title: Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.
abstract: The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.
authors.affiliation: Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305-5428, USA.
authors.forename: R
authors.initials: R
authors.lastname: Seifert
authors.forename: K
authors.initials: K
authors.lastname: Wenzel-Seifert
authors.forename: T W
authors.initials: TW
authors.lastname: Lee
authors.forename: U
authors.initials: U
authors.lastname: Gether
authors.forename: E
authors.initials: E
authors.lastname: Sanders-Bush
authors.forename: B K
authors.initials: BK
authors.lastname: Kobilka
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D012326:RNA Splicing; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology
chemical_list: D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs