The First Extracellular Domain of Corticotropin Releasing Factor-R1 Contains Major Binding Determinants for Urocortin and Astressin*

Abstract

The CRF receptors are members of a 7-transmembrane receptor family that includes GH-releasing hormone (GRF), calcitonin, vasoactive intestinal peptide (VIP), secretin, and PTH receptors. To determine the structural features of the CRF receptor that may influence ligand recognition, a series of mutant receptors was analyzed for binding to astressin, a CRF antagonist, and to urocortin, a CRF agonist. Mutant receptors included chimeras between the CRF-R1 and GRF-R or Activin IIB-R, a single membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using 125I-[DTyr1] astressin (Ast*) and 125I-[Tyr0]-rat urocortin (Ucn*). There was no binding to a chimeric receptor in which the first extracellular domain (E1c) (i.e. the N-terminal region) of the CRF-R1 was replaced by that of the GRF-R. The complementary chimera in which E1 domain of the GRF-R was replaced by that of the CRF-R1 bound astressin and urocortin with Ki values approximately 10 nM, compared with inhibitory binding dissociation constant (Ki) values of approximately 2-4 nM for the wild-type CRF-R1. The chimera in which E1 of the activin IIB receptor was replaced by E1 of the CRF-R1 bound astressin with a Ki approximately 4 nM. A chimera in which both the first and fourth extracellular domains of the CRF-R1 replaced the corresponding domains of the GRF-R bound astressin with Ki approximately 4 nM and urocortin with a Ki approximately 2 nM. A chimera in which all four extracellular domains of the CRF receptor replaced those of the GRF-R bound astressin and urocortin with Ki values approximately 4 nM and approximately 1 nM, respectively. In conclusion, the major determinants for high affinity binding of CRF agonists and antagonists to CRF-R1 are found in the first extracellular domain of the receptor.

The Isolated N-terminal Domain of the Glucagon-like Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1

“…The results are also very similar to the analogous corticotropin-releasing factor receptor fragment expressed in COSM6 cells, which showed no detectable binding in homologous competition binding assays when the agonist urocortin was used as tracer but bound with low nM affinity to the antagonist astressin when this peptide was used as the tracer in competition binding studies (33).…”

Expression, Purification, and Characterization of a Soluble Form of the First Extracellular Domain of the Human Type 1 Corticotropin Releasing Factor Receptor

“…In both cases, the K i values of astressin and urocortin for the bNT- CRFR are ϳ5-10-fold lower than those for the wild type receptor and also than those for the CRFR/activin-R chimera (21). The reduced affinity of astressin and urocortin for bNT-CRFR1 may be due to the absence of ligand interactions with the plasma membrane (46).…”

“…Interestingly, the ECD-1 of the Xenopus CRFR1 suffices to determine ligand specificity (23). Of special relevance for this work was our demonstration that a chimeric receptor in which the ECD-1 of CRFR1 replaced the ECD of the single transmembrane domain activin receptor displays high affinity binding for both a CRF antagonist and agonist (21). This observation prompted us to initiate a structural characterization of the isolated ECD-1 of CRFR1 with the goal of providing information on this important receptor-binding region.…”

“…Binding to Membrane Fractions-Transfection and binding to membrane fractions were performed as described (21). Background ccounts/ min (counts bound in absence of receptor) were subtracted from all counts/min.…”

“…Mutagenesis studies have identified regions of the CRF receptors that are implicated in differential recognition of agonists and antagonists, as well as in governing the ligand selectivity of the two types of receptors (21)(22)(23)(24)(25). We showed that a chimeric receptor in which the ECD-1 of CRFR1 replaced the ECD of the activin receptor, a single transmembrane receptor kinase (26), was capable of high affinity binding to both astressin and urocortin (21). The mode of receptor activation was explored by our study of a tethered peptide-receptor chimera in which the first 16 amino acids of CRF were substituted for the ECD-1 of CRFR1 (CRF (1-16)/R1 ⌬N ) (27).…”