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Molecular Endocrinology 11 (7): 980-985
Copyright © 1997 by The Endocrine Society

Localization of Ligand-Binding Domains of Human Corticotropin-Releasing Factor Receptor: A Chimeric Receptor Approach

Chen W. Liaw, Dimitri E. Grigoriadis, Timothy W. Lovenberg, Errol B. De Souza and Richard A. Maki

Departments of Molecular Neurobiology and Neuroscience, Neurocrine Biosciences, Inc., San Diego, California 92121


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175–178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266–268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRF is a 41-amino acid (aa) peptide (1) that functions as a neurohormone to integrate the electrophysiological, autonomic, and behavioral responses to stress (2, 3, 4). Two CRFR subtypes, CRFR1 and CRFR2, have recently been cloned and characterized (5, 6, 7, 8, 9, 10, 11, 12, 13). For the CRFR2, two alternatively spliced forms (CRFR2{alpha} and CRFR2ß) with different 5'-coding sequences have been identified (9). Both receptors belong to the superfamily of G protein-coupled receptors (GPCR) characterized by the presence of seven transmembrane domains.

For GPCRs that bind small molecule ligands such as catecholamines and acetylcholine, extensive mutagenesis studies have localized the ligand-binding sites to the transmembrane domains (14, 15, 16, 17). Since some of the peptide ligands are considerably larger, it is conceivable that the ligand-binding pocket of their receptors might extend beyond the transmembrane regions. Indeed, it has been shown that both the extracellular segments as well as the transmembrane domains of the neurokinin receptors are required for the high affinity binding of substance P and neurokinins (18, 19, 20).

Human CRFR1 and CRFR2{alpha} are 70% identical in aa sequence. The 30% difference in primary sequence translates into more than 2 orders of magnitude difference in EC50 value in stimulating intracellular cAMP for the peptide ligand rat/human CRF (r/hCRF), i.e. the EC50 values are ~0.16 and ~60 nM for CRFR1 and CRFR2{alpha}, respectively. Those different aa residues between CRFR1 and CRFR2{alpha} that cause such a shift in EC50 for r/hCRF presumably are important for the binding of r/hCRF and/or receptor activation. As there is a fairly good correlation between shift in EC50 value and change in binding affinity for CRFR1 and CRFR2{alpha}, the change in EC50 value is more likely to reflect a change in binding affinity than in receptor activation. Thus, as a first step toward localizing the regions of CRFR involved in r/hCRF binding, we constructed a series of chimeric receptors with various regions of the CRFR1 sequence replaced by the corresponding CRFR2{alpha} sequence. By determining the EC50 values of these chimeras for r/hCRF, we found three regions, one in the second extracellular domain (EC2), one at the junction of EC2 and transmembrane domain 3 (TM3), and one at the junction of EC3 and TM5, that caused shift in EC50 value and were likely to be important for the high affinity binding of r/hCRF.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To localize the regions of the CRFR involved in binding of r/hCRF, four chimeric receptors, R1334R2, R1243R2, R1228R2, and R1166R2, with increasing portions of CRFR1 C-terminal sequences replaced by corresponding CRFR2 sequences, were constructed (Fig. 1Go). As shown in Fig. 2Go and Table 1Go, R1334R2 and R1166R2 had similar EC50 values for r/hCRF to CRFR1 (~0.16 nM) and CRFR2{alpha} (~60 nM), respectively, whereas R1243R2 and R1228R2 had an intermediate EC50 value of ~1.5 nM. These results suggested that the sequence differences between CRFR1 and CRFR2{alpha} for the 166 N-terminal aa, the region between aa 228–243, and the C-terminal region starting at aa 334, did not significantly change the affinity of r/hCRF binding. On the other hand, there were two regions that influenced r/hCRF binding. A 40-fold increase in the EC50 value (1.5 to 63.8 nM) was observed when aa 166–228 of CRFR1 were replaced by the corresponding region of CRFR2{alpha}. The other region found to be important was between aa 243–334, which resulted in an increase in the EC50 value of approximately 1 order of magnitude (0.10 to 1.6 nM) when replaced by the corresponding region of CRFR2{alpha}.



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  		Figure 1. Schematic Representation with Sequence Alignment of Human CRFR1 and CRFR2{alpha}

The conserved aa are shown in solid circles. The divergent aa are shown in open symbols, with the aa of CRFR1 shown on the left and those of CRFR2{alpha} shown on the right. The aa residues that have been mutated are shown in squares, and the junctions of chimeric receptors are indicated by arrows. The numbers indicate the aa positions of those residues flanking the TM domains. All numberings are based on the sequence of CRFR1.

 


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  		Figure 2. Stimulation of Intracellular cAMP of Wild Type CRFR1, CRFR2{alpha}, and Four Chimeric Receptors by r/hCRF

VIP2.0Zc cells transiently transfected with various receptors were incubated with different concentrations of r/hCRF for 7 h, and cAMP-induced ß-galactosidase activity was measured. Each data point is the average of triplicate determinations. Each curve is representative of at least four independent experiments.

 

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  		Table 1. EC50 of r/hCRF Stimulation of Intracellular cAMP for Wild Type CRFR1, CRFR2{alpha} and Four Chimeric Receptors

 
To further map the region(s) involved in r/hCRF binding between aa 243–334, two chimeric receptors, R1266R2 and R1228R2268R1, were constructed. In the latter chimera, aa 229–268 of the CRFR1 sequence were replaced by the corresponding CRFR2{alpha} sequence. An intermediate EC50 value of R1266R2 (0.25 nM) between R1243R2 (1.6 nM) and R1334R2 (0.1 nM; Fig. 3Go) suggested that both regions, aa 244–266 and aa 267–334 were involved in r/hCRF binding. On the other hand, R1228R2268R1 had an EC50 value of 1.4 nM, approximately the same as that of R1243R2, suggesting that it was the region containing the CRFR2{alpha} sequence shared by the two chimeric receptors, i.e. the region from aa 244–268, that was responsible for the ~10-fold increase in the EC50 value over CRFR1. Point and double mutations were then introduced into each of the six aa that are different between CRFR1 and CRFR2{alpha} within aa 244–268. As shown in Table 2Go, substitution of three aa at the junction of the third EC domain and the fifth TM domain, Val266, Tyr267, and Thr268, each caused ~a 2- to 3-fold increase in the EC50 value when replaced by the corresponding CRFR2{alpha} aa (see Table 2Go, D266, D266L267, and D266V268). Triple mutations with all three aa replaced by corresponding aa of CRFR2{alpha} (D266L267V268 in Table 2Go) had an EC50 value of 1.5 nM, similar to that of R1243R2.



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  		Figure 3. Stimulation of Intracellular cAMP of Four Chimeric CRFR1/CRFR2{alpha} by r/hCRF

Each curve is representative of at least four independent experiments. The calculated EC50 values (mean ± SEM) for R1334R2, R1266R2, R1228R2268R1, and R1243R2, are 0.10 ± 0.02, 0.25 ± 0.04, 1.4 ± 0.2, and 1.6 ± 0.4 nM, respectively.

 

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  		Table 2. EC50 of r/hCRF Stimulation of Intracellular cAMP for CRFR1 Mutants with Mutations in EC3 and TM5

 
Four additional chimeric receptors, R1174R2, R1178R2, R1188R2, and R1191R2, were constructed to localize the region(s) involved in r/hCRF binding between aa 166–228. As shown in Table 3Go, the EC50 value of R1191R2 (1.3 nM) was similar to that of R1228R2 (1.5 nM), suggesting that none of the aa changes between CRFR1 and CRFR2{alpha} within the aa 192–228 region caused a significant change in the EC50 value. On the other hand, both R1178R2 and R1188R2 had an EC50 value (~9.7 nM) ~7-fold higher than that of R1191R2, yet significantly lower than that of either R1174R2 or R1166R2. These data suggested that the aa differences between CRFR1 and CRFR2{alpha} within the region aa 178–188 did not change the binding affinity for r/hCRF, while two regions, one between aa 174–178 and another between aa 188–191 were important for r/hCRF binding.


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  		Table 3. EC50 of r/hCRF Stimulation of Intracellular cAMP for Chimeric Receptors with Fusion Junctions in EC2

 
Point mutations of each of the three aa at aa 189, 190, and 191 to the corresponding CRFR2{alpha} aa showed that only mutation of the Arg residue at aa 189 to His (H189) had a slight effect on the EC50 value (see H189, C190, and I191 in Table 4Go). This shift in the EC50 value was much less than what was observed between R1188R2 and R1191R2. As the difference between H189 mutant receptor and R1188R2 chimeric receptor is that the R1188R2 chimera contains the CRFR2 sequence C-terminal to the mutation site, it is possible that some C-terminal CRFR2 sequence might be required for the full expression of this H189 mutant phenotype. One likely candidate for such a C-terminal sequence is aa 266–268, which was shown above to be important for r/hCRF binding. To address this, a point mutation at aa 189 was combined with the D266L267V268 triple mutation; the resulting mutant receptor H189DLV had an EC50 value of 12.3 nM (Table 4Go) and an 8-fold increase over that of D266L267V268 (Table 2Go) and was comparable to that of R1188R2 (Table 3Go).


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  		Table 4. EC50 of r/hCRF Stimulation of Intracellular cAMP for CRFR1 Mutants with Mutations in EC2 and at Junction of EC2 and TM3

 
Unexpectedly, although the R1174R2 chimera contains more CRFR1 sequences than the R1166R2 chimera and wild type CRFR2, it had a higher EC50 value than either R1166R2 or CRFR2 (see Discussion). The ~20-fold shift of EC50 values between R1174R2 and R1178R2 (Table 3Go) indicated that aa 175–178 was important for r/hCRF binding. However, when this region was replaced by corresponding CRFR2{alpha} sequence (R1174R2178R1), there was no change in the EC50 value (Table 4Go). Similar to the H189 mutation, when mutation of aa 175–178 was combined with the D266L267V268 triple mutation (R1174R2178DLVR1 in Table 4Go), the resulting mutant receptor had an EC50 value of ~11 nM, 7-fold higher than that of the D266L267V268 mutant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Chimeric receptors have been used extensively as an approach to map the regions that are critical for agonist/antagonist binding of GPCR (21, 22, 23, 24). The present study was designed to use such an approach to localize some of the regions of the CRFR involved in r/hCRF binding. This was followed by generating point mutations to more specifically map the sites within the receptor that are important for peptide ligand binding. The assay we used to monitor the affinities of various chimeric receptors for r/hCRF is a colorimetric assay that measures the induction of intracellular cAMP. As there is a fairly good correlation between the change in the EC50 of stimulation of intracellular cAMP and the shift in binding affinity for the two CRFR subtypes, as previously described (25), and the two CRFR have identical aa sequence in the third intracellular loop, which has been shown to be a critical determinant of receptor activation, the changes in EC50 of various chimeric receptors observed in the current study are more likely to correspond to changes in binding affinity than to those in functional activation. However, the possibility that the regions mapped in the current study that result in changes in EC50 may be involved in receptor activation cannot be excluded.

As CRFR1 has more than 2 orders of magnitude higher affinity for r/hCRF than CRFR2, it was expected that as more CRFR1 sequences were systematically replaced by the corresponding CRFR2 sequences, there would be a gradual decrease in the affinity for r/hCRF. This was generally the case, except in one instance where R1174R2 had a lower affinity for r/hCRF than R1166R2 or CRFR2. One possible explanation for this is that aa 166–174 are incompatible with some other region(s) of the receptor molecule in this particular chimeric environment, and the resulting conformational change directly or indirectly affects the binding affinity of the peptide ligand r/hCRF.

By monitoring the EC50 values of various CRF chimeric and mutant receptors, three regions that affected the binding affinity of r/hCRF have been localized. Two of the regions were within the second EC domain, i.e. aa 175–178 and His189 at the junction of EC2 and TM3, whereas the third region was at the junction of EC3 and TM5 involving three aa residues, Val266, Tyr267, and Thr268. A mutant CRFR1 with aa 266–268 replaced by the corresponding CRFR2{alpha} sequence had a 10-fold lower affinity for r/hCRF than the wild type CRFR1 and could completely account for the shift in the EC50 value of the R1228R2 chimera. Whether all three aa are directly interacting with r/hCRF peptide, or some residues are playing a more indirect role, such as maintaining the local conformation for ligand binding, is not known.

Although substitution of the aa 266–268 sequence to the corresponding CRFR2{alpha} sequence could completely account for the 10-fold shift of the EC50 value of chimeric receptor R1228R2, neither substitution of aa 175–178 nor mutation of His189 to the corresponding CRFR2{alpha} sequence lowered the affinity for r/hCRF to the same degree as predicted from chimeric receptors involving the two regions. That is, although R1178R2 had 20- and 6-fold higher affinities for r/hCRF than R1174R2 and R1166R2, respectively, the R1174R2178R1 mutant had essentially the same affinity for r/hCRF as wild type CRF1. Mutation of Arg189 to His increased the EC50 value by less than 2-fold, whereas comparison of EC50 values of R1188R2 and R1191R2 would predict approximately a 7-fold decrease in affinity. As the difference between the mutant and chimeric receptors is that chimeric receptors have more CRFR2 sequence C-terminal to the mutation sites, the two EC2 mutants were each combined with the downstream mutation involving aa 266–268. In both instances, the resulting mutants R1174R2178DLVR1 and H189DLV showed a 7- to 8-fold increase in EC50 value compared with the D266L267V268 mutant. One possible explanation for such results is that although aa 266–268 play a primary role in securing the binding of r/hCRF peptide, the roles of aa 174–178 and His189 are secondary, such that only when the interaction between peptide ligand r/hCRF and aa 266–268 of the receptor is weakened (as in the case of D266L267V268 mutant) do these two regions in EC2 play a significant role in binding r/hCRF.

Recently, a new member of the CRF peptide family, urocortin, was cloned from rats (26) and humans (25). With 45% sequence identity to CRF, urocortin shows only limited selectivity for CRFR1vs. CRFR2 (3- to 4-fold difference in affinity) (25), and thus, the shift in the EC50 value was also expected to be much smaller in magnitude for the chimeric receptor. Indeed, the three regions mapped in the current study each appeared to contribute about a 1.5- to 2-fold shift in EC50 value in response to urocortin (data not shown).

Finally, it is important to note that when the chimeric receptor approach was used to map the regions that are important for ligand binding, as in the current study, only those regions that are different between the two receptor subtypes used to construct the chimeras were examined. It is conceivable that some of those conserved aa between CRFR1 and CRFR2 are critical for the binding of r/hCRF, and the importance of those regions would be revealed only when a more extensive study involving chimeric receptors constructed from CRFR and a more distant GPCR family member is performed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
All chimeric and mutant receptors were constructed from human CRFR1 and CRFR2{alpha} by using either naturally occurring restriction enzyme sites or sites generated by PCR. Sequences derived from PCR and synthetic oligonucleotides were confirmed by DNA sequencing.

All receptors were transiently expressed in LVIP2.0Zc cells, a cell line containing a cAMP-responsive ß-galactosidase reporter gene (27), and the EC50 values of r/hCRF in increasing the levels of intracellular cAMP for various receptors were determined as previously described (28). Briefly, 24 h after transfection, the cells were plated in 96-well plates and, 1 day later, incubated with various concentrations of r/hCRF. The intracellular cAMP levels induced by CRF were then indirectly determined by assaying the ß-galactosidase activity as previously described (28).


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Brownstein and Monika König for providing the LVIP2.0Zc cell line, and Dr. Wylie Vale for the human CRFR1 receptor complementary DNA clone. We also thank Dr. Nick Ling for synthesizing r/hCRF, and Guy Barry for technical assistance.

This work was supported in part by SBIR grant from NINDS (R43 NS34203).


   FOOTNOTES
 
Address requests for reprints to: Chen W. Liaw, Ph.D., 3050 Science Park Road, San Diego, California 92121.

Received for publication February 6, 1997. Revision received March 13, 1997. Accepted for publication March 14, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of a 41 residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213:1394–1397[Free Full Text]
  2. De Souza EB, Nemeroff CB 1990 Corticotropin-Releasing Factor: Basic and Clinical Studies of a Neuropeptide. CRC Press, Boca Raton, FL
  3. Dunn AJ, Berridge CW 1990 Physiological and behavioral responses of corticotropin-releasing factor administration: is CRF a mediator of anxiety of stress responses? Brain Res Rev 15:71–100[CrossRef][Medline]
  4. Owens MJ, Nemeroff CB 1991 Physiology and pharmacology of corticotropin-releasing factor. Pharmacol Rev 43:425–473[Medline]
  5. Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning of a human corticotropin-releasing factor receptor. Proc Natl Acad Sci USA 90:8967–8971[Abstract/Free Full Text]
  6. Vita N, Laurent P, Lefort S, Chalon P, Lelias JM, Kaghad M, Le Fur G, Caput D, Ferrara P 1993 Primary structure and functional expression of mouse pituitary and human brain corticotropin releasing factor receptor. FEBS Lett 335:1–5[CrossRef][Medline]
  7. Chang C-P, Pearse IIRV, O’Connell S, Rosenfeld MG 1993 Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–1195[CrossRef][Medline]
  8. Perrin MH, Donaldson CJ, Chen R, Lewis KA, Vale WW 1993 Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor. Endocrinology 133:3058–3061[Abstract/Free Full Text]
  9. Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdorf T 1995 Cloning and characterization of a functionally distinct corticotropin-releasing-factor receptor subtype from rat brain. Proc Natl Acad Sci USA 92:836–840[Abstract/Free Full Text]
  10. Kishimoto T, Pearse IIRV, Lin CR, Rosenfeld MG 1995 A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle. Proc Natl Acad Sci USA 92:1108–1112[Abstract/Free Full Text]
  11. Perrin M, Donaldson C, Chen R, Blount A, Berggren T, Bilezikjian L, Sawchenko P, Vale W 1995 Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA 92:2969–2973[Abstract/Free Full Text]
  12. Stenzel P, Kesterson R, Yeung W, Cone RD, Rittenberg MB, Stenzel-Poore MP 1995 Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 9:637–645[Abstract/Free Full Text]
  13. Liaw CW, Lovenberg TW, Barry G, Oltersdorf T, Grigoriadis DE, De Souza EB 1996 Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid. Endocrinology 137:72–77[Abstract]
  14. Dixon RAF, Sigal IS, Rands E, Register RB, Candelore MR, Blake AD, Strader CD 1987 Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core. Nature 326:73–77[CrossRef][Medline]
  15. Wheatley M, Hulme EC, Birdsall NJM, Curtis CAM, Eveleigh P, Pedder EK, Poyner D 1988 Peptide mapping studies on muscarinic receptors: receptor structure and location of the ligand binding site. Trends Pharmacol Sci [Suppl] 00:19–24
  16. Strader CD, Sigal IS, Dixon RAF 1989 Structural basis of the beta-adrenergic receptor function. FASEB J 3:1825–1832[Abstract]
  17. Khorana HG 1992 Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J Biol Chem 267:1–4[Free Full Text]
  18. Fong TM, Huang R-RC Strader CD 1992 Localization of agonist and antagonist binding domains of the human neurokinin-1 receptor J Biol Chem 267:25664–25667[Abstract/Free Full Text]
  19. Huang R-RC, Yu H, Strader CD, Fong TM 1994 Localization of the ligand binding site of the neurokinin-1 receptor: interpretation of chimeric mutations and single-residue substitutions. Mol Pharmacol 45:690–695[Abstract]
  20. Huang R-RC, Yu H, Strader CD, Fong TM 1994 Interaction of substance P with the second and seventh transmembrane domains of the neurokinin-1 receptor. Biochemistry 33:3007–3013[CrossRef][Medline]
  21. Frielle T, Daniel KW, Caron MG, Lefkowitz RJ 1988 Structural basis of ß-adrenergic receptor subtype specificity studied with chimeric ß1/ß2-adrenergic receptors. Proc Natl Acad Sci USA 85:9494–9498[Abstract/Free Full Text]
  22. Gether U, Johansen TE, Snider RM, Lowe JA, Nakanishi S, Schwartz TW 1993 Different binding epitopes on the NK1 receptor for substance P and a non-peptide antagonist. Nature 362:345–348[CrossRef][Medline]
  23. Fong TM, Yu H, Strader CD 1992 Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580. J Biol Chem 267:25668–25671[Abstract/Free Full Text]
  24. Sachais BS, Snider RM, Lowe JAI II, Krause JE 1993 Molecular basis for the species selectivity of the substance P antagonist CP-96,345. J Biol Chem 268:2319–2323[Abstract/Free Full Text]
  25. Donaldson CJ, Sutton SW, Perrin MH, Corrigan AZ, Lewis KA, Rivier JE, Vaughan JM, Vale WW 1996 Cloning and characterization of human urocortin. Endocrinology 137:2167–2170[Abstract]
  26. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Rivier J, Sawchenko PE, Vale W 1995 Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378:287–292[CrossRef][Medline]
  27. König M, Mahan LC, Marsh JW, Fink JS, Brownstein MJ 1991 Method for identifying ligands that bind to cloned Gs- or Gi-coupled receptors. Mol Cell Neurosci 2:331–337
  28. Liaw CW, Grigoriadis DE, De Souza EB, Oltersdorf T 1994 Colorimetric assay for rapid screening of corticotropin releasing factor receptor ligands. J Mol Neurosci 5:83–92[Medline]



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A Soluble Form of the First Extracellular Domain of Mouse Type 2beta Corticotropin-releasing Factor Receptor Reveals Differential Ligand Specificity
J. Biol. Chem., April 25, 2003; 278(18): 15595 - 15600.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. R. J. Hoare, S. K. Sullivan, N. Ling, P. D. Crowe, and D. E. Grigoriadis
Mechanism of Corticotropin-Releasing Factor Type I Receptor Regulation by Nonpeptide Antagonists
Mol. Pharmacol., March 1, 2003; 63(3): 751 - 765.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Assil-Kishawi and A. B. Abou-Samra
Sauvagine Cross-links to the Second Extracellular Loop of the Corticotropin-releasing Factor Type 1 Receptor
J. Biol. Chem., August 30, 2002; 277(36): 32558 - 32561.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
F. M. Dautzenberg, J. Higelin, O. Brauns, B. Butscha, and R. L. Hauger
Five Amino Acids of the Xenopus laevis CRF (Corticotropin-Releasing Factor) Type 2 Receptor Mediate Differential Binding of CRF Ligands in Comparison with Its Human Counterpart
Mol. Pharmacol., May 1, 2002; 61(5): 1132 - 1139.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
G. Xu, C. Rabadan-Diehl, M. Nikodemova, P. Wynn, J. Spiess, and G. Aguilera
Inhibition of Corticotropin Releasing Hormone Type-1 Receptor Translation by an Upstream AUG Triplet in the 5' Untranslated Region
Mol. Pharmacol., March 1, 2001; 59(3): 485 - 492.
[Abstract] [Full Text]

Home page
 J. Pharmacol. Exp. Ther.Home page
F. M. Dautzenberg, G. Py-Lang, J. Higelin, C. Fischer, M. B. Wright, and G. Huber
Different Binding Modes of Amphibian and Human Corticotropin-Releasing Factor Type 1 and Type 2 Receptors: Evidence for Evolutionary Differences
J. Pharmacol. Exp. Ther., January 1, 2001; 296(1): 113 - 120.
[Abstract] [Full Text]

Home page
 Mol. Endocrinol.Home page
D. K. Grammatopoulos, H. S. Randeva, M. A. Levine, E. S. Katsanou, and E. W. Hillhouse
Urocortin, but Not Corticotropin-Releasing Hormone (CRH), Activates the Mitogen-Activated Protein Kinase Signal Transduction Pathway in Human Pregnant Myometrium: An Effect Mediated via R1{{alpha}} and R2{beta} CRH Receptor Subtypes and Stimulation of Gq-Proteins
Mol. Endocrinol., December 1, 2000; 14(12): 2076 - 2091.
[Abstract] [Full Text]

Home page
 Mol. Endocrinol.Home page
D. K. Grammatopoulos, Y. Dai, H. S. Randeva, M. A. Levine, E. Karteris, A. J. Easton, and E. W. Hillhouse
A Novel Spliced Variant of the Type 1 Corticotropin-Releasing Hormone Receptor with a Deletion in the Seventh Transmembrane Domain Present in the Human Pregnant Term Myometrium and Fetal Membranes
Mol. Endocrinol., December 1, 1999; 13(12): 2189 - 2202.
[Abstract] [Full Text]

Home page
 Mol. Endocrinol.Home page
V. I. DeAlmeida and K. E. Mayo
Identification of Binding Domains of the Growth Hormone-Releasing Hormone Receptor by Analysis of Mutant and Chimeric Receptor Proteins
Mol. Endocrinol., May 1, 1998; 12(5): 750 - 765.
[Abstract] [Full Text]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. M. Dautzenberg, S. Wille, R. Lohmann, and J. Spiess
Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: Implications for the ligand-binding site
PNAS, April 28, 1998; 95(9): 4941 - 4946.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
C. W. Liaw, D. E. Grigoriadis, M. T. Lorang, E. B. De Souza, and R. A. Maki
Localization of Agonist- and Antagonist-Binding Domains of Human Corticotropin-Releasing Factor Receptors
Mol. Endocrinol., December 1, 1997; 11(13): 2048 - 2053.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. H. Perrin, W. H. Fischer, K. S. Kunitake, A. G. Craig, S. C. Koerber, L. A. Cervini, J. E. Rivier, J. C. Groppe, J. Greenwald, S. M. Nielsen, et al.
Expression, Purification, and Characterization of a Soluble Form of the First Extracellular Domain of the Human Type 1 Corticotropin Releasing Factor Receptor
J. Biol. Chem., August 17, 2001; 276(34): 31528 - 31534.
[Abstract] [Full Text] [PDF]

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