Stable association of G proteins with beta 2AR is independent of the state of receptor activation.
beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.
Centre de Recherche, Institut de Cardiologie de Montréal, PQ, Canada.
M
M
Lachance
N
N
Ethier
G
G
Wolbring
P P
PP
Schnetkamp
T E
TE
Hébert
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D006801:Humans; D007545:Isoproterenol / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology
D000318:Adrenergic beta-Agonists; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018343:Receptors, Adrenergic, beta-2; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol