Regulation of the Coupling to Different G Proteins of Rat Corticotropin-releasing Factor Receptor Type 1 in Human Embryonic Kidney 293 Cells

Abstract 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 [35S]GTPγS binding. Contrary to the low potency response, the high potency response was of lower GTPγS 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 [35S]GTPγS-bound Gαs and Gαi subunits it is concluded that the high and low potency [35S]GTPγS binding stimulation reflected coupling to Gs and Gi proteins, respectively, only Gs coupling being homologously desensitized. Immunoprecipitation of [35S]GTPγS-bound Gαq/11 revealed additional coupling to Gq/11, which also was homologously desensitized. Although Gαq/11 coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of Gi in addition to Gq/11in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to Gs- and Gq/11-mediated signaling steps and desensitization and another leading to Gi -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.