title: 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 EC(50) values as measured by stimulation of [(35)S]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 [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]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.
authors.affiliation: Institute of Molecular Pharmacology, Robert-Rössle-Strasse 10, Berlin 13125, Germany.
authors.forename: Doreen
authors.initials: D
authors.lastname: Wietfeld
authors.forename: Nadja
authors.initials: N
authors.lastname: Heinrich
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: Klaus
authors.initials: K
authors.lastname: Fechner
authors.forename: Michael
authors.initials: M
authors.lastname: Beyermann
authors.forename: Michael
authors.initials: M
authors.lastname: Bienert
authors.forename: Hartmut
authors.initials: H
authors.lastname: Berger
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D029845:Amphibian Proteins; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D013997:Time Factors; D014162:Transfection
chemical_list: D029845:Amphibian Proteins; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C025727:sauvagine; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D003346:Corticotropin-Releasing Hormone; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases