Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells.
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.
Wietfeld
Doreen
D
Institute of Molecular Pharmacology, Robert-Rössle-Strasse 10, Berlin 13125, Germany.
Heinrich
Nadja
N
Furkert
Jens
J
Fechner
Klaus
K
Beyermann
Michael
M
Bienert
Michael
M
Berger
Hartmut
H
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
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