Background and purpose: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF1), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF1 by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF1 states for Gs and Gi coupling are similar or different.Experimental approach: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF1 to Gs- and Gi-protein in human embryonic kidney cells, using the [S-35]-GTP gamma S binding stimulation assay.Key results: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism.Conclusions and Implications: The results allow us to extend the two-domain model of CRF1 activation by assuming that CRF1 agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs- mediated physiological activities of CRF1.

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK) 293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC50 values as measured by stimulation of [S-35]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [S-35]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [S-35]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [S-35]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11) in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i)-mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.