequence, those skilled in the art can do little modification and acquisition comes to the same thing to hereinafter reported or similar substantially result to these sequences in this area.From a kind of sequence to another kind of sequence (as from rat receptor to people's acceptor or from people's acceptor A to people's acceptor B) use or be based upon on the basis of series arrangement correlation technique the traditional method of solution sequence box, use described technology to arrange the described sequence of contrast, to determine total zone.Mutation method disclosed herein does not also rely on this technology, but is based upon algorithmic method and the position distance of conservative proline residue in the TM6 district of people GPCR.In case grasp this method, just capable little modification and the acquisition result identical in fact (being the composing type activation) that this is made of those skilled in the art with result disclosed herein.Described modifying method is considered in the scope of disclosure thing.

Embodiment 1

Endogenous people GPCR

1. identifier GPCR

At search GenBank ^TMIdentify disclosed endogenous people GPCR on the basis of database information.When the described database of search, identify to obtain the cDNA clone shown in the following table (table C):

Table C

The lonely GPCR of disclosed people	The registration number of identifying	Open reading-frame (ORF) (base pair)	The contrast of homology GPCR	Nucleic acid SEQ.ID.N O.	Amino acid SEQ.ID.N O.
						hRUP28	?AC073957	??1,002bp	????hGPR30	????1	????2
hRUP29	?AC083865	??918bp	????hGPR18	????3	????4
						hRUP30	?AC055863	??1,125bp	????hBRB1	????5	????6
hRUP31	?AL356214	??1,086bp	????hGALR-1	????7	????8
						hRUP32	?AL513524	??1,038bp	????hPNR	????9	????10
hRUP33	?AL513524	??1,020bp	????GPR57 ????GPR58	????11	????12
						hRUP34	?AL513524	??1,029bp	????hPNR	????13	????14
hRUP35	?AC021089	??1,062bp	H κ-3 type opioid	????15	????16
						hRUP36	?AC090099	??969bp	????GPR90	????17	????18
hRUP37	?AC090099	??969bp	????hMRG	????19	????20

2. full-length clone

a.hRUP28(Seq.Id.Nos.1&2)

On the basis of using the GenBank data message, identify disclosed hRUP28.When the described database of search, identify the cDNA clone of registration number AC073957, this clone is the people's gene group sequence from karyomit(e) 7.

Use adult liver Marathon-Ready ^TMCDNA (Clontech) clones total length RUP28 with following primer by PCR as template:

5 '-CAGAGCTCTGGTGGCCACCTCTGTCC-3 ' (SEQ.ID.NO.:21; Sense strand, 5 ' initiator codon),

5 '-CTGCGTCCACCAGAGTCACGTCTCC-3 ' (SEQ.ID.NO.:22; Antisense strand, 3 ' terminator codon).

Use Advantage ^TMCDNA polysaccharase (Clontech) increases by following circulation in 50 μ l reactants, and wherein step 2 to 4 repeats 35 times: 95 ℃ 5 minutes; 94 ℃ of 30 second; 58 ℃ of 30 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 1.16kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:1 and the aminoacid sequence that SEQ.ID.NO.:2 infers.

b.hRUP29(Seq.Id.Nos.3&4)

On the basis of using the GenBank data message, identify disclosed hRUP29.When the described database of search, identify the cDNA clone of registration number AC0083865, this clone is the people's gene group sequence from karyomit(e) 7.

End user's genomic dna is cloned total length RUP29 with following primer by PCR as template:

5 '-GTATGCCTGGCCACAATACCTCCAGG-3 ' (SEQ.ID.NO.:23; Sense strand comprises initiator codon),

5 '-GTTTGTGGCTAACGGCACAAAACACAATTCC-3 ' (SEQ.ID.NO.:24; Antisense strand comprises terminator codon).

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase by following circulation in 50 μ l reactants, wherein step 2 to 4 repeats 35 times: 95 ℃ 5 minutes; 94 ℃ of 30 second; 54 ℃ of 30 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 930bp from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.EBiosystems) order-checking.

End user's white corpuscle and ovary Marathon-Ready ^TMCDNA (Clontech) rapid amplifying cDNA end (RACE) is determined accurate 5 ' end of RUP29 cDNA.Use has the RUP29 Auele Specific Primer (1) of following sequence:

5′-GGTACCACAATGACAATCACCAGCGTCC-3′(SEQ.ID.NO.：25)

Carry out first round PCR reaction, RUP29 Auele Specific Primer (2) with AP1 primer (Clontech) with following sequence:

5′-GGAACGTGAGGTACATGTGGATGTGCAGC-3′(SEQ.ID.NO.：26)

Carry out second with AP2 primer (Clontech) and take turns the PCR reaction.The product that separates described RACE reaction, the clone advances pCRII-TOPO carrier (Invitrogen) and order-checking.See the nucleotide sequence of SEQ.ID.NO.:3 and the putative amino acid sequence of SEQ.ID.NO.:4.

c.hRUP30(Seq.Id.Nos.5&6)

On the basis of using the GenBank data message, identify disclosed HRUP30.When the described database of search, identify the cDNA clone of registration number AC055863, this clone is the people's gene group sequence from karyomit(e) 17.

Following 5 ' RACE-PCR clone total length RUP30 that passes through: end user's pancreas Marathon-Ready ^TMCDNA (Clontech) uses following oligonucleotide as template

5 '-GCAGTGTAGCGGTCAACCGTGAGCAGG-3 ' (SEQ.ID.NO.:27; Sense strand comprises initiator codon) and AP1 primer (Clontech) carry out first round RT-PCR, use oligonucleotide:

5 '-TGAGCAGGATGGCGATCCAGACTGAGGCGTGG-3 ' (SEQ.ID.NO.:28; Antisense strand comprises terminator codon) and AP2 primer (Clontech) carry out second and take turns PCR.To clone into pCRII-TOPO carrier (Invitrogen) by the dna fragmentation that described 5 ' RACE-PCR produces, use SP6/T7 primer (Stratagene) order-checking.

According to the sequence of described 5 ' RACE product, end user's pancreas Marathon-Ready ^TMCDNA (Clontech) clones total length RUP30 with following primer by RT-PCR as template:

5 '-GAGGTACAGCTGGCGATGCTGACAG-3 ' (SEQ.ID.NO.:29; Sense strand, ATG are initiator codon),

5 '-GTGGCCATGAGCCACCCTGAGCTCC-3 ' (SEQ.ID.NO.:30; Antisense strand, 3 ' terminator codon).

Use Taq archaeal dna polymerase (Stratagene) to increase by following circulation in 50 μ l reactants, wherein step 2 to 4 repeats 35 times: 94 ℃ of 40 second; 94 ℃ of 20 second; 64 ℃ of 20 second; 72 ℃ 2 minutes; With 72 ℃ 5 minutes.

Isolate the PCR fragment of a 1.2kp from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) several clones that check order.See the nucleotide sequence of SEQ.ID.NO.:5 and the putative amino acid sequence of SEQ.ID.NO.:6.

d.hRUP31(Seq.Id.Nos.7&8)

On the basis of using the GenBank data message, identify disclosed HRUP31.When the described database of search, identify the cDNA clone of registration number AL356214, this clone is the people's gene group sequence from karyomit(e) 10.

End user's mammary gland Marathon-Ready ^TMCDNA (Clontech) clones total length RUP31 with following primer by RT-PCR as template:

5 '-GGAATGTCCACTGAATGCGCGCGG-3 ' (SEQ.ID.NO.:31; Sense strand comprises initiator codon),

5 '-AGCTCGCCAGGTGTGAGAAACTCGG-3 ' (SEQ.ID.NO.:32; Antisense strand, 3 ' terminator codon).

Use Advantage ^TMCDNA polysaccharase (Clontech) increases by following circulation in 50 μ l reactants, and wherein step 2 to 4 repeats 35 times: 94 ℃ of 40 second; 94 ℃ of 20 second; 66 ℃ of 20 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 5 minutes.

Isolate the PCR fragment of a 1.1kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:7 and the aminoacid sequence that SEQ.ID.NO.:8 infers.

e.hRUP32(Seq.Id.Nos.9&10)

On the basis of using the GenBank data message, identify disclosed HRUP32.When the described database of search, identify the cDNA clone of registration number AL513524, this clone is the people's gene group sequence from karyomit(e) 6.

End user's genomic dna (Clontech) is cloned total length RUP32 with following primer by PCR as template:

5 '-GCGTTATGAGCAGCAATTCATCCCTGCTGG-3 ' (SEQ.ID.NO.:33; Sense strand comprises initiator codon),

5 '-GTATCCTGAACTTCGTCTATACAACTGC-3 ' (SEQ.ID.NO.:34; Antisense strand).

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase by following circulation, wherein step 2 to 4 repeats 35 times: 94 ℃ 3 minutes; 94 ℃ of 20 second; 58 ℃ of 20 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 1.06kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:9 and the aminoacid sequence that SEQ.ID.NO.:10 infers.

f.hRUP33(Seq.Id.Nos.11&12)

On the basis of using the GenBank data message, identify disclosed HRUP33.When the described database of search, identify the cDNA clone of registration number AL513524, this clone is the people's gene group sequence from karyomit(e) 6.

End user's genomic dna (Clontech) is cloned total length RUP33 with following primer by PCR as template:

5 '-CCCTCAGGAATGATGCCCTTTTGCCACAA-3 ' (SEQ.ID.NO.:35; Sense strand comprises initiator codon),

5 '-ATCCATGTGGTTGGTGCATGTGGTTCGT-3 ' (SEQ.ID.NO.:36; Antisense strand).

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase by following circulation, wherein step 2 to 4 repeats 35 times: 94 ℃ 3 minutes; 94 ℃ of 20 second; 56 ℃ of 20 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 1.1kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:11 and the aminoacid sequence that SEQ.ID.NO.:12 infers.

g.hRUP32(Seq.Id.Nos.13&14)

On the basis of using the GenBank data message, identify disclosed HRUP34.When the described database of search, identify the cDNA clone of registration number AL513524, this clone is the people's gene group sequence from karyomit(e) 6.

End user's genomic dna (Clontech) is cloned total length RUP32 with following primer by PCR as template:

5 '-AAACAACAAACAGCAGAACCATGACCAGC-3 ' (SEQ.ID.NO.:37; Sense strand comprises initiator codon),

5 '-ACATAGAGACAAGTGACATGTGTGAACCAC-3 ' (SEQ.ID.NO.:38; Antisense strand).

Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase by following circulation, wherein step 2 to 4 repeats 35 times: 94 ℃ 3 minutes; 94 ℃ of 20 second; 60 ℃ of 20 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 1.27kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:13 and the aminoacid sequence that SEQ.ID.NO.:14 infers.

h.hRUP35(Seq.Id.Nos.15&16)

On the basis of using the GenBank data message, identify disclosed HRUP35.When the described database of search, identify the cDNA clone of registration number AC021089, this clone is the people's gene group sequence from karyomit(e) 16.

End user's fetal brain Marathon-Ready ^TMCDNA (Clontech) determines 5 ' sequence of RUP35 as template by 5 ' RACE-PCR.Use oligonucleotide

5 '-GGTATGAGACCGTGTGGTACTTGAGC-3 ' (SEQ.ID.NO.:39; Sense strand) and AP1 primer (Clontech) carry out first round RT-PCR, use oligonucleotide:

5 '-GTGGCAGACAGCGATATACTGTCAATGG-3 ' (SEQ.ID.NO.:40; Antisense strand) and AP2 primer (Clontech) carry out second and take turns PCR.To clone into pCRII-TOPO carrier (Invitrogen) by the dna fragmentation that described 5 ' RACE-PCR produces, use SP6/T7 primer (Stratagene) order-checking.

According to the sequence of described 5 ' RACE product, use human brain Marathon-Ready ^TMCDNA (Clontech) clones total length RUP30 with following primer by RT-PCR as template:

5 '-GCGCTCATGGAGCACACGCACGCCCAC-3 ' (SEQ.ID.NO.:41; Sense strand, ATG are initiator codons),

5 '-GAGGCAGTAGTTGCCACACCTATGG-3 ' (SEQ.ID.NO.:42; Antisense strand, 3 ' terminator codon).Use Advantage ^TMCDNA polysaccharase (Clontech) increases by following circulation in 100 μ l reactants, and wherein step 2 to 4 repeats 45 times: 95 ℃ 2 minutes; 95 ℃ of 20 second; 60 ℃ of 20 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 5 minutes.

Isolate the PCR fragment of a 1.0kp from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:15 and the putative amino acid sequence of SEQ.ID.NO.:16.

i.hRUP36(Seq.Id.Nos.17&18)

On the basis of using the GenBank data message, identify disclosed HRUP36.When the described database of search, identify the cDNA clone of registration number AC090099, this clone is the people's gene group sequence from karyomit(e) 11.

End user's genomic dna (Clontech) is cloned total length RUP36 with following primer by PCR as template:

5 '-CATCTGGTTTGTGTTCCCAGGGGCACCAG-3 ' (SEQ.ID.NO.:43; Justice chain, 5 ' initiator codon),

5 '-GACAGTGTTGCTCTCAAAGTCCCGTCTGACTG-3 ' (SEQ.ID.NO.:44; Antisense strand, 3 ' terminator codon).Use TaqPlus  PrecisionDNA polysaccharase (Stratagene) to increase by following circulation in 50 μ l reactants, wherein step 2 to 4 repeats 30 times: 95 ℃, and 5 minutes; 95 ℃ of 30 second; 70 ℃ of 30 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of a 1.0kb from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:17 and the aminoacid sequence that SEQ.ID.NO.:18 infers.

j.hRUP37(Seq.Id.Nos.19&20)

On the basis of using the GenBank data message, identify disclosed HRUP37.When the described database of search, identify the cDNA clone of registration number AC090099, this clone is the people's gene group sequence from karyomit(e) 11.

End user's genomic dna (Clontech) is cloned total length RUP37 with following primer by PCR as template:

5 '-CTGTTTCCAGGGTCATCAGACTGGG-3 ' (SEQ.ID.NO.:45; Sense strand),

5 '-GCAGCATTGCTCTCAAAGTCCTGTCTG-3 ' (SEQ.ID.NO.:46; Antisense strand).Use TaqPlus  Precision archaeal dna polymerase (Stratagene) to increase by following circulation, wherein step 2 to 4 repeats 35 times: 95 ℃ 5 minutes; 95 ℃ of 30 second; 62 ℃ of 30 second; 72 ℃ 1 minute 30 seconds; With 72 ℃ 7 minutes.

Isolate the PCR fragment of one 969 base pair from 1% sepharose, the clone advances pCRII-TOPO carrier (Invitrogen), uses ABI Big dyestuff to stop test kit (P.E.Biosystems) order-checking.See the nucleotide sequence of SEQ.ID.NO.:19 and the aminoacid sequence that SEQ.ID.NO.:20 infers.

Embodiment 2

Prepare non-endogenous composing type activatory GPCR

Those skilled in that art can select to be used for the technology of mutant nucleic acid sequence.It hereinafter is the method that is used to produce the non-endogenous form of above disclosed several people GPCR.Hereinafter disclosed sudden change is based on algorithmic method, the 16th amino acid (being positioned at the IC3 district of described GPCR) that begins apart from conservative proline(Pro) (or therefore endogenous conservative replacement) residue preferably sports L-Ala, Histidine, arginine or lysine residue thus, most preferably sports lysine residue.

1.Transformer Site-Directed ^TMMutagenesis

Can use for example Transformer Site-Directed ^TMMutagenesis kit (Clontech) according to manufacturer's guidance, prepares non-endogenous people GPCR from people GPCR.In some embodiments, use two kinds of mutagenic primers, preferably a kind of Methionin mutagenic oligonucleotide and a kind of selective marker oligonucleotide that produces lysine mutation.For convenient, (table D) marks the codon mutation that adds described people GPCR with standard form:

Table D

The acceptor sign	Codon mutation
		?hRUP28	?V274K
?hRUP29	?T249K
		?hRUP30	?R232K
?hRUP31	?M294K
		?hRUP32	?F220K
?hRUP34	?A238K
		?hRUP35	?Y215K
?hRUP36	?L294K
		?hRUP37	?T219K

Embodiment 3

Expression of receptor

Though those skilled in that art can use the various kinds of cell expressing protein, preferably utilize mammalian cell.Major cause is practicality, though that is: for example utilizing, yeast cell to express GPCR is possible, but this introduces the nonmammalian cell of acceptor coupling, genetic mechanism and the Secretory Pathway (not comprising these under the zymic situation really) that do not comprise mammlian system and evolve out in method, therefore, though the result who obtains in the nonmammalian cell may be useful, the result who more preferably uses mammalian cell to obtain.In mammalian cell, though can select the specific mammalian cell that be utilized according to technician's needs, especially preferred COS-7,293 and the 293T cell.

A. transient transfection

First day, inoculate 293 cells, 6 * 10 ⁶Cell/10cm dish.Second day, prepare two reaction tubess (every pipe contains a dishful of consumption): pipe A contains 4 μ g DNA (as the pCMV carrier in 0.5ml serum-free DMEM (GibcoBRL); Carry the pCMV carrier of receptor cdna etc.); Pipe B contains 24 μ l lipofection amine reagent (GibcoBRL) in 0.5ml serum-free DMEM.Put upside down several times mixing tube A and pipe B, at room temperature incubation 30-45 minute then.This mixture is called " transfection mixture ".293 cells with 1XPBS washing inoculation add 5ml serum-free DMEM then.In cell, add the described transfection mixture of 1ml, then at 37 ℃/5%CO ₂Incubation 4 hours.Siphon away and remove described transfection mixture, add 10ml DMEM/10% foetal calf serum subsequently.At 37 ℃/5%CO ₂The incubation cell.Behind the incubation 48 hours, harvested cell is used for analyzing.

B. stable clone

With about 12 * 10 ⁶293 cell inoculations are cultivated in containing one of 10% foetal calf serum and percentage Sodium.alpha.-ketopropionate, L-L-glutamic acid and antibiotic DME High Glucose substratum to 15cm tissue culturing plate.Inoculate (reaching about 80% converges) behind the 293 cell twenty four hours, with the described cell of 12 μ g DNA transfections.Described 12 μ g DNA are mixed with 60 μ l lipofection amine reagent and 2mL serum-free DME High Glucose substratum.Siphon away from described flat board and to remove described substratum, wash described cell once with serum free medium.Described DNA, lipofection amine reagent and substratum mixture and 10mL serum free medium are added described flat board., wash away and remove described substratum after four to five hours at 37 ℃ of incubations, add the substratum that 25ml contains serum then.Twenty four hours after the transfection siphons away once more and removes substratum, adds then and contains the fresh serum substratum.After the transfection 48 hours, siphon away and remove substratum, add the blood serum medium that contains that contains final concentration 500 μ g/mL Geneticins (G418 medicine) then.Select to contain in the transfectional cell the positive transfectional cell of G418 resistant gene then.Per four to five days replacing one subcultures in the chosen process.In the chosen process, culturing cell produces stable aggregate, or goes down to posterity to select stable clone.

Embodiment 4

Determine the mensuration of non-endogenous GPCR constitutive activity

Can make ins all sorts of ways estimates the constitutive activity of non-endogenous people GPCR.Be illustrative below; Those skilled in that art can determine those technology of the most suitable technician's needs.

1. film is in conjunction with mensuration: [ ³⁵S] GTP γ S mensuration

When g protein coupled receptor owing to part in conjunction with or composing type activation when being in active condition, described acceptor coupling G albumen, stimulate GDP to discharge and GTP subsequently in conjunction with described G albumen.Described G protein receptor mixture alpha subunit works as the GTP enzyme, and slowly hydrolysis GTP becomes GDP, generally described after this acceptor inactivation.The composing type activated receptors continues conversion GTP and GDP.Can utilize GTP the non-hydrolysable analogue [ ³⁵S] GTP γ S prove [ ³⁵S] GTP γ S strengthens and the combining of the composing type activated receptor of film expression.Use [ ³⁵S] GTP γ S includes but not limited to following in conjunction with measuring composing type activatory benefit: (i) it can be widely used in all g protein coupled receptors; (b) it is near the film surface, and this makes it can not pick up the molecule that (pick-up) influences the reaction of intracellular level connection.

Described mensuration utilize g protein coupled receptor stimulate [ ³⁵S] GTP γ S is in conjunction with the film of expressing associated receptor.Therefore, described mensuration can be used for direct authentication method, and screening is at the candidate compound of composing type activatory g protein coupled receptor.Described mensuration is general, can be applicable to the drug discovery of all g protein coupled receptors.

Described [ ³⁵S] GTP γ S was determined in the following composition incubation 1 hour: 20mM HEPES and 1 to about 20mM MgCl ₂Though (preferred 20mM, for adjust this amount at optimum result) pH7.4, contain about 0.3 to about 1.2nM[ ³⁵S] the binding buffer liquid (though preferred 1.2, can adjust this amount for optimum result) of GTP γ S and 12.5 to 75 μ g membranins are (as 293 cells of Gs fusion rotein as described in expressing; Can adjust this amount for optimization) and 10 μ MGDP (can be optimization change amount).Add wheat germ agglutinin pearl (25 μ l subsequently; Amersham), described mixture at room temperature continued incubation 30 minutes.Described pipe at room temperature in 1500xg centrifugal 5 minutes is subsequently counted with scintillometer then.

2. adenylate cyclase

Improvement and design is used for the Flash Plate based on raji cell assay Raji ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Cat.No.SMP004A), use it for thick plasma membrane.The FlashPlate hole can comprise the flicker encrusting substance, and described encrusting substance also comprises the specific antibody of discerning cAMP.By with the combining of radioactivity cAMP tracer direct competitive and described cAMP antibody, the quantitatively cAMP that in the hole, produces.Be the Short Description of measuring the method that the cAMP level changes in the full cell of expressing described acceptor below.

Gather in the crops transfectional cell behind the about twenty four hours of transient transfection.Sucking-off and discard substratum carefully.The gentle 10ml PBS that adds in every dish cell, careful then sucking-off.Every dull and stereotyped 1ml Sigma cell dissociation damping fluid and 3ml PBS of adding.With cell sucking-off flat board, described cell suspending liquid is collected into the conical centrifuge tube of 50ml with transfer pipet.Cell is at room temperature in 1, centrifugal 5 minutes of 100rpm.The resuspension cell precipitation is in the PBS of suitable volumes (about 3ml/ flat board) carefully.Use hemocyte instrument counting cells then, replenish adding PBS, obtain the cell (reaching the final volume in about 50 μ l/ holes) of sufficient amount.

According to producer instruct preparation and keep the cAMP standard and detect damping fluid (11ml detect damping fluid include 1 μ Ci tracer [ ¹²⁵I cAMP (50 μ l)]).The mensuration damping fluid that prepared fresh is used to screen, described mensuration damping fluid contain 50 μ l stimulates damping fluid, 3 μ l test compounds (12 μ M finally measure concentration) and 50 μ l cells, with described mensuration buffer preserving on ice up to use.In appropriate bore, add 50 μ l cAMP standards, add 50 μ l PBSA at H-11 and H12 hole then, begin to measure.In institute is porose, add 50 μ l and stimulate damping fluid.Use can distribute the application of sample instrument (pin tool) of 3 μ l compound solutions, adds DMSO (or selected candidate compound) in appropriate bore, and the final concentration and the 100 μ l that reach 12 μ M mensuration compound always measure volume.Add cell then in the hole, at room temperature incubation is 60 minutes.In the hole, add the detection mixture that 100 μ l contain spike cAMP then.Continue incubation dull and stereotyped 2 hours, and used Wallac MicroBeta then ^TMThe scintillometer counting.Every assay plate is production standard cAMP curve all, according to the value in described standard cAMP curve extrapolation cAMP/ hole.

3. the cAMP that is used for Gi coupling target GPCR based on cell

TSHR is G _sCoupling GPCR causes the cAMP accumulation when activation.By mutating acid residue 623 (be about to alanine residue and change into the Isoleucine residue) composing type activation TSHR.Expection G _iCoupled receptor suppresses adenylate cyclase, and therefore reduces the cAMP yield level, and this makes that measuring the cAMP level becomes possibility.Measure cAMP output and reduce (G _iCoupled receptor composing type activatory index) effective technology is: cotransfection is as most preferably non-endogenous composing type activation TSHR (TSHR-A623I) (or the endogenous composing type activation G of " signal toughener " _sCoupled receptor) and G _iThe target GPCR that connects sets up basic cAMP level.After the non-endogenous form of creating described Gi coupled receptor, use this non-endogenous form and the described signal toughener cotransfection of described target GPCR, can use described material to screen.When using cAMP to measure, can use this method effectively to produce signal; Preferably use this method at G _iCoupled receptor is directly identified candidate compound.Notice that for Gi coupling GPCR when this method of use, the inverse agonist of described target GPCR increases the cAMP signal, and agonist reduces the cAMP signal.

First day, inoculation 2 * 10 ⁴293 cells/well.Second day, prepare two reaction tubess (every pipe contains a dishful of consumption): pipe A is prepared as follows: mix every kind of acceptor 2ug DNA that mammalian cell is advanced in transfection, (CA) middle total amount contains 4 μ g DNA (as the pCMV carrier for Irvine Scientific, Irvine at 1.2ml serum-free DMEM; Carry the pCMV carrier of mutation T HSR (TSHR-A623I); TSHR-A623I and GPCR etc.); Pipe B is prepared as follows: mix 120 μ l lipofection amine reagent (Gibco BRL) in 1.2ml serum-free DMEM.Put upside down several times mixing tube A and pipe B, at room temperature incubation 30-45 minute then.This mixture is called " transfection mixture ".293 cells with 1XPBS washing inoculation add 10ml serum-free DMEM then.In cell, add the described transfection mixture of 2.4ml, then at 37 ℃/5%CO ₂Incubation 4 hours.Siphon away and remove described transfection mixture, add the 25mlDMEM/10% foetal calf serum subsequently.At 37 ℃/5%CO ₂The incubation cell.Behind the incubation 24 hours, harvested cell is used for analyzing.

Can be according to technician's needs, improvement and design is used for the FlashPlate based on raji cell assay Raji ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Cat.No.SMP004A), use it for thick plasma membrane.Flash Plate hole can comprise the flicker encrusting substance, and described encrusting substance also comprises the specific antibody of discerning cAMP.By with the combining of radioactivity cAMP tracer direct competitive and described cAMP antibody, the quantitatively cAMP that in the hole, produces.Be the Short Description of measuring the method that the cAMP level changes in the full cell of expressing described acceptor below.

Gather in the crops transfectional cell behind the about twenty four hours of transient transfection.Sucking-off and discard substratum carefully.The gentle 10ml PBS that adds in every dish cell, careful then sucking-off.Every dull and stereotyped 1ml Sigma cell dissociation damping fluid and 3ml PBS of adding.With cell sucking-off flat board, described cell suspending liquid is collected into the conical centrifuge tube of 50ml with transfer pipet.Cell is at room temperature in 1, centrifugal 5 minutes of 100rpm.The resuspension cell precipitation is in the PBS of suitable volumes (about 3ml/ flat board) carefully.Use hemocyte instrument counting cells then, replenish adding PBS, obtain the cell (reaching the final volume in about 50 μ l/ holes) of sufficient amount.

According to producer instruct preparation and keep the cAMP standard and detect damping fluid (11ml detect damping fluid include 1 μ Ci tracer [ ¹²⁵I cAMP (50 μ l)]).The mensuration damping fluid that prepared fresh is used to screen, described mensuration damping fluid contain 50 μ l stimulates damping fluid, 3 μ l test compounds (12 μ M finally measure concentration) and 50 μ l cells, with described mensuration buffer preserving on ice up to use.In appropriate bore, add 50 μ l cAMP standards, add 50 μ l PBSA at H-11 and H12 hole then, begin to measure.In institute is porose, add 50 μ l and stimulate damping fluid.Use can distribute the application of sample instrument of 3 μ l compound solutions, adds selected compound (as TSH) in appropriate bore, and the final concentration and the 100 μ l that reach 12 μ M mensuration compound always measure volume.Add cell then in the hole, at room temperature incubation is 60 minutes.In the hole, add the detection mixture that 100 μ l contain spike cAMP then.Continue incubation dull and stereotyped 2 hours, and used WallacMicroBeta scintillometer counting then.Every assay plate is production standard cAMP curve all, according to the value in described standard cAMP curve extrapolation cAMP/ hole.

4. based on the mensuration of reporter

The a.CRE-LUC reporter is measured (Gs-coupled receptor)

With 293 cells and 293T cell with every hole 2 * 10 ⁴The density of cell is seeded on 96 orifice plates, carries out transfection according to the guidance of manufacturer with lipofection amine reagent (BRL) in second day.Be prepared as follows the DNA/ lipid mixt that is used for per 6 hole transfections: (described 260ng plasmid DNA comprised 200ng 8xCRE-Luc and reported that plasmid, 50ng contain the pCMV of endogenous reporter or non-endogenous reporter or independent pCMV and 10ng GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)) gentle mixing of 2 μ l lipids that will be in the 260ng plasmid DNA in the 100 μ l DMEM and 100 μ l DMEM.Be prepared as follows described 8XCRE-Luc report plasmid: (71/+51) clone enters the BglV-HindIII site of p β gal-Basic plasmid (Clontech), obtains carrier S RIF-β-gal with rat somatostatin promotor.Obtain eight (8) copy of cAMP response element by PCR by adenovirus template AdpCF126CCRE8 (seeing 7 Human Gene Therapy 1883 (1996)), the clone enters the Kpn-BglV site of SRIF-β-gal carrier, produces 8xCRE-β-gal and reports carrier.Replace the beta-galactosidase gene that 8xCRE-β-gal reports carrier with the luciferase genes that derives from pGL3-carrier is carrier (Promega) HindIII-BamHI site, produce 8xCRE-Luc report plasmid.At room temperature incubation diluted described DNA/ lipid mixt with 400 μ l DMEM after 30 minutes, and every hole adds the described dilution of 100 μ l.Incubation is after 4 hours in cell culture incubator, and every hole adds the DMEM that 100 μ l contain 10%FCS.Described transfectional cell changed to the DMEM that 200 μ l/ holes contain 10%FCS in second day.After eight hours, with the PBS hole flushing once, be replaced by 100 μ l/ holes then and do not contain phenol red DMEM.Used LucLite in second day ^TMReporter gene is measured test kit (Packard) and is measured luciferase activity according to manufacturer's guidance, at 1450 MicroBeta ^TMFlicker luminescent counter (Wallac) is gone up reading.

The b.AP1 report is measured (G _qBind receptor)

The method that detects the Gq hormesis is based on following known features: depend on G _qPhospholipase C cause the gene activation that in promotor, contains the AP1 element.Can utilize Pathdetect ^TMAP-1 cis report system's (Stratagene, catalog number (Cat.No.) 219073) adopts the method for above measuring statement about the CREB report, and just the composition of calcium phosphate precipitation is 410ng pAP1-Luc, 80ng pCMV-expression of receptor plasmid and 20ng CMV-SEAP.

The c.SRF-LUC report is measured (Gq bind receptor)

Detect G _qThe method that stimulates is based on following known features: depend on G _qPhospholipase C cause the gene activation that in promotor, contains serum response factor.Can utilize Pathdetect ^TMSRF-Luc reports system (Stratagene), measures G in as the COS7 cell _qCoupling activity.Use Mammalian Transfection ^TMTest kit (Stratagene, catalog number (Cat.No.) 200285) according to manufacturer's guidance, is expressed the plasmid transfection cell with the appointment of the plasmid composition of described system and encode endogenous or non-endogenous GPCR.In brief, the guidance according to the manufacturer mixes 410ng SRF-Luc, 80ng pCMV-expression of receptor plasmid and 20ngCMV-SEAP (secretor type alkaline phosphatase expression plasmid in calcium phosphate precipitation; Measure transfectional cell alkaline phosphatase activities compared with the control in the substratum, the difference of transfection efficiency between sample for reference).With 3 holes of half described precipitation evenly distribute, in serum free medium, kept in touch 24 hours with described cell to 96 orifice plates.Last 5 hours, according to indication, with 1 μ l angiotonin incubation cell.Lysing cell uses Luclite then ^TMTest kit (Packard, catalog number (Cat.No.) 6016911) and " Trilux 1450 Microbeta " liquid scintillation luminescent counter (Wallac) are measured luciferase activity according to manufacturer's guidance.Use GraphPad Prism ^TM(2.0a GraphPadSoftware Inc.) analytical data.

D. IP in the cell ₃(G is measured in accumulation _qBind receptor)

The 1st day, the cell inoculation that will comprise described acceptor (endogenous and/or non-endogenous) generally was 1 * 10 to 24 orifice plates ⁵Cells/well (can optimize this value).The 2nd day, following transfectional cell: at first mix 0.25ug DNA in the 50 μ l serum-free DMEM/ holes and 2 μ l lipofection amine in the 50 μ l serum-free DMEM/ holes.Gentle described solution, at room temperature incubation 15-30 minute then of mixing.Use 0.5ml PBS and 400 μ l serum free medium washed cells subsequently, mix and add described cell then with transfection media.Described cell is at 37 ℃/5%CO ₂Incubation 3-4 hour, remove described transfection media then, be replaced by the conventional substratum in 1ml/ hole.The 3rd day, use ³H-inositol labeled cell.In brief, remove described substratum, with 0.5ml PBS washed cell.Every then hole adds 0.5ml does not have inositol/serum free medium (GIBCO BRL) and 0.25 μ Ci ³The H-inositol, with cell at 37 ℃/5%CO ₂Be incubated overnight 16-18 hour.The 4th day, with 0.5ml PBS washed cell, add 0.45ml and measure substratum, or add 0.4ml and measure the final concentration that substratum and 50 μ l 10x ketanserin (ket) reach 10 μ M, described 0.45ml measures substratum and contains no inositol/serum free medium, 10 μ Mpargyline, 10mM lithium chloride.Described cell is subsequently 37 ℃ of incubations 30 minutes.With 0.5ml PBS washed cell, every hole adds fresh ice-cold stop bath (the 1M KOH of 200 μ l; The 18mM Sodium Tetraborate; 3.8mM EDTA).Described solution keeps 5-10 minute (or up to lysis) on ice, add the fresh ice-cold neutralization solutions of 200 μ l (7.5%HCL) neutralization then.Subsequently described lysate is transferred to the 1.5ml Eppendorf tube, every pipe adds 1ml chloroform/methanol (1: 2).In 15 seconds of the described solution of eddy oscillating, the upper strata is loaded into Biorad AG1-X8 mutually ^TMAnionite-exchange resin (100-200 order).At first water is washed described resin with 1: 1.25 W/V, then the 0.9ml upper strata is loaded on the post mutually.Wash post with 10ml 5mM inositol and 10ml 5mM Sodium Tetraborate/60mM sodium formiate.The inositoltriphosphoric acid wash-out is gone into to contain the flicker tubule of 10ml flicker mixture with 2ml 0.1M formic acid/1M ammonium formiate.Following regeneration pillar: with 10ml0.1M formic acid/3M ammonium formiate washing, clean twice, be stored in the water under 4 ℃ then with distilled water.

With reference to figure 1.In Fig. 1, with following albumen rotaring redyeing 293 cell: the G of six aminoacid deletion _qAlbumen " G _q(del) "; With G _iThe G that albumen merges _qAlbumen " G _q(del)/G _i"; Endogenous RUP32; And have G _q(del) RUP32 (" RUP32+G _q(del)/G _i").According to measure R UP32 and G _q(del)/G _iThe IP of cotransfection ₃The accumulation, these data are pointed out: RUP32 not with G _qThe endogenous coupling of albumen.Yet, as RUP32 and G _q(del)/G _iDuring the fusion rotein cotransfection, RUP32 has to and G _qAlbumen coupling.RUP27+G _q(del)/G _iCompare the IP3 accumulation with endogenous RUP32 and increase about nine (9) doubly.These data show: described Gq ₍Del)/G _iFusion construct can with the GPCR cotransfection, and can be used for screening agonist or inverse agonist.

Referring to Fig. 2.In Fig. 2, use RUP35 and RUP36 acceptor rotaring redyeing 293 cell, and compare with contrast pCMV.These data are pointed out: RUP35 and RUP36 are endogenous composing type activatory.RUP35 compares endocellular phosphorus acid inositol with pCMV increase about six (6) doubly, and RUP36 compares with pCMV increases about four (4) doubly.

Embodiment 5

The preparation fusion rotein

A.GPCR:G _sFusion construct

Can followingly finish the design of composing type activation GPCR-G albumen fusion construct: through engineering approaches rat G Protein G s α (microscler formula; Itoh, H. etc., 83 PNAS 3776 (1986)) 5 ' terminal and 3 ' end, comprise HindIII (5 '-AAGCTT-3 ') sequence thereon.After confirming correct sequence (comprising flank HindIII sequence), carry out subclone, with the described complete sequence into described carrier that shuttles back and forth by the HindIII restriction site that uses pcDNA3.1 (-) (Invitrogen, catalog number (Cat.No.) V795-20).After subclone advances pcDNA3.1 (-), confirm described G _sThe correct orientation of α sequence.Confirm then to comprise described rat G in the HindIII sequence _sThe modification pcDNA3.1 (-) of α gene; This carrier is later on as " general " G _sThe α protein carrier.Described pcDNA3.1 (-) carrier comprises multiple well-known restriction site in upstream, HindIII site, therefore advantageously provides at described G _sThe ability that endogenous composing type has the encoding sequence of active GPCR is inserted in the albumen upstream.Can utilize with quadrat method and make other " general " G protein carrier, can certainly utilize other commercialization carrier known to the skilled or patent carrier.In some embodiments, important criterion is that the sequence of described GPCR meets frame in the upstream of described G protein sequence and with described G protein sequence.

Transcribed spacer between described G albumen and described GPCR in the restriction site is optional.There are adopted primer and antisense primer to comprise XbaI and EcoRV restriction site respectively, make between described G albumen and described GPCR, to have transcribed spacer (owing to described restriction site).

Utilize PCR to guarantee that each receptor sequence merges into disclosed described G above then _sThe α universal support, method below wherein using: add the cDNA of 100ng coding GPCR in each pipe, each pipe comprises every kind of primer of 2 μ l (justice and antisense are arranged), 3 μ l 10mM dNTP, 10 μ l10XTaqPlus ^TMPrecision damping fluid, 1 μ l TaqPlus ^TMPrecision polysaccharase (Stratagene:#600211) and 80 μ l water.Temperature of reaction and cycling time at described GPCR are as follows, and wherein step 2 repeats 35 times to step 4: 94 ℃ 1 minute; 94 ℃ of 30 second; 62 ℃ of 20 second; 72 ℃ 1 minute 40 seconds; Then 72 ℃ 5 minutes.The PCR product is electrophoresis on 1% sepharose, then purifying.Digest described purified product with XbaI and EcoRV, the required insertion fragment of purifying connects to advance described G _sEach restriction site of universal support.Transform the back and separate positive colony, confirm by restriction enzyme digestion then; Finish the expression of using 293 cells according to the method for above statement.Order-checking GPCR-G _sEach positive colony of fusion rotein is confirmed its exactness.

B.G _q(six amino acid disappearance)/G _iFusion construct

The following G that finishes _q(del)/G _iThe design of fusion construct: remove G α q subunit N-terminal six (6) individual amino acid (amino acid 2 to 7, sequence TLESIM (SEQ.ID.NO.:47), and the proteic corresponding amino acid of G α i (sequence D CGLF (SEQ.ID.NO.:49)) replaces C-terminal five (5) individual amino acid (sequence EYNLV (SEQ.ID.NO.:48)).Use plasmid 63313 as template, primer obtains this fusion construct by PCR below using:

5′-gatcAAGCTTCCATGGCGTGCTGCCTGAGCGAGG-3′

(SEQ.ID.NO.:50) and

5′-

gatcGGATCCTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCA

GGATGGTG-3 ' (SEQ.ID.NO.:51), described plasmid 63313 comprises the mouse G α q wild-type form of carrying the hemagglutinin mark.The Nucleotide that lowercase is represented is transcribed spacer.

Utilize TaqPlus  Precision archaeal dna polymerase (Stratagene) by following cyclic amplification, wherein step 2 to 4 repeats 35 times: 95 ℃ 2 minutes; 95 ℃ of 20 second; 56 ℃ of 20 second; 72 ℃ 2 minutes; Then 72 ℃ 7 minutes.Described PCR product cloning is advanced pCRII-TOPO carrier (Invitrogen), use ABI Big Dye Terminator test kit (P.E.Biosystems) order-checking.By 2 step cloning process, the TOPO of self-contained described fusion construct sequence clone's insertion fragment is shuttled back and forth into the HindIII/BamHI site of expression vector pcDNA3.1 (+) in the future.

Embodiment 6

The tissue distribution of disclosed people GPCR: RT-PCR

Use RT-PCR and confirm to express, confirm the tissue distribution of several Novel Human GPCR.The oligonucleotide that is utilized is that GPCR is specific, and (MTC is Clontech) as template for the many tissue cDNA combinations of end user.In 40 μ l reactants, use TaqDNA polysaccharase (Stratagene) according to manufacturer's guidance.The described reactant of 20 μ l is loaded on 1.5% sepharose, analyzes described RT-PCR product.The primer that following table E lists acceptor, cycling condition and utilized, and list and described receptor related typical disease/disorder.

Table E

The acceptor title	Cycling condition minute ('), second (") circulation 2-4 repeats 35 times	5 ' primer (SEQ.ID.NO.)	3 ' primer (SEQ.ID.NO.)	Dna fragmentation	Tissue expression
						??hRUP28	94 ℃ 5 minutes; 94 ℃ of 30 second; 58 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	??GTCCTCACTGG ??TGGCCATGTAC ??TCC(52)	??CTGCGTCCA ??CCAGAGTCA ??CGTCTCC ??(53)	??710bp	The heart, kidney, liver, lung and pancreas
??hRUP29	94 ℃ 5 minutes; 94 ℃ of 30 second; 58 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	??CTTGGATGTTT ??GGGCTGCCCTT ??CTGC(54)	??GTTTGTGGC ??TAACGGCAC ??AAAACACAA ??TTCC(55)	??690bp	White corpuscle and ovary
						??hRUP30	94 ℃ 2 minutes; 94 ℃ of 15 second; 58 ℃ of 20 second, 72 ℃ of 1 minute and 72 ℃ 10 minutes	??CTGCTCACGGT ??TGACCGCTACA ??CTGC(56)	??GTGGCCATG ??AGCCACCCT ??GAGCTCC ??(57)	??690bp	Pancreas
??hRUP31	95 ℃ 4 minutes; 95 ℃ 1 minute; 52 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	??CTTCTTCTCCG ??ACGTCAAGG ??(58)	??CCAAATCAG ??TGTGCAAAT ??CG(59)	??56bp	Colon, lung, pancreas, thymus gland; The hippocampus of pallium, brain and adipocyte
						??hRUP32	95 ℃ 4 minutes; 95 ℃ 1 minute; 52 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	??TGAATGGGTCC ??TGTGTGAAA ??(60)	??CAACGGTCT ??GACAACCTC ??CT(61)	??527bp	Thymus gland

hRUP34	95 ℃ 4 minutes; 95 ℃ 1 minute; 52 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	TTGCTGTGATG TGGCATTTTG (62)	CAGGAAGCC CATAAAGGC ATCAA(63)	534bp	Peripheral blood leucocyte (" PBL "), prostate gland and kidney
						hRUP35	95 ℃ 4 minutes; 95 ℃ 1 minute; 52 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	ACATCACCTGC TTCCTGACC (64)	CCAGCATCT TGATGCAGT GT(65)	557bp	Thalamus
hRUP37	95 ℃ 4 minutes; 95 ℃ 1 minute; 52 ℃ of 30 second, 72 ℃ of 1 minute and 72 ℃ 7 minutes	CCATCTCCAAA ATCCTCAGTC (66)	GCTGTTAAG AGCGGACAG GAAA(67)	517bp	Testis, pallium and hippocampus

Include but not limited to the receptor related disease and the disorder that are arranged in these tissues or zone: cardiac disorders and disease are (as thrombosis, myocardial infarction, atherosclerosis, myocardosis), ephrosis/disorder is (as renal failure, renal tubular acidosis, renal glycosuria, diabetes insipidus,nephrogenic, cystinuria, multicystic kidney disease), the eosinophilia, leukocytosis, oligoleukocythemia, ovarian cancer, sexual dysfunction, polycystic ovarian syndrome, pancreatitis and carcinoma of the pancreas, irritable bowel syndrome, colorectal carcinoma, the CrohnShi disease, ulcerative colitis, diverticulitis, chronic obstructive pulmonary disease (COPD), Cysticfibrosis, pneumonia, pulmonary hypertension, pulmonary tuberculosis and lung cancer, the ParkinsonShi disease, dyskinesia and ataxia, learning memory disorder, eating disorder is (as apocleisis, exessive appetite etc.), fat, cancer, thymoma, myasthenia gravis, circulation system disease, prostate cancer, prostatitis, ephrosis/disorder is (as renal failure, renal tubular acidosis, renal glycosuria, diabetes insipidus,nephrogenic, cystinuria, multicystic kidney disease), obstacle is handled and is waken up in sensorimotor, anancastic obstacle, carcinoma of testis, priapism, prostatitis, hernia, endocrine regulation, sexual dysfunction, transformation reactions, depressed, psychosis, migraine, anti-stream, schizophrenia, ulcer, bronchospasm, epilepsy, prostatomegaly, anxiety, rhinitis, angor and glaucoma.Therefore, method of the present invention can be used for diagnosis and/or treat these and other disease and disorder.

Embodiment 7

Method: directly identify inverse agonist and agonist

A.[35S] GTP γ S mensuration

Can be used as for example candidate compound of inverse agonist though used the activated GPCR of endogenous composing type directly to identify, owing to do not understand the difference in aggravating to measure fully.In some embodiments, above disclosed gpcr fusion protein is also used with non-endogenous composing type activatory GPCR.When using described albumen, difference seems basicly stable in measuring, and obtains effective signal to noise ratio thus.Its good result is to allow to identify more reliably candidate compound.Therefore, in some embodiments, preferably when directly identifying, use gpcr fusion protein, and when using described fusion rotein, measuring method below utilizing.

1. membrane prepare

Preferably be prepared as follows and comprise the film that target group forms active lonely gpcr fusion protein and is used for directly identifying the candidate compound that can be used as inverse agonist or agonist:

A. material

" film strikes off damping fluid " comprises 20mM HEPES and 10mM EDTA, and pH 7.4; " film lavation buffer solution " comprises 20mM HEPES and 0.1mM EDTA, and pH 7.4; " binding buffer liquid " comprises 20mM HEPES, 100mM NaCl and 10mM MgCl ₂, pH7.4.

B. method

All substances all are kept on ice in the entire method process.At first siphon away substratum, clean, siphon away PBS then with the cold PBS of 10ml from the monolayer cell that converges.After this, add the 5ml film and strike off the damping fluid cast-off cells; Then cell extract is shifted into 50ml centrifuge tube (under 4 ℃ 20, centrifugal 17 minutes of 000rpm).After this, siphon away supernatant, with pellet resuspended in 30ml film lavation buffer solution, under 4 ℃ 20, centrifugal 17 minutes of 000rpm.Siphon away supernatant, with pellet resuspended in binding buffer liquid.Use Brinkman Polytron then ^TMThe precipitation of the described resuspension of homogenizer homogenate (broken 15-20 second, form suspension) up to material.This material is referred to herein as " membranin ".

2.Bradford protein determination

After the homogenate, measure the protein concentration of described film, for example use Bradford protein determination method to measure that (diluted protein is to about 1.5mg/ml, use after five equilibrium and freezing (80 ℃) are waited for; When freezing, using method is as follows: measure that day, the freezing membranin that thaws under the room temperature, vortex vibration then, with Polytron about 12 * 1, the about 5-10 of 000rpm homogenate second; Attention is when repeatedly preparing, and described homogenizer should fully clean between the different prepared products of homogenate).

A. material

Binding buffer liquid (as indicated above); The Bradford dye reagent; Bradford protein standard (Biorad, catalog number (Cat.No.) 500-0006) is used in guidance according to the manufacturer.

B. method

Prepare two parts of pipes, a pipe comprises film, and another part pipe is " blank " in contrast.Every pipe comprises 800 μ l binding buffer liquid.After this, every pipe adds 10 μ l Bradford protein standards (1mg/ml), and a pipe adds 10 μ l membranins (not being blank pipe) therein.After this, every pipe adds 200 μ l Bradford dye reagents, the vortex vibration.After five minutes, vortex vibration two pipes once more, with wherein substance transfer to cuvette.Use CECIL 3041 spectrophotometers at 595 pairs of described cuvette readings of wavelength.

3. directly identify and measure

A. material

The GDP damping fluid contains 37.5ml binding buffer liquid and 2mg GDP (Sigma, catalog number (Cat.No.) G-7127), and serial dilution obtains the concentration (ultimate density of GDP is 0.1 μ M GDP in every hole) of 0.2 μ M GDP in binding buffer liquid; The final volume in every hole is 200 μ l, comprise in membranin in candidate compound, 100 μ l GDP damping fluids (ultimate density 0.1 μ M GDP), the 50 μ l binding buffer liquid and the 50 μ l binding buffer liquid [ ³⁵S] GTP γ S (0.6nM) (every 10ml binding buffer liquid 2.5 μ l[ ³⁵S] GTP γ S).

B. method

Preferably use 96 orifice plates screening candidate compound (can be freezing) at-80 ℃.Simple homogenate membranin (or the film that carries expression vector is in contrast, and described expression vector does not conform to described gpcr fusion protein) up to becoming suspension.Use Bradford protein determination method for example mentioned above to measure protein concentration then.Dilute membranin (and contrast) to 0.25mg/ml (finally measuring concentration 12.5 μ g/ holes) with binding buffer liquid.After this, each Wallac Scintistrip ^TM(Wallac) hole adds 100 μ l GDP damping fluids.Use 5 μ l application of sample instruments transferase 45 μ l candidate compound (promptly account for 5 μ l, constitute 1: 40 ratio, make that the final screening concentration of described candidate compound is 10 μ M) in described hole then total mensuration among the volume 200 μ l.In addition, for avoiding polluting, all clean the application of sample instrument after each transfer step in three ponds, described three ponds fill water (1X), ethanol (1X) and water (2X), and unnecessary liquid is got rid of from the application of sample instrument in each back of cleaning, and dries described instrument with paper and Kim rag.After this, every hole adds 50 μ l membranins (also utilize control wells, this hole comprises the film that does not have described gpcr fusion protein), under the room temperature incubation 5-10 minute.After this, every hole add in the 50 μ l binding buffer liquid [ ³⁵S] GTP γ S (0.6nM), incubation 60 minutes under the room temperature (same, as in the present embodiment, to cover dull and stereotyped with paillon foil) in shaking table then.By under 22 ℃ centrifugal described dull and stereotyped 15 minutes, stop measuring with 4000RPM.Siphon away liquid in the flat board with 8 passage manifolds, with dull and stereotyped cap seal mouth.Described then flat board uses setting " Prot.#37 " reading (according to manufacturer's guidance) on Wallac 1450.

B. ring-type AMP measures

Directly the another kind of measuring method of evaluation candidate compound can use based on the mensuration of cyclase and finish.Except that direct evaluation, can utilize the method for this measuring method as independence, confirm according to mentioned above [ ³⁵S] result that obtains of GTP γ S method.

Preferably, utilize improvement Flash Plate according to following method ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Catalog number (Cat.No.) SMP004A) directly identifies the candidate compound that can be used as GPCR inverse agonist and agonist.

About three days results transfectional cells after the transfection.Homogenate is containing 20mM HEPES, pH7.4 and 10mM MgCl ₂The cell of damping fluid inner suspension, the preparation film.Use BrinkrnanPolytron ^TMIn homogenate on ice about 10 seconds.The homogenate that obtains under 4 ℃ in 49, centrifugal 15 minutes of 000Xg.The pellet resuspended that obtains is in containing 20mM HEPES, in the damping fluid of pH 7.4 and 0.1mMEDTA, 10 seconds of homogenate, then under 4 ℃ in 49, centrifugal 15 minutes of 000Xg.The precipitation that obtains is kept at-80 ℃ up to use.In that day of direct evaluation and screening, at room temperature slowly the described film of thawing precipitates, and is resuspended to contain 20mM HEPES, pH 7.4 and 10mM MgCl ₂Damping fluid, make that final protein concentration is 0.60mg/ml (film of described resuspension place on ice up to use).

According to manufacturer's guidance preparation and keep the cAMP standard and detect damping fluid (11ml detect damping fluid include 2 μ Ci tracers [ ¹²⁵I cAMP (100 μ l)]).The mensuration damping fluid that prepared fresh is used to screen, described damping fluid contains 20mM HEPES, and pH 7.4,10mM MgCl ₂, 20mM phosphocreatine (Sigma), 0.1 unit/ml creatine phosphokinase (Sigma), 50 μ MGTP (Sigma) and 0.2mM ATP (Sigma); Measure buffer preserving on ice up to use.

Preferably the candidate compound that will identify as mentioned (if freezing, then at room temperature thawing) adds (3 μ l/ holes in the hole of 96 orifice plates; Finally measure concentration 12 μ M), and add 40 μ l membranins (30 μ g/ hole) and 50 μ l mensuration damping fluid.Described mixture is mild stirring incubation 30 minutes at room temperature.

Behind the incubation, every hole adds 100 μ l and measures damping fluid, incubation 2-24 hour then.Then at Wallac MicroBeta ^TMUse " Prot.#31 " to read plate (according to manufacturer's guidance) on the plate reading machine.

C. melanophore screening assay

Can followingly identify the method for GPCR candidate agonist or inverse agonist: introduce the test cell of pigment cell system, described test cell can be tackled particular stimulation and disperse or assemble their pigment, and expresses the external source clone of the described GPCR of coding.If the activation of described GPCR induces pigment to disperse, then the initial state that pigment handles is set is that pigment accumulates in the test cell to stimulator (as light).Yet if the activation of described GPCR induces pigment to assemble, the initial state that pigment deposition is set with the stimulator irritation cell is that pigment disperses.Described test cell is contacted with described compound, determine that the initial state whether intracellular pigment processing is handled from pigment changes.The pigment cell that causes with described GPCR coupling candidate compound is dispersed in and seems darker on the culture dish, and that chromatophorous gathering seems to cry is bright.

Material and method be according to U.S. Patent number 5,462, and 856 and the disclosure of U.S. Patent number 6,051,386, these two parts of publications are attached to herein by reference.

Though for utilizing the purpose of endogenous and non-endogenous people GPCR, those skilled in that art can utilize various expression vectors, the preferred in some embodiments carrier that utilizes is pCMV.The microbial preservation budapest treaty that this carrier is used for patented procedure according to international recognition is specified in and was preserved in American type culture collection (ATCC) (10801 University Blvd. on October 13rd, 1998, Manassas, VA 20110-2209 USA).Described DNA is confirmed to be great-hearted through the ATCC test.ATCC gives following preserving number: ATCC#203351 to pCMV.

Except that indicating specially, the reference of quoting in this patent file comprises common unsettled and related application all intactly is attached to herein by reference.Top open scheme and following claim are included in modification and the expansion to disclosed invention in technician's limit of power.

Sequence table

<110>Arena?Pharmaceuticals，Inc.

＜120〉endogenous and non-endogenous composing type activation human G protein-coupled receptor

<130>AREN-0309

<150>09/170,496

<151>1998-10-13

<150>PCT/US99/23938

<151>1998-10-13

<150>60/253,404

<151>2000-11-27

<150>60/255,366

<151>2000-12-12

<150>60/270,286

<151>2001-02-20

<150>60/282,365

<151>2001-04-06

<150>60/270,266

<151>2001-02-20

<150>60/282,032

<151>2001-04-06

<150>60/282,358

<151>2001-04-06

<150>60/282,356

<151>2001-04-06

<150>60/290,917

<151>2001-05-14

<150>60/309,208

<151>2001-07-31

<160>67

＜170〉PatentIn version 3 .1

<210>1

<211>1002

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>1

atgtggagct?gcagctggtt?caacggcaca?gggctggtgg?aggagctgcc?tgcctgccag?????60

gacctgcagc?tggggctgtc?actgttgtcg?ctgctgggcc?tggtggtggg?cgtgccagtg????120

ggcctgtgct?acaacgccct?gctggtgctg?gccaacctac?acagcaaggc?cagcatgacc????180

atgccggacg?tgtactttgt?caacatggca?gtggcaggcc?tggtgctcag?cgccctggcc????240

cctgtgcacc?tgctcggccc?cccgagctcc?cggtgggcgc?tgtggagtgt?gggcggcgaa????300

gtccacgtgg?cactgcagat?ccccttcaat?gtgtcctcac?tggtggccat?gtactccacc????360

gccctgctga?gcctcgacca?ctacatcgag?cgtgcactgc?cgcggaccta?catggccagc????420

gtgtacaaca?cgcggcacgt?gtgcggcttc?gtgtggggtg?gcgcgctgct?gaccagcttc????480

tcctcgctgc?tcttctacat?ctgcagccat?gtgtccaccc?gcgcgctaga?gtgcgccaag????540

atgcagaacg?cagaagctgc?cgacgccacg?ctggtgttca?tcggctacgt?ggtgccagca????600

ctggccaccc?tctacgcgct?ggtgctactc?tcccgcgtcc?gcagggagga?cacgcccctg????660

gaccgggaca?cgggccggct?ggagccctcg?gcacacaggc?tgctggtggc?caccgtgtgc????720

acgcagtttg?ggctctggac?gccacactat?ctgatcctgc?tggggcacac?ggtcatcatc????780

tcgcgaggga?agcccgtgga?cgcacactac?ctggggctac?tgcactttgt?gaaggatttc????840

tccaaactcc?tggccttctc?cagcagcttt?gtgacaccac?ttctctaccg?ctacatgaac????900

cagagcttcc?ccagcaagct?ccaacggctg?atgaaaaagc?tgccctgcgg?ggaccggcac????960

tgctccccgg?accacatggg?ggtgcagcag?gtgctggcgt?ag??????????????????????1002

<210>2

<211>333

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>2

Met?Trp?Ser?Cys?Ser?Trp?Phe?Asn?Gly?Thr?Gly?Leu?Val?Glu?Glu?Leu

1???????????????5???????????????????10??????????????????15

Pro?Ala?Cys?Gln?Asp?Leu?Gln?Leu?Gly?Leu?Ser?Leu?Leu?Ser?Leu?Leu

20??????????????????25??????????????????30

Gly?Leu?Val?Val?Gly?Val?Pro?Val?Gly?Leu?Cys?Tyr?Asn?Ala?Leu?Leu

35??????????????????40??????????????????45

Val?Leu?Ala?Asn?Leu?His?Ser?Lys?Ala?Ser?Met?Thr?Met?Pro?Asp?Val

50??????????????????55??????????????????60

Tyr?Phe?Val?Asn?Met?Ala?Val?Ala?Gly?Leu?Val?Leu?Ser?Ala?Leu?Ala

65??????????????????70??????????????????75??????????????????80

Pro?Val?His?Leu?Leu?Gly?Pro?Pro?Ser?Ser?Arg?Trp?Ala?Leu?Trp?Ser

85??????????????????90??????????????????95

Val?Gly?Gly?Glu?Val?His?Val?Ala?Leu?Gln?Ile?Pro?Phe?Asn?Val?Ser

100?????????????????105?????????????????110

Ser?Leu?Val?Ala?Met?Tyr?Ser?Thr?Ala?Leu?Leu?Ser?Leu?Asp?His?Tyr

115?????????????????120?????????????????125

Ile?Glu?Arg?Ala?Leu?Pro?Arg?Thr?Tyr?Met?Ala?Ser?Val?Tyr?Asn?Thr

130?????????????????135?????????????????140

Arg?His?Val?Cys?Gly?Phe?Val?Trp?Gly?Gly?Ala?Leu?Leu?Thr?Ser?Phe

145?????????????????150?????????????????155?????????????????160

Ser?Ser?Leu?Leu?Phe?Tyr?Ile?Cys?Ser?His?Val?Ser?Thr?Arg?Ala?Leu

165?????????????????170?????????????????175

Glu?Cys?Ala?Lys?Met?Gln?Asn?Ala?Glu?Ala?Ala?Asp?Ala?Thr?Leu?Val

180?????????????????185?????????????????190

Phe?Ile?Gly?Tyr?Val?Val?Pro?Ala?Leu?Ala?Thr?Leu?Tyr?Ala?Leu?Val

195?????????????????200?????????????????205

Leu?Leu?Ser?Arg?Val?Arg?Arg?Glu?Asp?Thr?Pro?Leu?Asp?Arg?Asp?Thr

210?????????????????215?????????????????220

Gly?Arg?Leu?Glu?Pro?Ser?Ala?His?Arg?Leu?Leu?Val?Ala?Thr?Val?Cys

225?????????????????230?????????????????235?????????????????240

Thr?Gln?Phe?Gly?Leu?Trp?Thr?Pro?His?Tyr?Leu?Ile?Leu?Leu?Gly?His

245?????????????????250?????????????????255

Thr?Val?Ile?Ile?Ser?Arg?Gly?Lys?Pro?Val?Asp?Ala?His?Tyr?Leu?Gly

260?????????????????265?????????????????270

Leu?Leu?His?Phe?Val?Lys?Asp?Phe?Ser?Lys?Leu?Leu?Ala?Phe?Ser?Ser

275?????????????????280?????????????????285

Ser?Phe?Val?Thr?Pro?Leu?Leu?Tyr?Arg?Tyr?Met?Asn?Gln?Ser?Phe?Pro

290?????????????????295?????????????????300

Ser?Lys?Leu?Gln?Arg?Leu?Met?Lys?Lys?Leu?Pro?Cys?Gly?Asp?Arg?His

305?????????????????310?????????????????315?????????????????320

Cys?Ser?Pro?Asp?His?Met?Gly?Val?Gln?Gln?Val?Leu?Ala

325?????????????????330

<210>3

<211>918

<212>DNA

＜213〉people

<400>3

atgcctggcc?acaatacctc?caggaattcc?tcttgcgatc?ctatagtgac?accccactta?????60

atcagcctct?acttcatagt?gcttattggc?gggctggtgg?gtgtcatttc?cattcttttc????120

ctcctggtga?aaatgaacac?ccggtcagtg?accaccatgg?cggtcattaa?cttggtggtg????180

gtccacagcg?tttttctgct?gacagtgcca?tttcgcttga?cctacctcat?caagaagact????240

tggatgtttg?ggctgccctt?ctgcaaattt?gtgagtgcca?tgctgcacat?ccacatgtac????300

ctcacgttcc?tattctatgt?ggtgatcctg?gtcaccagat?acctcatctt?cttcaagtgc????360

aaagacaaag?tggaattcta?cagaaaactg?catgctgtgg?ctgccagtgc?tggcatgtgg????420

acgctggtga?ttgtcattgt?ggtacccctg?gttgtctccc?ggtatggaat?ccatgaggaa????480

tacaatgagg?agcactgttt?taaatttcac?aaagagcttg?cttacacata?tgtgaaaatc????540

atcaactata?tgatagtcat?ttttgtcata?gccgttgctg?tgattctgtt?ggtcttccag????600

gtcttcatca?ttatgttgat?ggtgcagaag?ctacgccact?ctttactatc?ccaccaggag????660

ttctgggctc?agctgaaaaa?cctatttttt?ataggggtca?tccttgtttg?tttccttccc????720

taccagttct?ttaggatcta?ttacttgaat?gttgtgacgc?attccaatgc?ctgtaacagc????780

aaggttgcat?tttataacga?aatcttcttg?agtgtaacag?caattagctg?ctatgatttg????840

cttctctttg?tctttggggg?aagccattgg?tttaagcaaa?agataattgg?cttatggaat????900

tgtgttttgt?gccgttag??????????????????????????????????????????????????918

<210>4

<211>305

<212>PRT

＜213〉people

<400>4

Met?Pro?Gly?His?Asn?Thr?Ser?Arg?Asn?Ser?Ser?Cys?Asp?Pro?Ile?Val

1???????????????5???????????????????10??????????????????15

Thr?Pro?His?Leu?Ile?Ser?Leu?Tyr?Phe?Ile?Val?Leu?Ile?Gly?Gly?Leu

20??????????????????25??????????????????30

Val?Gly?Val?Ile?Ser?Ile?Leu?Phe?Leu?Leu?Val?Lys?Met?Asn?Thr?Arg

35??????????????????40??????????????????45

Ser?Val?Thr?Thr?Met?Ala?Val?Ile?Asn?Leu?Val?Val?Val?His?Ser?Val

50??????????????????55??????????????????60

Phe?Leu?Leu?Thr?Val?Pro?Phe?Arg?Leu?Thr?Tyr?Leu?Ile?Lys?Lys?Thr

65??????????????????70??????????????????75??????????????????80

Trp?Met?Phe?Gly?Leu?Pro?Phe?Cys?Lys?Phe?Val?Ser?Ala?Met?Leu?His

85??????????????????90??????????????????95

Ile?His?Met?Tyr?Leu?Thr?Phe?Leu?Phe?Tyr?Val?Val?Ile?Leu?Val?Thr

100?????????????????105?????????????????110

Arg?Tyr?Leu?Ile?Phe?Phe?Lys?Cys?Lys?Asp?Lys?Val?Glu?Phe?Tyr?Arg

115?????????????????120?????????????????125

Lys?Leu?His?Ala?Val?Ala?Ala?Ser?Ala?Gly?Met?Trp?Thr?Leu?Val?Ile

130?????????????????135?????????????????140

Val?Ile?Val?Val?Pro?Leu?Val?Val?Ser?Arg?Tyr?Gly?Ile?His?Glu?Glu

145?????????????????150?????????????????155?????????????????160

Tyr?Asn?Glu?Glu?His?Cys?Phe?Lys?Phe?His?Lys?Glu?Leu?Ala?Tyr?Thr

165?????????????????170?????????????????175

Tyr?Val?Lys?Ile?Ile?Asn?Tyr?Met?Ile?Val?Ile?Phe?Val?Ile?Ala?Val

180?????????????????185?????????????????190

Ala?Val?Ile?Leu?Leu?Val?Phe?Gln?Val?Phe?Ile?Ile?Met?Leu?Met?Val

195?????????????????200?????????????????205

Gln?Lys?Leu?Arg?His?Ser?Leu?Leu?Ser?His?Gln?Glu?Phe?Trp?Ala?Gln

210?????????????????215?????????????????220

Leu?Lys?Asn?Leu?Phe?Phe?Ile?Gly?Val?Ile?Leu?Val?Cys?Phe?Leu?Pro

225?????????????????230?????????????????235?????????????????240

Tyr?Gln?Phe?Phe?Arg?Ile?Tyr?Tyr?Leu?Asn?Val?Val?Thr?His?Ser?Asn

245?????????????????250?????????????????255

Ala?Cys?Asn?Ser?Lys?Val?Ala?Phe?Tyr?Asn?Glu?Ile?Phe?Leu?Ser?Val

260?????????????????265?????????????????270

Thr?Ala?Ile?Ser?Cys?Tyr?Asp?Leu?Leu?Leu?Phe?Val?Phe?Gly?Gly?Ser

275?????????????????280?????????????????285

His?Trp?Phe?Lys?Gln?Lys?Ile?Ile?Gly?Leu?Trp?Asn?Cys?Val?Leu?Cys

290?????????????????295?????????????????300

Arg

305

<210>5

<211>1125

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>5

atgctgacag?ggagctgcgg?ggaccctcag?aaaaagccac?aggtgaccca?ggactcaggg?????60

ccccagagca?tggggcttga?gggacgagag?acagctggcc?agccacgagt?gaccctgctg????120

cccacgccca?acgtcagcgg?gctgagccag?gagtttgaaa?gccactggcc?agagatcgca????180

gagaggtccc?cgtgtgtggc?tggcgtcatc?cctgtcatct?actacagtgt?cctgctgggc????240

ttggggctgc?ctgtcagcct?cctgaccgca?gtggccctgg?cgcgccttgc?caccaggacc????300

aggaggccct?cctactacta?ccttctggcg?ctcacagcct?cggatatcat?catccaggtg????360

gtcatcgtgt?tcgcgggctt?cctcctgcag?ggagcagtgc?tggcccgcca?ggtgccccag????420

gctgtggtgc?gcacggccaa?catcctggag?tttgctgcca?accacgcctc?agtctggatc????480

gccatcctgc?tcacggttga?ccgctacact?gccctgtgcc?accccctgca?ccatcgggcc????540

gcctcgtccc?caggccggac?ccgccgggcc?attgctgctg?tcctgagtgc?tgccctgttg????600

accggcatcc?ccttctactg?gtggctggac?atgtggagag?acaccgactc?acccagaaca????660

ctggacgagg?tcctcaagtg?ggctcactgt?ctcactgtct?atttcatccc?ttgtggcgtg????720

ttcctggtca?ccaactcggc?catcatccac?cggctacgga?ggaggggccg?gagtgggctg????780

cagccccggg?tgggcaagag?cacagccatc?ctcctgggca?tcaccacact?gttcaccctc????840

ctgtgggcgc?cccgggtctt?cgtcatgctc?taccacatgt?acgtggcccc?tgtccaccgg????900

gactggaggg?tccacctggc?cttggatgtg?gccaatatgg?tggccatgct?ccacacggca????960

gccaacttcg?gcctctactg?ctttgtcagc?aagactttcc?gggccactgt?ccgacaggtc???1020

atccacgatg?cctacctgcc?ctgcactttg?gcatcacagc?cagagggcat?ggcggcgaag???1080

cctgtgatgg?agcctccggg?actccccaca?ggggcagaag?tgtag???????????????????1125

<210>6

<211>374

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>6

Met?Leu?Thr?Gly?Ser?Cys?Gly?Asp?Pro?Gln?Lys?Lys?Pro?Gln?Val?Thr

1???????????????5???????????????????10??????????????????15

Gln?Asp?Ser?Gly?Pro?Gln?Ser?Met?Gly?Leu?Glu?Gly?Arg?Glu?Thr?Ala

20??????????????????25??????????????????30

Gly?Gln?Pro?Arg?Val?Thr?Leu?Leu?Pro?Thr?Pro?Asn?Val?Ser?Gly?Leu

35??????????????????40??????????????????45

Ser?Gln?Glu?Phe?Glu?Ser?His?Trp?Pro?Glu?Ile?Ala?Glu?Arg?Ser?Pro

50??????????????????55??????????????????60

Cys?Val?Ala?Gly?Val?Ile?Pro?Val?Ile?Tyr?Tyr?Ser?Val?Leu?Leu?Gly

65??????????????????70??????????????????75??????????????????80

Leu?Gly?Leu?Pro?Val?Ser?Leu?Leu?Thr?Ala?Val?Ala?Leu?Ala?Arg?Leu

85??????????????????90??????????????????95

Ala?Thr?Arg?Thr?Arg?Arg?Pro?Ser?Tyr?Tyr?Tyr?Leu?Leu?Ala?Leu?Thr

100?????????????????105?????????????????110

Ala?Ser?Asp?Ile?Ile?Ile?Gln?Val?Val?Ile?Val?Phe?Ala?Gly?Phe?Leu

115?????????????????120?????????????????125

Leu?Gln?Gly?Ala?Val?Leu?Ala?Arg?Gln?Val?Pro?Gln?Ala?Val?Val?Arg

130?????????????????135?????????????????140

Thr?Ala?Asn?Ile?Leu?Glu?Phe?Ala?Ala?Asn?His?Ala?Ser?Val?Trp?Ile

145?????????????????150?????????????????155?????????????????160

Ala?Ile?Leu?Leu?Thr?Val?Asp?Arg?Tyr?Thr?Aia?Leu?Cys?His?Pro?Leu

165?????????????????170?????????????????175

His?His?Arg?Ala?Ala?Ser?Ser?Pro?Gly?Arg?Thr?Arg?Arg?Ala?Ile?Ala

180?????????????????185?????????????????190

Ala?Val?Leu?Ser?Ala?Ala?Leu?Leu?Thr?Gly?Ile?Pro?Phe?Tyr?Trp?Trp

195?????????????????200?????????????????205

Leu?Asp?Met?Trp?Arg?Asp?Thr?Asp?Ser?Pro?Arg?Thr?Leu?Asp?Glu?Val

210?????????????????215?????????????????220

Leu?Lys?Trp?Ala?His?Cys?Leu?Thr?Val?Tyr?Phe?Ile?Pro?Cys?Gly?Val

225?????????????????230?????????????????235?????????????????240

Phe?Leu?Val?Thr?Asn?Ser?Ala?Ile?Ile?His?Arg?Leu?Arg?Arg?Arg?Gly

245?????????????????250?????????????????255

Arg?Ser?Gly?Leu?Gln?Pro?Arg?Val?Gly?Lys?Ser?Thr?Ala?Ile?Leu?Leu

260?????????????????265?????????????????270

Gly?Ile?Thr?Thr?Leu?Phe?Thr?Leu?Leu?Trp?Ala?Pro?Arg?Val?Phe?Val

275?????????????????280?????????????????285

Met?Leu?Tyr?His?Met?Tyr?Val?Ala?Pro?Val?His?Arg?Asp?Trp?Arg?Val

290?????????????????295?????????????????300

His?Leu?Ala?Leu?Asp?Val?Ala?Asn?Met?Val?Ala?Met?Leu?His?Thr?Ala

305?????????????????310?????????????????315?????????????????320

Ala?Asn?Phe?Gly?Leu?Tyr?Cys?Phe?Val?Ser?Lys?Thr?Phe?Arg?Ala?Thr

325?????????????????330?????????????????335

Val?Arg?Gln?Val?Ile?His?Asp?Ala?Tyr?Leu?Pro?Cys?Thr?Leu?Ala?Ser

340?????????????????345?????????????????350

Gln?Pro?Glu?Gly?Met?Ala?Ala?Lys?Pro?Val?Met?Glu?Pro?Pro?Gly?Leu

355?????????????????360?????????????????365

Pro?Thr?Gly?Ala?Glu?Val

370

<210>7

<211>1086

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>7

atgtccactg?aatgcgcgcg?ggcagcgggc?gacgcgccct?tgcgcagcct?ggagcaagcc?????60

aaccgcaccc?gctttccctt?cttctccgac?gtcaagggcg?accaccggct?ggtgctggcc????120

gcggtggaga?caaccgtgct?ggtgctcatc?tttgcagtgt?cgctgctggg?caacgtgtgc????180

gccctggtgc?tggtggcgcg?ccgacgacgc?cgcggcgcga?ctgcctgcct?ggtactcaac????240

ctcttctgcg?cggacctgct?cttcatcagc?gctatccctc?tggtgctggc?cgtgcgctgg????300

actgaggcct?ggctgctggg?ccccgttgcc?tgccacctgc?tcttctacgt?gatgaccctg????360

agcggcagcg?tcaccatcct?cacgctggcc?gcggtcagcc?tggagcgcat?ggtgtgcatc????420

gtgcacctgc?agcgcggcgt?gcggggtcct?gggcggcggg?cgcgggcagt?gctgctggcg????480

ctcatatggg?gctattcggc?ggtcgccgct?ctgcctctat?gcgtcttctt?ccgagtcgtc????540

ccgcaacggc?tccccggcgc?cgaccaggaa?atttcgattt?gcacactgat?ttggcccacc????600

attcctggag?agatctcgtg?ggatgtctct?tttgttactt?tgaacttctt?ggtgccagga????660

ctggtcattg?tgatcagtta?ctccaaaatt?ttacagatca?caaaggcatc?aaggaagagg????720

ctcacggtaa?gcctggccta?ctcggagagc?caccagatcc?gcgtgtccca?gcaggacttc????780

cggctcttcc?gcaccctctt?cctcctcatg?gtctccttct?tcatcatgtg?gagccccatc????840

atcatcacca?tcctcctcat?cctgatccag?aacttcaagc?aagacctggt?catctggccg????900

tccctcttct?tctgggtggt?ggccttcaca?tttgctaatt?cagccctaaa?ccccatcctc????960

tacaacatga?cactgtgcag?gaatgagtgg?aagaaaattt?tttgctgctt?ctggttccca???1020

gaaaagggag?ccattttaac?agacacatct?gtcaaaagaa?atgacttgtc?gattatttct???1080

ggctaa??????????????????????????????????????????????????????????????1086

<210>8

<211>361

<212>PRT

＜213〉artificial sequence

<220>

＜323〉novel sequences

<400>8

Met?Ser?Thr?Glu?Cys?Ala?Arg?Ala?Ala?Gly?Asp?Ala?Pro?Leu?Arg?Ser

1???????????????5???????????????????10??????????????????15

Leu?Glu?Gln?Ala?Asn?Arg?Thr?Arg?Phe?Pro?Phe?Phe?Ser?Asp?Val?Lys

20???????????????????25??????????????????30

Gly?Asp?His?Arg?Leu?Val?Leu?Ala?Ala?Val?Glu?Thr?Thr?Val?Leu?Val

35??????????????????40??????????????????45

Leu?Ile?Phe?Ala?Val?Ser?Leu?Leu?Gly?Asn?Val?Cys?Ala?Leu?Val?Leu

50??????????????????55??????????????????60

Val?Ala?Arg?Arg?Arg?Arg?Arg?Gly?Ala?Thr?Ala?Cys?Leu?Val?Leu?Asn

65??????????????????70??????????????????75??????????????????80

Leu?Phe?Cys?Ala?Asp?Leu?Leu?Phe?Ile?Ser?Ala?Ile?Pro?Leu?Val?Leu

85??????????????????90??????????????????95

Ala?Val?Arg?Trp?Thr?Glu?Ala?Trp?Leu?Leu?Gly?Pro?Val?Ala?Cys?His

100?????????????????105?????????????????110

Leu?Leu?Phe?Tyr?Val?Met?Thr?Leu?Ser?Gly?Ser?Val?Thr?Ile?Leu?Thr

115?????????????????120?????????????????125

Leu?Ala?Ala?Val?Ser?Leu?Glu?Arg?Met?Val?Cys?Ile?Val?His?Leu?Gln

130?????????????????135?????????????????140

Arg?Gly?Val?Arg?Gly?Pro?Gly?Arg?Arg?Ala?Arg?Ala?Val?Leu?Leu?Ala

145?????????????????150?????????????????155?????????????????160

Leu?Ile?Trp?Gly?Tyr?Ser?Ala?Val?Ala?Ala?Leu?Pro?Leu?Cys?Val?Phe

165?????????????????170?????????????????175

Phe?Arg?Val?Val?Pro?Gln?Arg?Leu?Pro?Gly?Ala?Asp?Gln?Glu?Ile?Ser

180?????????????????185?????????????????190

Ile?Cys?Thr?Leu?Ile?Trp?Pro?Thr?Ile?Pro?Gly?Glu?Ile?Ser?Trp?Asp

195?????????????????200?????????????????205

Val?Ser?Phe?Val?Thr?Leu?Asn?Phe?Leu?Val?Pro?Gly?Leu?Val?Ile?Val

210?????????????????215?????????????????220

Ile?Ser?Tyr?Ser?Lys?Ile?Leu?Gln?Ile?Thr?Lys?Ala?Ser?Arg?Lys?Arg

225?????????????????230?????????????????235?????????????????240

Leu?Thr?Val?Ser?Leu?Ala?Tyr?Ser?Glu?Ser?His?Gln?Ile?Arg?Val?Ser

245?????????????????250?????????????????255

Gln?Gln?Asp?Phe?Arg?Leu?Phe?Arg?Thr?Leu?Phe?Leu?Leu?Met?Val?Ser

260?????????????????265?????????????????270

Phe?Phe?Ile?Met?Trp?Ser?Pro?Ile?Ile?Ile?Thr?Ile?Leu?Leu?Ile?Leu

275?????????????????280?????????????????285

Ile?Gln?Asn?Phe?Lys?Gln?Asp?Leu?Val?Ile?Trp?Pro?Ser?Leu?Phe?Phe

290?????????????????295?????????????????300

Trp?Val?Val?Ala?Phe?Thr?Phe?Ala?Asn?Ser?Ala?Leu?Asn?Pro?Ile?Leu

305?????????????????310?????????????????315?????????????????320

Tyr?Asn?Met?Thr?Leu?Cys?Arg?Asn?Glu?Trp?Lys?Lys?Ile?Phe?Cys?Cys

325?????????????????330?????????????????335

Phe?Trp?Phe?Pro?Glu?Lys?Gly?Ala?Ile?Leu?Thr?Asp?Thr?Ser?Val?Lys

340?????????????????345?????????????????350

Arg?Asn?Asp?Leu?Ser?Ile?Ile?Ser?Gly

355?????????????????360

<210>9

<211>1038

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>9

atgagcagca?attcatccct?gctggtggct?gtgcagctgt?gctacgcgaa?cgtgaatggg?????60

tcctgtgtga?aaatcccctt?ctcgccggga?tcccgggtga?ttctgtacat?agtgtttggc????120

tttggggctg?tgctggctgt?gtttggaaac?ctcctggtga?tgatttcaat?cctccatttc????180

aagcagctgc?actctccgac?caattttctc?gttgcctctc?tggcctgcgc?tgatttcttg????240

gtgggtgtga?ctgtgatgcc?cttcagcatg?gtcaggacgg?tggagagctg?ctggtatttt????300

gggaggagtt?tttgtacttt?ccacacctgc?tgtgatgtgg?cattttgtta?ctcttctctc????360

tttcacttgt?gcttcatctc?catcgacagg?tacattgcgg?ttactgaccc?cctggtctat????420

cctaccaagt?tcaccgtatc?tgtgtcagga?atttgcatca?gcgtgtcctg?gatcctgccc????480

ctcatgtaca?gcggtgctgt?gttctacaca?ggtgtctatg?acgatgggct?ggaggaatta????540

tctgatgccc?taaactgtat?aggaggttgt?cagaccgttg?taaatcaaaa?ctgggtgttg????600

acagattttc?tatccttctt?tatacctacc?tttattatga?taattctgta?tggtaacata????660

tttcttgtgg?ctagacgaca?ggcgaaaaag?atagaaaata?ctggtagcaa?gacagaatca????720

tcctcagaga?gttacaaagc?cagagtggcc?aggagagaga?gaaaagcagc?taaaaccctg????780

ggggtcacag?tggtagcatt?tatgatttca?tggttaccat?atagcattga?ttcattaatt????840

gatgccttta?tgggctttat?aacccctgcc?tgtatttatg?agatttgctg?ttggtgtgct????900

tattataact?cagccatgaa?tcctttgatt?tatgctttat?tttacccatg?gtttaggaaa????960

gcaataaaag?ttattgtaac?tggtcaggtt?ttaaagaaca?gttcagcaac?catgaatttg???1020

ttttctgaac?atatataa?????????????????????????????????????????????????1038

<210>10

<211>345

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>10

Met?Ser?Ser?Asn?Ser?Ser?Leu?Leu?Val?Ala?Val?Gln?Leu?Cys?Tyr?Ala

1???????????????5???????????????????10??????????????????15

Asn?Val?Asn?Gly?Ser?Cys?Val?Lys?Ile?Pro?Phe?Ser?Pro?Gly?Ser?Arg

20??????????????????25??????????????????30

Val?Ile?Leu?Tyr?Ile?Val?Phe?Gly?Phe?Gly?Ala?Val?Leu?Ala?Val?Phe

35??????????????????40??????????????????45

Gly?Asn?Leu?Leu?Val?Met?Ile?Ser?Ile?Leu?His?Phe?Lys?Gln?Leu?His

50??????????????????55??????????????????60

Ser?Pro?Thr?Asn?Phe?Leu?Val?Ala?Ser?Leu?Ala?Cys?Ala?Asp?Phe?Leu

65??????????????????70??????????????????75??????????????????80

Val?Gly?Val?Thr?Val?Met?Pro?Phe?Ser?Met?Val?Arg?Thr?Val?Glu?Ser

85??????????????????90??????????????????95

Cys?Trp?Tyr?Phe?Gly?Arg?Ser?Phe?Cys?Thr?Phe?His?Thr?Cys?Cys?Asp

100?????????????????105?????????????????110

Val?Ala?Phe?Cys?Tyr?Ser?Ser?Leu?Phe?His?Leu?Cys?Phe?Ile?Ser?Ile

115?????????????????120?????????????????125

Asp?Arg?Tyr?Ile?Ala?Val?Thr?Asp?Pro?Leu?Val?Tyr?Pro?Thr?Lys?Phe

130?????????????????135?????????????????140

Thr?Val?Ser?Val?Ser?Gly?Ile?Cys?Ile?Ser?Val?Ser?Trp?Ile?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Leu?Met?Tyr?Ser?Gly?Ala?Val?Phe?Tyr?Thr?Gly?Val?Tyr?Asp?Asp?Gly

165?????????????????170?????????????????175

Leu?Glu?Glu?Leu?Ser?Asp?Ala?Leu?Asn?Cys?Ile?Gly?Gly?Cys?Gln?Thr

180?????????????????185?????????????????190

Val?Val?Asn?Gln?Asn?Trp?Val?Leu?Thr?Asp?Phe?Leu?Ser?Phe?Phe?Ile

195?????????????????200?????????????????205

Pro?Thr?Phe?Ile?Met?Ile?Ile?Leu?Tyr?Gly?Asn?Ile?Phe?Leu?Val?Ala

210?????????????????215?????????????????220

Arg?Arg?Gln?Ala?Lys?Lys?Ile?Glu?Asn?Thr?Gly?Ser?Lys?Thr?Glu?Ser

225?????????????????230?????????????????235?????????????????240

Ser?Ser?Glu?Ser?Tyr?Lys?Ala?Arg?Val?Ala?Arg?Arg?Glu?Arg?Lys?Ala

245?????????????????250?????????????????255

Ala?Lys?Thr?Leu?Gly?Val?Thr?Val?Val?Ala?Phe?Met?Ile?Ser?Trp?Leu

260?????????????????265?????????????????270

Pro?Tyr?Ser?Ile?Asp?Ser?Leu?Ile?Asp?Ala?Phe?Met?Gly?Phe?Ile?Thr

275?????????????????280?????????????????285

Pro?Ala?Cys?Ile?Tyr?Glu?Ile?Cys?Cys?Trp?Cys?Ala?Tyr?Tyr?Asn?Ser

290?????????????????295?????????????????300

Ala?Met?Asn?Pro?Leu?Ile?Tyr?Ala?Leu?Phe?Tyr?Pro?Trp?Phe?Arg?Lys

305?????????????????310?????????????????315?????????????????320

Ala?Ile?Lys?Val?Ile?Val?Thr?Gly?Gln?Val?Leu?Lys?Asn?Ser?Ser?Ala

325?????????????????330?????????????????335

Thr?Met?Asn?Leu?Phe?Ser?Glu?His?Ile

340?????????????????345

<210>11

<211>1020

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>11

atgatgccct?tttgccacaa?tataattaat?atttcctgtg?tgaaaaacaa?ctggtcaaat?????60

gatgtccgtg?cttccctgta?cagtttaatg?gtgctcataa?ttctgaccac?actcgttggc????120

aatctgatag?ttattgtttc?tatatcacac?ttcaaacaac?ttcatacccc?aacaaattgg????180

ctcattcatt?ccatggccac?tgtggacttt?cttctggggt?gtctggtcat?gccttacagt????240

atggtgagat?ctgctgagca?ctgttggtat?tttggagaag?tcttctgtaa?aattcacaca????300

agcaccgaca?ttatgctgag?ctcagcctcc?attttccatt?tgtctttcat?ctccattgac????360

cgctactatg?ctgtgtgtga?tccactgaga?tataaagcca?agatgaatat?cttggttatt????420

tgtgtgatga?tcttcattag?ttggagtgtc?cctgctgttt?ttgcatttgg?aatgatcttt????480

ctggagctaa?acttcaaagg?cgctgaagag?atatattaca?aacatgttca?ctgcagagga????540

ggttgctctg?tcttctttag?caaaatatct?ggggtactga?cctttatgac?ttctttttat????600

atacctggat?ctattatgtt?atgtgtctat?tacagaatat?atcttatcgc?taaagaacag????660

gcaagattaa?ttagtgatgc?caatcagaag?ctccaaattg?gattggaaat?gaaaaatgga????720

atttcacaaa?gcaaagaaag?gaaagctgtg?aagacattgg?ggattgtgat?gggagttttc????780

ctaatatgct?ggtgcccttt?ctttatctgt?acagtcatgg?acccttttct?tcactacatt????840

attccaccta?ctttgaatga?tgtattgatt?tggtttggct?acttgaactc?tacatttaat????900

ccaatggttt?atgcattttt?ctatccttgg?tttagaaaag?cactgaagat?gatgctgttt????960

ggtaaaattt?tccaaaaaga?ttcatccagg?tgtaaattat?ttttggaatt?gagttcatag???1020

<210>12

<211>339

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>12

Met?Met?Pro?Phe?Cys?His?Asn?Ile?Ile?Asn?Ile?Ser?Cys?Val?Lys?Asn

1???????????????5???????????????????10??????????????????15

Asn?Trp?Ser?Asn?Asp?Val?Arg?Ala?Ser?Leu?Tyr?Ser?Leu?Met?Val?Leu

20??????????????????25??????????????????30

Ile?Ile?Leu?Thr?Thr?Leu?Val?Gly?Asn?Leu?Ile?Val?Ile?Val?Ser?Ile

35??????????????????40??????????????????45

Ser?His?Phe?Lys?Gln?Leu?His?Thr?Pro?Thr?Asn?Trp?Leu?Ile?His?Ser

50??????????????????55??????????????????60

Met?Ala?Thr?Val?Asp?Phe?Leu?Leu?Gly?Cys?Leu?Val?Met?Pro?Tyr?Ser

65??????????????????70??????????????????75??????????????????80

Met?Val?Arg?Ser?Ala?Glu?His?Cys?Trp?Tyr?Phe?Gly?Glu?Val?Phe?Cys

85??????????????????90??????????????????95

Lys?Ile?His?Thr?Ser?Thr?Asp?Ile?Met?Leu?Ser?Ser?Ala?Ser?Ile?Phe

100?????????????????105?????????????????110

His?Leu?Ser?Phe?Ile?Ser?Ile?Asp?Arg?Tyr?Tyr?Ala?Val?Cys?Asp?Pro

115?????????????????120?????????????????125

Leu?Arg?Tyr?Lys?Ala?Lys?Met?Asn?Ile?Leu?Val?Ile?Cys?Val?Met?Ile

130?????????????????135?????????????????140

Phe?Ile?Ser?Trp?Ser?Val?Pro?Ala?Val?Phe?Ala?Phe?Gly?Met?Ile?Phe

145?????????????????150?????????????????155?????????????????160

Leu?Glu?Leu?Asn?Phe?Lys?Gly?Ala?Glu?Glu?Ile?Tyr?Tyr?Lys?His?Val

165?????????????????170?????????????????175

His?Cys?Arg?Gly?Gly?Cys?Ser?Val?Phe?Phe?Ser?Lys?Ile?Ser?Gly?Val

180?????????????????185?????????????????190

Leu?Thr?Phe?Met?Thr?Ser?Phe?Tyr?Ile?Pro?Gly?Ser?Ile?Met?Leu?Cys

195?????????????????200?????????????????205

Val?Tyr?Tyr?Arg?Ile?Tyr?Leu?Ile?Ala?Lys?Glu?Gln?Ala?Arg?Leu?Ile

210?????????????????215?????????????????220

Ser?Asp?Ala?Asn?Gln?Lys?Leu?Gln?Ile?Gly?Leu?Glu?Met?Lys?Asn?Gly

225?????????????????230?????????????????235?????????????????240

Ile?Ser?Gln?Ser?Lys?Glu?Arg?Lys?Ala?Val?Lys?Thr?Leu?Gly?Ile?Val

245?????????????????250?????????????????255

Met?Gly?Val?Phe?Leu?Ile?Cys?Trp?Cys?Pro?Phe?Phe?Ile?Cys?Thr?Val

260?????????????????265?????????????????270

Met?Asp?Pro?Phe?Leu?His?Tyr?Ile?Ile?Pro?Pro?Thr?Leu?Asn?Asp?Val

275?????????????????280?????????????????285

Leu?Ile?Trp?Phe?Gly?Tyr?Leu?Asn?Ser?Thr?Phe?Asn?Pro?Met?Val?Tyr

290?????????????????295?????????????????300

Ala?Phe?Phe?Tyr?Pro?Trp?Phe?Arg?Lys?Ala?Leu?Lys?Met?Met?Leu?Phe

305?????????????????310?????????????????315?????????????????320

Gly?Lys?Ile?Phe?Gln?Lys?Asp?Ser?Ser?Arg?Cys?Lys?Leu?Phe?Leu?Glu

325?????????????????330?????????????????335

Leu?Ser?Ser

<210>13

<211>1029

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>13

atgaccagca?atttttccca?acctgttgtg?cagctttgct?atgaggatgt?gaatggatct?????60

tgtattgaaa?ctccctattc?tcctgggtcc?cgggtaattc?tgtacacggc?gtttagcttt????120

gggtctttgc?tggctgtatt?tggaaatctc?ttagtaatga?cttctgttct?tcattttaag????180

cagctgcact?ctccaaccaa?ttttctcatt?gcctctctgg?cctgtgctga?cttcttggta????240

ggtgtgactg?tgatgctttt?cagcatggtc?aggacggtgg?agagctgctg?gtattttgga????300

gccaaatttt?gtactcttca?cagttgctgt?gatgtggcat?tttgttactc?ttctgtcctc????360

cacttgtgct?tcatctgcat?cgacaggtac?attgtggtta?ctgatcccct?ggtctatgct????420

accaagttca?ccgtgtctgt?gtcgggaatt?tgcatcagcg?tgtcctggat?tctgcctctc????480

acgtacagcg?gtgctgtgtt?ctacacaggt?gtcaatgatg?atgggctgga?ggaattagta????540

agtgctctca?actgcgtagg?tggctgtcaa?attattgtaa?gtcaaggctg?ggtgttgata????600

gattttctgt?tattcttcat?acctaccctt?gttatgataa?ttctttacag?taagattttt????660

cttatagcta?aacaacaagc?tataaaaatt?gaaactacta?gtagcaaagt?agaatcatcc????720

tcagagagtt?ataaaatcag?agtggccaag?agagagagga?aagcagctaa?aaccctgggg????780

gtcacggtac?tagcatttgt?tatttcatgg?ttaccgtata?cagttgatat?attaattgat????840

gcctttatgg?gcttcctgac?ccctgcctat?atctatgaaa?tttgctgttg?gagtgcttat????900

tataactcag?ccatgaatcc?tttgatttat?gctctatttt?atccttggtt?taggaaagcc????960

ataaaactta?ttttaagtgg?agatgtttta?aaggctagtt?catcaaccat?tagtttattt???1020

ttagaataa???????????????????????????????????????????????????????????1029

<210>14

<211>342

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>14

Met?Thr?Ser?Asn?Phe?Ser?Gln?Pro?Val?Val?Gln?Leu?Cys?Tyr?Glu?Asp

1???????????????5???????????????????10??????????????????15

Val?Asn?Gly?Ser?Cys?Ile?Glu?Thr?Pro?Tyr?Ser?Pro?Gly?Ser?Arg?Val

20??????????????????25??????????????????30

Ile?Leu?Tyr?Thr?Ala?Phe?Ser?Phe?Gly?Ser?Leu?Leu?Ala?Val?Phe?Gly

35??????????????????40??????????????????45

Asn?Leu?Leu?Val?Met?Thr?Ser?Val?Leu?His?Phe?Lys?Gln?Leu?His?Ser

50??????????????????55??????????????????60

Pro?Thr?Asn?Phe?Leu?Ile?Ala?Ser?Leu?Ala?Cys?Ala?Asp?Phe?Leu?Val

65??????????????????70??????????????????75??????????????????80

Gly?Val?Thr?Val?Met?Leu?Phe?Ser?Met?Val?Arg?Thr?Val?Glu?Ser?Cys

85??????????????????90??????????????????95

Trp?Tyr?Phe?Gly?Ala?Lys?Phe?Cys?Thr?Leu?His?Ser?Cys?Cys?Asp?Val

100?????????????????105?????????????????110

Ala?Phe?Cys?Tyr?Ser?Ser?Val?Leu?His?Leu?Cys?Phe?Ile?Cys?Ile?Asp

115?????????????????120?????????????????125

Arg?Tyr?Ile?Val?Val?Thr?Asp?Pro?Leu?Val?Tyr?Ala?Thr?Lys?Phe?Thr

130?????????????????135?????????????????140

Val?Ser?Val?Ser?Gly?Ile?Cys?Ile?Ser?Val?Ser?Trp?Ile?Leu?Pro?Leu

145?????????????????150?????????????????155?????????????????160

Thr?Tyr?Ser?Gly?Ala?Val?Phe?Tyr?Thr?Gly?Val?Asn?Asp?Asp?Gly?Leu

165?????????????????170?????????????????175

Glu?Glu?Leu?Val?Ser?Ala?Leu?Asn?Cys?Val?Gly?Gly?Cys?Gln?Ile?Ile

180?????????????????185?????????????????190

Val?Ser?Gln?Gly?Trp?Val?Leu?Ile?Asp?Phe?Leu?Leu?Phe?Phe?Ile?Pro

195?????????????????200?????????????????205

Thr?Leu?Val?Met?Ile?Ile?Leu?Tyr?Ser?Lys?Ile?Phe?Leu?Ile?Ala?Lys

210?????????????????215?????????????????220

Gln?Gln?Ala?Ile?Lys?Ile?Glu?Thr?Thr?Ser?Ser?Lys?Val?Glu?Ser?Ser

225?????????????????230?????????????????235?????????????????240

Ser?Glu?Ser?Tyr?Lys?Ile?Arg?Val?Ala?Lys?Arg?Glu?Arg?Lys?Ala?Ala

245?????????????????250?????????????????255

Lys?Thr?Leu?Gly?Val?Thr?Val?Leu?Ala?Phe?Val?Ile?Ser?Trp?Leu?Pro

260?????????????????265?????????????????270

Tyr?Thr?Val?Asp?Ile?Leu?Ile?Asp?Ala?Phe?Met?Gly?Phe?Leu?Thr?Pro

275?????????????????280?????????????????285

Ala?Tyr?Ile?Tyr?Glu?Ile?Cys?Cys?Trp?Ser?Ala?Tyr?Tyr?Asn?Ser?Ala

290?????????????????295?????????????????300

Met?Asn?Pro?Leu?Ile?Tyr?Ala?Leu?Phe?Tyr?Pro?Trp?Phe?Arg?Lys?Ala

305?????????????????310?????????????????315?????????????????320

Ile?Lys?Leu?Ile?Leu?Ser?Gly?Asp?Val?Leu?Lys?Ala?Ser?Ser?Ser?Thr

325?????????????????330?????????????????335

Ile?Ser?Leu?Phe?Leu?Glu

340

<210>15

<211>1062

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>15

atggagcaca?cgcacgccca?cctcgcagcc?aacagctcgc?tgtcttggtg?gtcccccggc?????60

tcggcctgcg?gcttgggttt?cgtgcccgtg?gtctactaca?gcctcttgct?gtgcctcggt????120

ttaccagcaa?atatcttgac?agtgatcatc?ctctcccagc?tggtggcaag?aagacagaag????180

tcctcctaca?actatctctt?ggcactcgct?gctgccgaca?tcttggtcct?ctttttcata????240

gtgtttgtgg?acttcctgtt?ggaagatttc?atcttgaaca?tgcagatgcc?tcaggtcccc????300

gacaagatca?tagaagtgct?ggaattctca?tccatccaca?cctccatatg?gattactgta????360

ccgttaacca?ttgacaggta?tatcgctgtc?tgccacccgc?tcaagtacca?cacggtctca????420

tacccagccc?gcacccggaa?agtcattgta?agtgtttaca?tcacctgctt?cctgaccagc????480

atcccctatt?actggtggcc?caacatctgg?actgaagact?acatcagcac?ctctgtgcat????540

cacgtcctca?tctggatcca?ctgcttcacc?gtctacctgg?tgccctgctc?catcttcttc????600

atcttgaact?caatcattgt?gtacaagctc?aggaggaaga?gcaattttcg?tctccgtggc????660

tactccacgg?ggaagaccac?cgccatcttg?ttcaccatta?cctccatctt?tgccacactt????720

tgggcccccc?gcatcatcat?gattctttac?cacctctatg?gggcgcccat?ccagaaccgc????780

tggctggtgc?acatcatgtc?cgacattgcc?aacatgctag?cccttctgaa?cacagccatc????840

aacttcttcc?tctactgctt?catcagcaag?cggttccgca?ccatggcagc?cgccacgctc????900

aaggctttct?tcaagtgcca?gaagcaacct?gtacagttct?acaccaatca?taacttttcc????960

ataacaagta?gcccctggat?ctcgccggca?aactcacact?gcatcaagat?gctggtgtac???1020

cagtatgaca?aaaatggaaa?acctataaaa?gtatccccgt?ga??????????????????????1062

<210>16

<21l>353

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>16

Met?Glu?His?Thr?His?Ala?His?Leu?Ala?Ala?Asn?Ser?Ser?Leu?Ser?Trp

1???????????????5???????????????????10??????????????????15

Trp?Ser?Pro?Gly?Ser?Ala?Cys?Gly?Leu?Gly?Phe?Val?Pro?Val?Val?Tyr

20??????????????????25??????????????????30

Tyr?Ser?Leu?Leu?Leu?Cys?Leu?Gly?Leu?Pro?Ala?Asn?Ile?Leu?Thr?Val

35??????????????????40??????????????????45

Ile?Ile?Leu?Ser?Gln?Leu?Val?Ala?Arg?Arg?Gln?Lys?Ser?Ser?Tyr?Asn

50??????????????????55??????????????????60

Tyr?Leu?Leu?Ala?Leu?Ala?Ala?Ala?Asp?Ile?Leu?Val?Leu?Phe?Phe?Ile

65??????????????????70??????????????????75??????????????????80

Val?Phe?Val?Asp?Phe?Leu?Leu?Glu?Asp?Phe?Ile?Leu?Asn?Met?Gln?Met

85??????????????????90??????????????????95

Pro?Gln?Val?Pro?Asp?Lys?Ile?Ile?Glu?Val?Leu?Glu?Phe?Ser?Ser?Ile

100?????????????????105?????????????????110

His?Thr?Ser?Ile?Trp?Ile?Thr?Val?Pro?Leu?Thr?Ile?Asp?Arg?Tyr?Ile

115?????????????????120?????????????????125

Ala?Val?Cys?His?Pro?Leu?Lys?Tyr?His?Thr?Val?Ser?Tyr?Pro?Ala?Arg

130?????????????????135?????????????????140

Thr?Arg?Lys?Val?Ile?Val?Ser?Val?Tyr?Ile?Thr?Cys?Phe?Leu?Thr?Ser

145?????????????????150?????????????????155?????????????????160

Ile?Pro?Tyr?Tyr?Trp?Trp?Pro?Asn?Ile?Trp?Thr?Glu?Asp?Tyr?Ile?Ser

165?????????????????170?????????????????175

Thr?Ser?Val?His?His?Val?Leu?Ile?Trp?Ile?His?Cys?Phe?Thr?Val?Tyr

180?????????????????185?????????????????190

Leu?Val?Pro?Cys?Ser?Ile?Phe?Phe?Ile?Leu?Asn?Ser?Ile?Ile?Val?Tyr

195?????????????????200?????????????????205

Lys?Leu?Arg?Arg?Lys?Ser?Asn?Phe?Arg?Leu?Arg?Gly?Tyr?Ser?Thr?Gly

210?????????????????215?????????????????220

Lys?Thr?Thr?Ala?Ile?Leu?Phe?Thr?Ile?Thr?Ser?Ile?Phe?Ala?Thr?Leu

225?????????????????230?????????????????235?????????????????240

Trp?Ala?Pro?Arg?Ile?Ile?Met?Ile?Leu?Tyr?His?Leu?Tyr?Gly?Ala?Pro

245?????????????????250?????????????????255

Ile?Gln?Asn?Arg?Trp?Leu?Val?His?Ile?Met?Ser?Asp?Ile?Ala?Asn?Met

260?????????????????265?????????????????270

Leu?Ala?Leu?Leu?Asn?Thr?Ala?Ile?Asn?Phe?Phe?Leu?Tyr?Cys?Phe?Ile

275?????????????????280?????????????????285

Ser?Lys?Arg?Phe?Arg?Thr?Met?Ala?Ala?Ala?Thr?Leu?Lys?Ala?Phe?Phe

290?????????????????295?????????????????300

Lys?Cys?Gln?Lys?Gln?Pro?Val?Gln?Phe?Tyr?Thr?Asn?His?Asn?Phe?Ser

305?????????????????310?????????????????315?????????????????320

Ile?Thr?Ser?Ser?Pro?Trp?Ile?Ser?Pro?Ala?Asn?Ser?His?Cys?Ile?Lys

325?????????????????330?????????????????335

Met?Leu?Val?Tyr?Gln?Tyr?Asp?Lys?Asn?Gly?Lys?Pro?Ile?Lys?Val?Ser

340?????????????????345?????????????????350

Pro

<210>17

<211>969

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>17

atggatccaa?ccgtcccagt?cttcggtaca?aaactgacac?caatcaacgg?acgtgaggag?????60

actccttgct?acaatcagac?cctgagcttc?acggtgctga?cgtgcatcat?ttcccttgtc????120

ggactgacag?gaaacgcggt?agtgctctgg?ctcctgggct?accgcatgcg?caggaacgct????180

gtctccatct?acatcctcaa?cctggccgca?gcagacttcc?tcttcctcag?cttccagatt????240

atacgttcgc?cattacgcct?catcaatatc?agccatctca?tccgcaaaat?cctcgtttct????300

gtgatgacct?ttccctactt?tacaggcctg?agtatgctga?gcgccatcag?caccgagcgc????360

tgcctgtctg?ttctgtggcc?catctggtac?cgctgccgcc?gccccacaca?cctgtcagcg????420

gtcgtgtgtg?tcctgctctg?gggcctgtcc?ctgctgttta?gtatgctgga?gtggaggttc????480

tgtgacttcc?tgtttagtgg?tgctgattct?agttggtgtg?aaacgtcaga?tttcatccca????540

gtcgcgtggc?tgattttttt?atgtgtggtt?ctctgtgttt?ccagcctggt?cctgctggtc????600

aggatcctct?gtggatcccg?gaagatgccg?ctgaccaggc?tgtacgtgac?catcctgctc????660

acagtgctgg?tcttcctcct?ctgcggcctg?cccttcggca?ttctgggggc?cctaatttac????720

aggatgcacc?tgaatttgga?agtcttatat?tgtcatgttt?atctggtttg?catgtccctg????780

tcctctctaa?acagtagtgc?caaccccatc?atttacttct?tcgtgggctc?ctttaggcag????840

cgtcaaaata?ggcagaacct?gaagctggtt?ctccagaggg?ctctgcagga?caagcctgag????900

gtggataaag?gtgaagggca?gcttcctgag?gaaagcctgg?agctgtcggg?aagcagattg????960

gggccatga????????????????????????????????????????????????????????????969

<210>18

<211>322

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>18

Met?Asp?Pro?Thr?Val?Pro?Val?Phe?Gly?Thr?Lys?Leu?Thr?Pro?Ile?Asn

1???????????????5???????????????????10??????????????????15

Gly?Arg?Glu?Glu?Thr?Pro?Cys?Tyr?Asn?Gln?Thr?Leu?Ser?Phe?Thr?Val

20??????????????????25??????????????????30

Leu?Thr?Cys?Ile?Ile?Ser?Leu?Val?Gly?Leu?Thr?Gly?Asn?Ala?Val?Val

35??????????????????40??????????????????45

Leu?Trp?Leu?Leu?Gly?Tyr?Arg?Met?Arg?Arg?Asn?Ala?Val?Ser?Ile?Tyr

50??????????????????55??????????????????60

Ile?Leu?Asn?Leu?Ala?Ala?Ala?Asp?Phe?Leu?Phe?Leu?Ser?Phe?Gln?Ile

65??????????????????70??????????????????75??????????????????80

Ile?Arg?Ser?Pro?Leu?Arg?Leu?Ile?Asn?Ile?Ser?His?Leu?Ile?Arg?Lys

85??????????????????90??????????????????95

Ile?Leu?Val?Ser?Val?Met?Thr?Phe?Pro?Tyr?Phe?Thr?Gly?Leu?Ser?Met

100?????????????????105?????????????????110

Leu?Ser?Ala?Ile?Ser?Thr?Glu?Arg?Cys?Leu?Ser?Val?Leu?Trp?Pro?Ile

115?????????????????120?????????????????125

Trp?Tyr?Arg?Cys?Arg?Arg?Pro?Thr?His?Leu?Ser?Ala?Val?Val?Cys?Val

130?????????????????135?????????????????140

Leu?Leu?Trp?Gly?Leu?Ser?Leu?Leu?Phe?Ser?Met?Leu?Glu?Trp?Arg?Phe

145?????????????????150?????????????????155?????????????????160

Cys?Asp?Phe?Leu?Phe?Ser?Gly?Ala?Asp?Ser?Ser?Trp?Cys?Glu?Thr?Ser

165?????????????????170?????????????????175

Asp?Phe?Ile?Pro?Val?Ala?Trp?Leu?Ile?Phe?Leu?Cys?Val?Val?Leu?Cys

180?????????????????185?????????????????190

Val?Ser?Ser?Leu?Val?Leu?Leu?Val?Arg?Ile?Leu?Cys?Gly?Ser?Arg?Lys

195?????????????????200?????????????????205

Met?Pro?Leu?Thr?Arg?Leu?Tyr?Val?Thr?Ile?Leu?Leu?Thr?Val?Leu?Val

210?????????????????215?????????????????220

Phe?Leu?Leu?Cys?Gly?Leu?Pro?Phe?Gly?Ile?Leu?Gly?Ala?Leu?Ile?Tyr

225?????????????????230?????????????????235?????????????????240

Arg?Met?His?Leu?Asn?Leu?Glu?Val?Leu?Tyr?Cys?His?Val?Tyr?Leu?Val

245?????????????????250?????????????????255

Cys?Met?Ser?Leu?Ser?Ser?Leu?Asn?Ser?Ser?Ala?Asn?Pro?Ile?Ile?Tyr

260?????????????????265?????????????????270

Phe?Phe?Val?Gly?Ser?Phe?Arg?Gln?Arg?Gln?Asn?Arg?Gln?Asn?Leu?Lys

275?????????????????280?????????????????285

Leu?Val?Leu?Gln?Arg?Ala?Leu?Gln?Asp?Lys?Pro?Glu?Val?Asp?Lys?Gly

290?????????????????295?????????????????300

Glu?Gly?Gln?Leu?Pro?Glu?Glu?Ser?Leu?Glu?Leu?Ser?Gly?Ser?Arg?Leu

305?????????????????310?????????????????315?????????????????320

Gly?Pro

<210>19

<211>969

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>19

atggattcaa?ccatcccagt?cttgggtaca?gaactgacac?caatcaacgg?acgtgaggag?????60

actccttgct?acaagcagac?cctgagcttc?acggggctga?cgtgcatcgt?ttcccttgtt????120

gcgctgacag?gaaacgcggt?tgtgctctgg?ctcctgggct?gccgcatgcg?caggaacgct????180

gtctccatct?acatcctcaa?cctggtcgcg?gccgacttcc?tcttccttag?cggccacatt????240

atatgttcgc?cgttacgcct?catcaatatc?cgccatccca?tctccaaaat?cctcagtcct????300

gtgatgacct?ttccctactt?tataggccta?agcatgctga?gcgccatcag?caccgagcgc????360

tgcctgtcca?tcctgtggcc?catctggtac?cactgccgcc?gccccagata?cctgtcatcg????420

gtcatgtgtg?tcctgctctg?ggccctgtcc?ctgctgcgga?gtatcctgga?gtggatgttc????480

tgtgacttcc?tgtttagtgg?tgctgattct?gtttggtgtg?aaacgtcaga?tttcattaca????540

atcgcgtggc?tggttttttt?atgtgtggtt?ctctgtgggt?ccagcctggt?cctgctggtc????600

aggattctct?gtggatcccg?gaagatgccg?ctgaccaggc?tgtacgtgac?catcctcctc????660

acagtgctgg?tcttcctcct?ctgtggcctg?ccctttggca?ttcagtgggc?cctgttttcc????720

aggatccacc?tggattggaa?agtcttattt?tgtcatgtgc?atctagtttc?cattttcctg????780

tccgctctta?acagcagtgc?caaccccatc?atttacttct?tcgtgggctc?ctttaggcag????840

cgtcaaaata?ggcagaacct?gaagctggtt?ctccagaggg?ctctgcagga?cacgcctgag????900

gtggatgaag?gtggagggtg?gcttcctcag?gaaaccctgg?agctgtcggg?aagcagattg????960

gagcagtga????????????????????????????????????????????????????????????969

<210>20

<211>322

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>20

Met?Asp?Ser?Thr?Ile?Pro?Val?Leu?Gly?Thr?Glu?Leu?Thr?Pro?Ile?Asn

1???????????????5???????????????????10??????????????????15

Gly?Arg?Glu?Glu?Thr?Pro?Cys?Tyr?Lys?Gln?Thr?Leu?Ser?Phe?Thr?Gly

20??????????????????25??????????????????30

Leu?Thr?Cys?Ile?Val?Ser?Leu?Val?Ala?Leu?Thr?Gly?Asn?Ala?Val?Val

35??????????????????40??????????????????45

Leu?Trp?Leu?Leu?Gly?Cys?Arg?Met?Arg?Arg?Asn?Ala?Val?Ser?Ile?Tyr

50??????????????????55??????????????????60

Ile?Leu?Asn?Leu?Val?Ala?Ala?Asp?Phe?Leu?Phe?Leu?Ser?Gly?His?Ile

65??????????????????70??????????????????75??????????????????80

Ile?Cys?Ser?Pro?Leu?Arg?Leu?Ile?Asn?Ile?Arg?His?Pro?Ile?Ser?Lys

85??????????????????90??????????????????95

Ile?Leu?Ser?Pro?Val?Met?Thr?Phe?Pro?Tyr?Phe?Ile?Gly?Leu?Ser?Met

100?????????????????105?????????????????110

Leu?Ser?Ala?Ile?Ser?Thr?Glu?Arg?Cys?Leu?Ser?Ile?Leu?Trp?Pro?Ile

115?????????????????120?????????????????125

Trp?Tyr?His?Cys?Arg?Arg?Pro?Arg?Tyr?Leu?Ser?Ser?Val?Met?Cys?Val

130?????????????????135?????????????????140

Leu?Leu?Trp?Ala?Leu?Ser?Leu?Leu?Arg?Ser?Ile?Leu?Glu?Trp?Met?Phe

145?????????????????150?????????????????155?????????????????160

Cys?Asp?Phe?Leu?Phe?Ser?Gly?Ala?Asp?Ser?Val?Trp?Cys?Glu?Thr?Ser

165?????????????????170?????????????????175

Asp?Phe?Ile?Thr?Ile?Ala?Trp?Leu?Val?Phe?Leu?Cys?Val?Val?Leu?Cys

180?????????????????185?????????????????190

Gly?Ser?Ser?Leu?Val?Leu?Leu?Val?Arg?Ile?Leu?Cys?Gly?Ser?Arg?Lys

195?????????????????200?????????????????205

Met?Pro?Leu?Thr?Arg?Leu?Tyr?Val?Thr?Ile?Leu?Leu?Thr?Val?Leu?Val

210?????????????????215?????????????????220

Phe?Leu?Leu?Cys?Gly?Leu?Pro?Phe?Gly?Ile?Gln?Trp?Ala?Leu?Phe?Ser

225?????????????????230?????????????????235?????????????????240

Arg?Ile?His?Leu?Asp?Trp?Lys?Val?Leu?Phe?Cys?His?Val?His?Leu?Val

245?????????????????250?????????????????255

Ser?Ile?Phe?Leu?Ser?Ala?Leu?Asn?Ser?Ser?Ala?Asn?Pro?Ile?Ile?Tyr

260?????????????????265?????????????????270

Phe?Phe?Val?Gly?Ser?Phe?Arg?Gln?Arg?Gln?Asn?Arg?Gln?Asn?Leu?Lys

275?????????????????280?????????????????285

Leu?Val?Leu?Gln?Arg?Ala?Leu?Gln?Asp?Thr?Pro?Glu?Val?Asp?Glu?Gly

290?????????????????295?????????????????300

Gly?Gly?Trp?Leu?Pro?Gln?Glu?Thr?Leu?Glu?Leu?Ser?Gly?Ser?Arg?Leu

305?????????????????310?????????????????315?????????????????320

Glu?Gln

<210>21

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>21

cagagctctg?gtggccacct?ctgtcc??????????????????????????????????????????26

<210>22

<211>25

<212>DMA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>22

ctgcgtccac?cagagtcacg?tctcc???????????????????????????????????????????25

<210>23

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>23

gtatgcctgg?ccacaatacc?tccagg??????????????????????????????????????????26

<210>24

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>24

gtttgtggct?aacggcacaa?aacacaattc?c????????????????????????????????????31

<210>25

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>25

ggtaccacaa?tgacaatcac?cagcgtcc????????????????????????????????????????28

<210>26

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>26

ggaacgtgag?gtacatgtgg?atgtgcagc???????????????????????????????????????29

<210>27

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>27

gcagtgtagc?ggtcaaccgt?gagcagg?????????????????????????????????????????27

<210>28

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>28

tgagcaggat?ggcgatccag?actgaggcgtgg????????????????????????????????????32

<210>29

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>29

gaggtacagc?tggcgatgct?gacag???????????????????????????????????????????25

<210>30

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>30

gtggccatga?gccaccctga?gctcc???????????????????????????????????????????25

<210>31

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>31

ggaatgtcca?ctgaatgcgc?gcgg????????????????????????????????????????????24

<210>32

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>32

agctcgccag?gtgtgagaaa?ctcgg???????????????????????????????????????????25

<210>33

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>33

gcgttatgag?cagcaattca?tccctgctgg??????????????????????????????????????30

<210>34

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>34

gtatcctgaa?cttcgtctat?acaactgc????????????????????????????????????????28

<210>35

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>35

ccctcaggaa?tgatgccctt?ttgccacaa???????????????????????????????????????29

<210>36

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>36

atccatgtgg?ttggtgcatg?tggttcgt????????????????????????????????????????28

<210>37

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>37

aaacaacaaa?cagcagaacc?atgaccagc???????????????????????????????????????29

<210>38

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>38

acatagagac?aagtgacatg?tgtgaaccac??????????????????????????????????????30

<210>39

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>39

ggtatgagac?cgtgtggtac?ttgagc??????????????????????????????????????????26

<210>40

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>40

gtggcagaca?gcgatatacc?tgtcaatgg???????????????????????????????????????29

<210>41

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>41

gcgctcatgg?agcacacgca?cgcccac?????????????????????????????????????????27

<210>42

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>42

gaggcagtag?ttgccacacc?tatgg???????????????????????????????????????????25

<210>43

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>43

catctggttt?gtgttcccag?gggcaccag???????????????????????????????????????29

<210>44

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>44

gacagtgttg?ctctcaaagt?cccgtctgac?tg???????????????????????????????????32

<210>45

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>45

ctgtttccag?ggtcatcaga?ctggg???????????????????????????????????????????25

<210>46

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>46

gcagcattgc?tctcaaagtc?ctgtctg?????????????????????????????????????????27

<210>47

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>47

Thr?Leu?Glu?Ser?Ile?Met

1???????????????5

<210>48

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>48

Glu?Tyr?Asn?Leu?Val

1???????????????5

<210>49

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>49

Asp?Cys?Gly?Leu?Phe

1???????????????5

<210>50

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>50

gatcaagctt?ccatggcgtg?ctgcctgagc?gagg?????????????????????????????????34

<210>51

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>51

gatcggatcc?ttagaacagg?ccgcagtcct?tcaggttcag?ctgcaggatg?gtg????????????53

<210>52

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>52

gtcctcactg?gtggccatgt?actcc???????????????????????????????????????????25

<210>53

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>53

ctgcgtccac?cagagtcacg?tctcc???????????????????????????????????????????25

<210>54

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>54

cttggatgtt?tgggctgccc?ttctgc??????????????????????????????????????????26

<210>55

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>55

gtttgtggct?aacggcacaa?aacacaattc?c????????????????????????????????????31

<210>56

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>56

ctgctcacgg?ttgaccgcta?cactgc??????????????????????????????????????????26

<210>57

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>57

gtggccatga?gccaccctga?gctcc???????????????????????????????????????????25

<210>58

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>58

cttcttctcc?gacgtcaagg?????????????????????????????????????????????????20

<210>59

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>59

cttcttctcc?gacgtcaagg?????????????????????????????????????????????????20

<210>60

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>60

cttcttctcc?gacgtcaagg?????????????????????????????????????????????????20

<210>61

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>61

caacggtctg?acaacctcct?????????????????????????????????????????????????20

<210>62

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>62

ttgctgtgat?gtggcatttt?g???????????????????????????????????????????????21

<210>63

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>63

caggaagccc?ataaaggcat?caa?????????????????????????????????????????????23

<210>64

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>64

acatcacctg?cttcctgacc?????????????????????????????????????????????????20

<210>65

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>65

ccagcatctt?gatgcagtgt?????????????????????????????????????????????????20

<210>66

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>66

ccatctccaa?aatcctcagt?c???????????????????????????????????????????????21

<210>67

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉novel sequences

<400>67

gctgttaaga?gcggacagga?aa??????????????????????????????????????????????22

Claims (40)

1. g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:2.

2. the g protein coupled receptor of claim 1, described acceptor is non-endogenous composition activated form acceptor.

3. plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:1.

4. host cell, described host cell comprises the plasmid of claim 3.

5. g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:4.

6. the g protein coupled receptor of claim 5, described acceptor is non-endogenous composition activated form acceptor.

7. plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:3.

8. host cell, described host cell comprises the plasmid of claim 7.

9. g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:6.

10. the g protein coupled receptor of claim 9, described acceptor is non-endogenous composition activated form acceptor.

11. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:5.

12. a host cell, described host cell comprises the plasmid of claim 11.

13. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:8.

14. the g protein coupled receptor of claim 13, described acceptor are non-endogenous composition activated form acceptor.

15. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:7.

16. a host cell, described host cell comprises the plasmid of claim 15.

17. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:10.

18. the g protein coupled receptor of claim 17, described acceptor are non-endogenous composition activated form acceptor.

19. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:9.

20. a host cell, described host cell comprises the plasmid of claim 19.

21. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:12.

22. the g protein coupled receptor of claim 21, described acceptor are non-endogenous composition activated form acceptor.

23. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:11.

24. a host cell, described host cell comprises the plasmid of claim 23.

25. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:14.

26. the g protein coupled receptor of claim 25, described acceptor are non-endogenous composition activated form acceptor.

27. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:13.

28. a host cell, described host cell comprises the plasmid of claim 27,

29. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:16.

30. the g protein coupled receptor of claim 29, described acceptor are non-endogenous composition activated form acceptor.

31. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:15.

32. a host cell, described host cell comprises the plasmid of claim 31.

33. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:18.

34. the g protein coupled receptor of claim 33, described acceptor are non-endogenous composition activated form acceptor.

35. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:17.

36. a host cell, described host cell comprises the plasmid of claim 35.

37. a g protein coupled receptor, described acceptor is by the amino acid sequence encode of SEQ.ID.NO.:20.

38. the g protein coupled receptor of claim 37, described acceptor are non-endogenous composition activated form acceptor.

39. a plasmid, described plasmid comprises the cDNA of a kind of carrier and SEQ.ID.NO.:19.

40. a host cell, described host cell comprises the plasmid of claim 39.

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