Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8967-8971, October 1993 Biochemistry Expression cloning of a human corticotropin-releasing- factor receptor (G protein-coupled receptor/yDthalamic-oitulitarv-aldrenocortlcal Iax/cyclic AMP/peptide receptor) RUOPING CHEN, KATHY A. LEWIS, MARILYN H. PERRIN*, AND WYLIE W. VALE The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037 Contributed by Wylie W. Vale, July 19, 1993

ABSTRACT Corticotropin-releasing factor (CRF) is the Here we present the expression cloning and functional principal neuroregulator of the hypothalamic-pituitary- expression of a cDNA encoding a CRF receptor from a adrenocortical axis and plays an important role In coordinating human Cushing corticotropic tumor. We also report a the endocrine, autonomic, and behavioral responses to variant of the receptor produced by alternative splicing.t and immune chaLenge. We report here the cloning of a cDNA coding for a CRF receptor from a human corticotropic tumor MATERIALS AND library. The cloned cDNA encodes a 415- protein METHODS comprisng seven putative membrane-saning domains and Is Library Construction. A human corticotropic adenoma structurally related to the calcitonin/vasoactive intestnal pep- removed from a patient with Cushing disease was generously tide/growth hormone-releasing hormone subfamlly of G pro- provided by Mary Lee Vance and the neurosurgery unit tein-coupled receptors. The receptor expressed in COS cells headed by Edward Laws (University of Virginia School of binds rat/human CRF with high affnity (Kd = 3.3 t 0.45 nM) Medicine). Total RNA was prepared by guanidine extraction and specficity and is functionally coupled to adenylate cyclase. and poly(A)+ RNA was obtained with oligotex-dt (Qiagen). The CRF antagonist a-heICRF-(9-41) inhibits the CRF- The corresponding cDNA was ligated into pcDNA1 (Invit- stimulated increase in intracellular cAMP. Northern blot anal- rogen), yielding a library of =1.5 x 106 primary recombi- ysis reveals that the CRF receptor is expressed in the rat nants, 80%6 of which contained inserts. A AZAP II (Strata- pituitary and as well as in the mouse AtT20 corticotropic gene) library was synthesized from the same human Cushing cells. We also describe an alternatively spliced form of the tumor cDNA by using Not I/EcoRI adapters. receptor which indudes an insert of 29 amino acids in the ffrst Expression Cloning. Expression screening of the pcDNAl intracellular loop. library was carried out as reported (19). Binding to trans- fected COS-M6 cells was assessed by incubation with 1 x 106 cpm of [125I]iodotyrosyl ovine CRF (Peninsula Laboratories) Corticotropin-releasing factor (CRF) (1) is a 41-amino acid [iodinated as described (16)] in 0.7 ml ofbinding buffer [0.1% peptide isolated from the by virtue ofits ability ovalbumin in HDB (25 mM Hepes/137 mM NaCl/5 mM to stimulate the production of adrenocorticotropic hormone KCI/0.7 mM Na2HPO4, pH 7.5)] for 90 min at 21°C. (ACTH) and other products of the Phage Library Screening. A 1.2-kb Pst I fraginent in the . Anatomic, functional, and pharmacologic coding region ofhct-0 was used to screen the AZAP II library studies have suggested numerous physiologic roles for CRF by standard methods. Double-stranded sequencing was per- within the brain, adrenals, , gastrointestinal tract, formed by the dideoxy chain-termination method with the , and sites of inflammation (2, 3). A subset of CRF Sequenase kit (United States Biochemical). neurons may coordinate many of the endocrine, autonomic, Radioreceptor Assay of Cloned Receptor. Two days after and behavioral responses to stress and may be involved in the transfection with 10-20 ug of plasmid DNA, the COS-M6 pathophysiology of affective disorders. Moreover, CRF ap- cells were washed with HDB and detached by incubation pears to be a key intermediary in the communication between with 0.5 mM EDTA in HDB for 15 min at 21°C. The cells were the immune systems and the central nervous and endocrine washed twice with HDB and homogenized in 5% sucrose. systems (4-6). The neuropeptide and its binding sites are The homogenate was centrifuged at 600 x g for 5 min, after broadly distributed throughout the central nervous system as which the supernatant was centrifuged at 40,000 x g for 20 well as in some peripheral sites, reflecting the role CRF may min. The resulting membrane homogenate, P2, was resus- play in endocrine, autonomic, and behavioral aspects of the pended at 1-4 mg/ml in 10%o sucrose and used in the binding stress response, as well as in such pathophysiologic states as assay as described (16). Dissociation constants were calcu- depression, anxiety, and anorexia nervosa. lated from relative potencies by using the ALLFIT program CRF exerts its effects by initially binding to a membrane- (20) and determined from competitive displacement assays bound receptor. High-affinity CRF binding sites have been with rat/human CRF (r/hCRF) as the standard. characterized in the rat (7) and human (8) pituitary, rat (9) and cAMP Accumulation Assay. COS-M6 cells were transfected human (10) brain, and on AtT20 cells (11), a mouse corti- as described (21). At least 2 hr before treatments, the medium cotropic tumor cell line. The CRF receptor is coupled to was changed to contain 0.1% serum. After a 30-min prein- adenylate cyclase (12-15), probably through a GTP-binding cubation in medium with or without 0.1 mM 3-isobutyl-1- protein (16). Although a high-affinity soluble binding protein for CRF was recently isolated from human plasma (17) and Abbreviations: ACTH, adrenocorticotropic hormone; CRF, corti- cloned (18), the membrane receptor for this important neu- cotropin-releasing factor; r/hCRF, rat/human CRF; GnRH, - ropeptide was not identified. otropin-releasing hormone; GRF, growth hormone-releasing factor; IBMX, 3-isobutyl-1-methylxanthine; VIP, vasoactive intestinal pep- tide. The publication costs ofthis article were defrayed in part by page charge *To whom correspondence should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank database (accession nos. L23332 and L23333). 8967 Downloaded by guest on October 1, 2021 8968 Biochemistry: Chen et aL Proc. Natl. Acad. Sci. USA 90 (1993) methylxanthine (IBMX), test substances were added and new putative phosphorylation sites are introduced in the incubated for 30 min at 37°C. The cells were extracted as alternatively spliced form of the receptor. described (12). cAMP levels were determined in duplicate Binding Characteristics ofthe Cloned Receptor. The cloned from triplicate wells with an RIA kit (Biomedical Technolo- CRF receptor hct-0 exhibited appropriate pharmacologic gies, Stoughton, MA). specificity, with high affinity for r/hCRF (Fig. 3) [Kd = 3.3 Northern Blot Analysis. Poly(A)+ RNA was size-frac- ± 0.45 nM (n = 4)], ovine CRF [Kd = 8.3 (4-16) nM (n = 1)], tionated in a denaturing formaldehyde/agarose gel and trans- and for the antagonist (2) a-helCRF-(9-41) [Kd = 1.0 ± 0.10 ferred to nitrocellulose paper. The blot was prehybridized for nM (n = 2)] and low affinity for the biologically impotent 15 min at 68°C in the QuikHyb hybridization solution (Strat- analog [Ala14]ovine CRF [Kd> 300 nM (n = 2)]. hGRF-(l- agene) plus salmon sperm DNA (100 ,ug/ml) and hybridized 40)-OH, salmon calcitonin, and VIP were inactive in the in the same solution at 68°C for 30 min to a 1.3-kb Pst I cDNA receptor assay. Cells transfected with the rat gonadotropin- fragment labeled by random priming (Amersham). The Pst I releasing hormone (GnRH) receptor (21) showed no binding fragment includes the majority of the coding region of CRF- of CRF. The CRF-R1 clone displayed similar affinity to that R1. The blot was washed twice at 21°C in 2x SSPE/0.1% SDS of hct-0 [Kd = 3.8 ± 0.20 nM (n = 1)] for r/hCRF. for 15 min and once at 60°C in 0.2x SSPE/0.1% SDS for 30 Intracellular cAMP Accumulation in Cells Transfected with min (lx SSPE is 0.18 M NaCl/10 mM sodium phosphate, pH the Cloned Receptor. Consistent with earlier observations 7.4/1 mM EDTA). implicating adenylate cyclase in the transduction mechanism ofthe native CRF receptor (12-15), COS-M6 cells transfected with the hct-0 clone responded to r/hCRF with 10- to 20-fold RESULTS increases in intracellular cAMP, whereas COS-M6 cells of transfected with the rat GnRH receptor did not respond at all. Cloning CRF-Rl. From a screen of =400,000 indepen- The response was dose-dependent (Fig. 4A), and the half- dent clones, 1 positive pool was found. A subdivision of this maximal dose of =3 nM CRF corresponded to the Kd pool yielded a single clone, hct-0, that expressed high-affinity obtained from the radioreceptor assay and to the EC50 binding when transfected into COS-M6 cells. The sequence determined for CRF-stimulated cAMP production by rat ofthis and ofsubsequent clones revealed the inclusion oftwo anterior pituitary cells (12). hGRF-(1-40)-OH, salmon calci- putative intronic sequences separated by an exon. To obtain tonin, and VIP had no effect on cAMP levels in the cells other clones that did not include the introns, a phage library transfected with the cloned receptor (Fig. 4B). The CRF created from the same tumor was screened at high stringency antagonist a-helCRF-(9-41) blocked the induction of cAMP with a probe generated from the original clone. Three posi- by r/hCRF (Fig. 5). A comparison ofthe two clones hct-0 and tives were identified, two of which, CRF-R1 and CRF-R2, CRF-R1 showed the same dose-response of r/hCRF- were sequenced and found to lack introns. stimulated intracellular cAMP accumulation and the same Structural Characteristics of the CRF Receptor. Hydropa- lack of response to GRF, calcitonin, and VIP. thy analysis (22) ofthe CRF-R1 amino acid sequence, shown Tissue Distribution of the Cloned Receptor. Autoradio- in Fig. 1, indicates eight hydrophobic regions of :20 amino graphic and biochemical techniques have detected the native acids corresponding to a possible signal peptide at the N CRF receptor in the pituitary (7) and various brain regions terminus and seven putative transmembrane domains. (9). Northern blot analysis of poly(A)+ RNA using the CRF-R1 contains a 2584-bp insert with a 1245-bp open reading CRF-R1 probe revealed the presence ofa 2.7-kb mRNA in rat frame encoding a 415-amino acid protein. Sequence analysis brain and pituitary and in mouse AtT20 corticotropic cells (Fig. 2) shows that the CRF receptor appears to belong to the (Fig. 6). calcitonin/vasoactive intestinal peptide (VIP)/growth hor- mone-releasing factor (GRF) family of G protein-coupled receptors (23-29). At the amino acid level, the percent DISCUSSION identity to the human GRF receptor is 31%, to the rat secretin Using an expression cloning approach we have isolated a receptor, 32%, to the rat VIP receptor, 30%, and to the CRF receptor cDNA from a human Cushing tumor. When the porcine calcitonin receptor, 25%. However, there is signifi- binding assay was used to monitor receptor expression it was cantly greater homology throughout the transmembrane do- largely masked by high nonspecific binding of the CRF mains, so that the CRF receptor is 44%, 46%, 43%, and 38% radioligand. Substitution of ovalbumin for bovine serum identical with the GRF, secretin, VIP, and calcitonin recep- albumin was found to reduce the nonspecific binding of tors, respectively, in those regions. 125I-labeled ovine CRF, allowing positive cells to be detected. Five potential N-glycosylation sites (Asn-Xaa-Ser/Thr) in Based on a hydropathy analysis, the receptor has a hydro- the N terminus are shown in Fig. 2. Also shown are potential phobic domain at the N terminus that may encode a signal protein kinase C phosphorylation sites in the first and second peptide. There are seven other hydrophobic regions charac- intracellular loops and in the C-terminal tail, as well as a teristic ofmembrane-spanning domains. After removal ofthe casein kinase II and phosphorylation site in putative signal peptide, the receptor is predicted to have a the third intracellular loop (30). The third clone, CRF-R2, is molecular weight of 44,000. an alternatively spliced form of the receptor in which 29 There are six conserved cysteines in the calcitonin/VIP/ amino acids are inserted into the first intracellular loop. No GRF family ofreceptors. These cysteines may be involved in the folding ofthe extracellular domain allowing binding ofthe ligands. The conservation of all these cysteines in the CRF receptor may serve the same purpose or may merely reflect common ancestry. Although r/hCRF binds with high affinity to the soluble CRF-binding protein (CRF-BP) (17, 18) it has no sequence homology with the cloned receptor. Because the affinity of ovine CRF is nearly 100 times lower for the human CRF-BP 50 100 150 200 250 300 350 400 than for the cloned receptor, there must be different struc- Amino acid residue tural determinants for binding CRF ligands. This difference in specificity was exploited in the expression cloning by using FIG. 1. Kyte-Doolittle (22) hydrophobicity analysis of CRF-R1. an ovine analog of CRF as the radioligand to avoid detecting Downloaded by guest on October 1, 2021 Biochemistry: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 8969

- - -- GG H P 0 L R L V K A L LL L G L N P V S A S L Q D Q Hl- - - |C E S L 33 hCRF-Rl - hGRF-R MID R R M W G A HIVIF C|S P L P T V L G H M H P E ------ICIDF I 31 rSecret in-R |IILSTMRP IRLISLLIL RWLWL T K AKH T V G V P P R L - - - - ICIDV R 36 rVIP-R KJR P P S P P H V R W L C VLJAjA L A C A LR P A G S Q A A S P Q H E IC jYY 40 v v hCRF-Rl IS L A S7| ------INISI- la LQC N A S V D L I GI 52 hGRF-R T Q L R E D E S A C L Q A A E E - - - - M P ]L | -CP D|TwDG L - 63 rSecret in-R R V L L E E R A H C L Q Q L S K E K K G A L G P E T A S I- - ICE G L W IDIN M - 74 rVIP-R Q QI E I Q R Q Q C L E E A Q L E - - - E [ET [-T iCS- K M W DN L - 71

v hCRF-Rl T C W P R S P A G L V V R P C P A F F Y G V R Y N T T N N - - R E C L A 89 hGRF-R LC W TAGGEVTLPDP - - S HFSSE S -rTA V KIRIDC T I 0-S L F R N T Q 98 rSecret in-R ,C P S A P A ERT V QC PK - - L M L S N K N- 109 rVIP-R ITC W P TT BR |GQA VIL D IC P L I E--Q L F A P I H - a Y N I S RS CT E 107

hCRF-Rl __W_A_A - ( - C Q E I L N E E K K|- S K V H - Y H V A V I I 122 hGRF-R T O Wi S E P F P-P P V A C - - P V - P L E L L A E E E S - - Y UK I 131 rSecret in-R D a w S E TF P R P - D L A C G V N I - N N S F N E R R HA - - Y L L K L K V 144 rVIP-R E 0 - W S Q L E P G P Z]H I A C G L N D R A S S L D E Q Q Q T K F Y N T V - K T 146

hCRF-Rl INY - IL a H C I S L V A L L VA F V L F L R L R S I R C L R N II N W N L I S AI 161 hGRF-R I Y T V O S V A L E3V A I T I L VA LR R L H C P R N V T Q LF T T 171 rSecret in-R M Y T V 0Y SS L A M L L V A L S I L CS FR R L H C T R N YIN M H L F V S 184 rVIP-R G Y T I 0 JS V aS|MA I L S L F R K L H C T R N Y IIN M H L F M S 185

hCRF-Rl IP L RN A T W P V V Q L T MI - - a P E V H l- - - |S N V G W C R L V T AA Y N I 196 hGRF-R IL K A G R V|F|L KD A A L F H D D T D H C S F TV L - C K VS VIA ASH 210 rSecret in-R F I L A L S N|FI K D A V L F S SID DuT Y C D A H K V - CIK L VIM I F F 223 rVIP-R F I L R A T A V I K D M A L F N G[ I D H C S E A S V G - CK AA V V F FQ 224 -3 hCRF-Rl IY F H V T N FF N M F G * G C YL H T A I V L T Y S T D R L R K - |WM F I C IaI 235 hGRF-R F AFAMNFS~WLLaT M FNw LLA|EYLYLIHCLLASITSPSRLLAS R It A~JLLA0J 249 rSecret in-R YC I M AN WL LV|LY L H TL L A I S F F S E R|- K Y LQA I3V L.L 262 rVIP-R MCV MAN F F L V MGG|L YLYT L LA V SFF SE R - KY F JG YILL a01 263 -4 hCRF-Rl W O V P F P I I V A W A I GKL Y Y D N E K C W F G K R P G V Y T D Y I Y G P 275 hGRF-R VOL P V L F T G TWV CIKLA F E D I AICVW-DL D D T S P Y WWW PK|G 288 rSecret in-R |OWO P A I F V A L|W A IT R H F L E WT G C W|-D I N A N A S V W W V I R GP 301 rVIP-R VW V PS V FWET I[JT VV R I1F ED F G CW- D TI INS S LW WI K 302 -5 00 hCRF-Rl IMIL V L L I Jl F I F L F N X V RIt L M T K L R A S I T S |- - |ETIQY R K A Vl 313 hGRF-R VLS V G VIG LF LN IR LXS RL|ER P AQ GUSL HTQ W R L S 328 rSecret in-R V I L S I L I N * I F|F I N I L R I L R |A L P T Q E R G SITN H Y|K R L A 341 rVIP-R ILLS I LVN VL F I I R L VQR L R P P D I G K ND S S S R L A 342 6 - hCRF-Rl K A T L V L L P L L O S T Y M L F F V N P G E D E V S R V V F I Y F N S F L E S 353 N G - - L 366 hGRF-R KT LIPLP LF YI INFLPD A G L G I R L P L E L KST FL--LIG- - rSecret in-R |K|S |T LFLLIL L |L|I |P L|F 1°OIH1YIVIVANFSLPEDAEQS|H |YI V 7 A F S |P E D - A M E V Q L E]F EL A JG | 378 rVIP-R KS T L|L L IP L|F G1 IH V M V A F F P D N F K A Q V K M V F E L V - - V G 380

hCRF-Rl Q a FF V S V F Y C F L N S Z V R S A I R K R W H R W_Q_D_K H|-ls I R A R V A 392 hGRF-R |FQ OF VA I L CVLNIVRTEI S KHG - HIDPEE - LLP AWRT 404 rSecret in-R F Q 0LV V AWL Y C F L N|G| V|Q L E V QWKVK R H L Q E F P LWP V A F 418 rVIP-R Q ° F|V V A I L Y C * L N G 3 V Q A E L E]R K W R H L Q G V L G W S S K S 420

hCRF-RlhCRF-Rl R A M SI PP-PTT S P T RVIFII-KV S F H S------IKQSTAV|41Q S TA V 415 hGRF-R L T S M C 423 rSecretin-R N N S F - - S N A LJN G P T H s-T K A S T E Q S R S - r]P R A S I I 449 rVIP-R Q H P W -G G S N G A T C S T Q V S M L T R V S P S A R RS SSFQAEVSLL1 459 FIG. 2. Sequence comparison ofthe human (h) CRF receptor clone CRF-R1 with human GRF, rat (r) secretin, and rat VIP receptors. Putative transmembrane regions 1-7 are overlined. Potential glycosylation sites (v) and phosphorylation sites for protein kinase C (*), casein kinase II (o), and protein kinase A (o) are indicated in the CRF-R1 sequence. The position ofthe 29-amino acid insertion resulting from alternative splicing is indicated by the arrow. The sequence of the 29-amino acid insertion is PGCTHWGDQADGALEVGAPWSGAPFQVRR. the CRF-BP. It is interesting that some brain regions and pituitary and brain CRF receptors are glycoproteins that, after anterior pituitary corticotropes express both the CRF recep- deglycosylation, display an apparent molecular weight of tor and the CRF-BP (31). The functional relationship between 40,000-45,000. This is in agreement with the predicted mo- the binding protein and the receptor remains to be deter- lecular weight of the receptor deduced from the cDNA se- mined. quence. At this time, we cannot rule out the existence of The CRF receptor has five potential glycosylation sites. additional receptor types for CRF or related peptides in the Their presence may explain the fact that the apparent molec- brain or elsewhere. ular size of the pituitary receptor seen in crosslinking exper- There are potential phosphorylation sites in each of the iments (32) is larger than that predicted from the size of the three intracellular loops, as well as in the C terminus. The cloned receptor. Further, it has been shown (33) that both the protein kinase C phosphorylation sites may be involved in Downloaded by guest on October 1, 2021 8970 Biochemistry: Chen et aL Proc. Natl. Acad. Sci. USA 90 (1993)

x 2 n0 0r.a) E E C) CL 0 0.01 0.1 1 10 100 Cu 1 T r/hCRF, nM .5 C)E 40- B 13~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 CL

30 I 0 0.01 0.1 1 10 100 1000 r/hCRF, nM FIG. 3. Competitive displacement by r/hCRF of 125I-labeled 20- [Nle21,Tyr32]ovine CRF bound to membranes prepared from COS-M6 cells transfected with hct-0 (m) or rat GnRH receptor (o). Data are from one representative experiment repeated at least four 10 - times. modulating the as evidenced by the potentiation of CRF-stimulated cAMP accumulation and O I lIriI I ACTH release (34, 35) by activators ofprotein kinase C, such 'I& & .A\I kp as phorbol esters and . Protein kinase A phos- e phorylation of residues in the third intracellular loop and the C terminus has been implicated in the desensitization and down-regulation of other G protein-coupled receptors (36). Whether the phosphorylation sites in the CRF receptor are FIG. 4. Stimulation of intracellular cAMP accumulation in COS-M6 cells transfected with hct-0. Data are from one represen- involved in the observed CRF desensitization (37) remains to tative experiment repeated at least three times. (A) Stimulation by be investigated. Consistent with the coupling of the CRF r/hCRF of cAMP accumulation in cells pretreated (m) or not treated receptor to the adenylate cyclase system, the third intracel- (o) with IBMX. (B) cAMP accumulation aftertreatment with r/hCRF lular loop of CRF-R1 contains a sequence similar to the (100 nM) or non-CRF peptides (100 nM) in cells pretreated with G,-activating region found in the third intracellular loop ofthe IBMX. P2-adrenergic receptor (38). Our original clone contains an in-frame insertion at a point itary and brain as the primary target sites of CRF action and identified as a consensus sequence for an intron-exon splice the identification of CRF receptors in these tissues. junction; consequently, the CRF receptor gene contains at In conclusion, we have cloned from a human pituitary least two introns. The gene for the GRF receptor contains tumor a cDNA encoding a 415-amino acid protein that dis- many introns within the coding region (23). Alternative plays the functional properties of a physiological CRF re- splicing events produce an insertion of 41 amino acids in the third intracellular loop of the rat GRF receptor (23, 25), and 3n a deletion in the first extracellular loop of the mouse GRF 0) receptor (25). In the only alternative form of the CRF 25 - receptor identified thus far, 29 amino acids are inserted in the first intracellular loop. It is possible that alternative RNA 20 - processing may generate two different forms of the CRF E' receptor. The functional significance of these forms remains CL02115 - to be elucidated. Because the CRF receptor was cloned from a human cu 1 pituitary tumor, the receptor may include mutations com- C.) pared to the normal receptor. However, the affinity and specificity of the cloned receptor are similar to those of the C normal rat pituitary receptors, as are the transduction of o CRF-stimulated accumulation of intracellular cAMP and the _ antagonism by the potent CRF antagonist a-helCRF-(9-41). 0 0.1 1 10 When expression of the cloned CRF receptor was exam- r/hCRF, nM ined by Northern analysis (Fig. 6), a major transcript of 2.7 FIG. 5. Inhibition by a-helCRF-(9-41) of r/hCRF-stimulated kb was detected in the mouse corticotropic cell line AtT20 intracellular cAMP accumulation in COS-M6 cells transfected with and in rat brain and pituitary, but not in rat heart. These hct-0 and pretreated with IBMX. r/hCRF was added without (open results are consistent with the previous studies of the pitu- bars) or with (filled bars) 2 IAM a-helCRF-(9-41). Downloaded by guest on October 1, 2021 Biochemistry: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 8971 7. Wynn, P. C., Aguilera, G., Morell, J. & Catt, K. J. (1983) Biochem. Biophys. Res. Commun. 110, 602-608. moI 8. DeSouza, E. B., Perrin, M. H., Whitehouse, P. J., Rivier, J., < m LXI Vale, W. W. & Kuhar, M. J. (1985) 40, 419-422. 9. DeSouza, E. B., Perrin, M. H., Insel, T. R., Rivier, J., Vale, W. W. & Kuhar, M. J. (1984) Science 224, 1449-1451. 10. DeSouza, E. B., Whitehouse, P. J., Kuhar, M. J., Price, D. L. & Vale, W. W. (1986) Nature (London) 319, 593-595. 11. Rosendale, B. E., Jarrett, D. B. & Robinson, A. G. (1987) 120, 2357-2366. 12. Bilezikjian, L. M. & Vale, W. W. (1983) Endocrinology 113, 657-662. 28S - 13. Labrie, F., Veilleux, R., Lefevre, G., Coy, D. H., Sueiras- Diaz, J. & Schally, A. V. (1982) Science 216, 1007-1008. 2.7Kb - 14. Reisine, T., Heisler, S., Hook, V. Y. & Axelrod, J. (1982) 18S- Biochem. Biophys. Res. Commun. 108, 1251-1257. 15. Aguilera, G., Harwood, J. P., Wilson, J. X., Morell, J., Brown, J. H. & Catt, K. J. (1983) J. Biol. Chem. 258, 8039-8045. 16. Perrin, M. H., Haas, Y., Rivier, J. E. & Vale, W. W. (1986) Endocrinology 118, 1171-1179. 17. Behan, D. P., Linton, E. A. & Lowry, P. J. (1989) J. Endo- crinol. 122, 23-31. 18. Potter, E., Behan, D. P., Fischer, W. H., Linton, E. A., Lowry, P. J. & Vale, W. W. (1991) Nature (London) 349, 423-426. FiG. 6. Expression of CRF receptor as shown by Northern blot 19. Mathews, L. & Vale, W. (1991) Cell 65, 973-982. analysis of poly(A)+ RNA (4 ug) from AtT20 cells and rat brain, 20. DeLean, A., Munson, P. J. & Rodbard, D. (1978) Am. J. pituitary, and heart. The autoradiograph shown is a 5-hr exposure. Physiol. 235, E97-E102. Positions of28S and 18S rRNA and size ofthe mature CRF-R mRNA 21. Perrin, M. H., Bilezikjian, L. M., Hoeger, C., Donaldson, are indicated. C. J., Rivier, J., Haas, Y. & Vale, W. W. (1993) Biochem. Biophys. Res. Commun. 191, 1139-1144. ceptor-namely, high-affinity CRF binding and transduction 22. Kyte, J. & Doolittle, R. (1982) J. Mol. Biol. 157, 105-132. of the accumulation of cAMP in response to CRF. The 23. Mayo, K. E. (1992) Mol. Endocrinol. 6, 1734-1744. sequence confirms that the receptor belongs to a G protein- 24. Gaylinn, B. D., Harrison, J. K., Zysk, J. R., Lyons, C. E., coupled receptor family characterized by seven transmem- Lynch, K. R. & Thorner, M. 0. (1993) Mol. Endocrinol. 7, brane domains. The cloning of the human CRF receptor fills 77-84. a major gap in our knowledge ofthe signaling molecules ofthe 25. Lin, C., Lin, S.-C., Chang, C.-P. & Rosenfeld, M. G. (1992) axis and Nature (London) 360, 765-768. hypothalamic-pituitary-adrenocortical provides 26. Lin, H. Y., Tonja, L. H., Flannery, M. S., Aruffo, A., Kaji, tools to further explore the mode of action of CRF and its E. H., Gorn, A., Kolakowski, L. F., Jr., Lodish, H. F. & target cells. Goldring, S. R. (1991) Science 254, 1022-1024. 27. Ishihara, T., Nakamura, S., Kaziro, Y., Takahashi, T., Taka- R.C., K.A.L., and M.H.P. should be considered co-first authors hashi, K. & Nagata, S. 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