Ligand Affinity for Amino-Terminal and Juxtamembrane Domains of the Corticotropin Releasing Factor Type I Receptor: Regulation by G-Protein and Nonpeptide Antagonists
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- 作者：Sam R. J. Hoare ; Sue K. Sullivan ; David A. Schwarz ; Nicholas Ling ; Wylie W. Vale ; Paul D. Crowe ; and Dimitri E. Grigoriadis
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：April 6, 2004
- 年：2004
- 卷：43
- 期：13
- 页码：3996 - 4011
- 全文大小：267K
- 年卷期：v.43,no.13(April 6, 2004)
- ISSN：1520-4995
文摘
Peptide ligands bind the CRF1 receptor by a two-domain mechanism: the ligand's carboxyl-terminal portion binds the receptor's extracellular N-terminal domain (N-domain) and the ligand's amino-terminal portion binds the receptor's juxtamembrane domain (J-domain). Little quantitative informationis available regarding this mechanism. Specifically, the microaffinity of the two interactions and theircontribution to overall ligand affinity are largely undetermined. Here we measured ligand interactionwith N- and J-domains expressed independently, the former (residues 1-118) fused to the activin IIBreceptor's membrane-spanning 
-helix (CRF1-N) and the latter comprising residues 110-415 (CRF1-J).We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- andJ-domains. Peptide agonist affinity for CRF1-N was only 1.1-3.5-fold lower than affinity for the wholereceptor (CRF1-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonistinteraction with CRF1-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weakerthan with CRF1-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonistaffinity for CRF1-J and CRF1-R was indistinguishable, indicating the compounds bind predominantly theJ-domain. Agonist activation of CRF1-J was fully blocked by nonpeptide antagonist, suggesting antagonismresults from inhibition of agonist-J-domain interaction. G-protein coupling with CRF1-R (forming RG)increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction withthe J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptideagonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffectedby R-G coupling. These findings extend the two-domain model quantitatively and are consistent with asimple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist withmoderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowingweak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced byreceptor-G-protein interaction and inhibited by nonpeptide antagonist.
-helix (CRF1-N) and the latter comprising residues 110-415 (CRF1-J).We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- andJ-domains. Peptide agonist affinity for CRF1-N was only 1.1-3.5-fold lower than affinity for the wholereceptor (CRF1-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonistinteraction with CRF1-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weakerthan with CRF1-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonistaffinity for CRF1-J and CRF1-R was indistinguishable, indicating the compounds bind predominantly theJ-domain. Agonist activation of CRF1-J was fully blocked by nonpeptide antagonist, suggesting antagonismresults from inhibition of agonist-J-domain interaction. G-protein coupling with CRF1-R (forming RG)increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction withthe J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptideagonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffectedby R-G coupling. These findings extend the two-domain model quantitatively and are consistent with asimple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist withmoderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowingweak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced byreceptor-G-protein interaction and inhibited by nonpeptide antagonist.