title: Differential coupling of beta3A- and beta3B-adrenergic receptors to endogenous and chimeric Galphas and Galphai.
abstract: Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G(alpha)q with the COOH-terminal heptapeptide of G(alpha)s or G(alpha)i were used to assess the relative coupling of beta(3)-adrenergic receptor (beta(3)-AR) splice variants (beta(3A) and beta(3B)) to G(alpha)s and G(alpha)i. The G(alpha)q/s and G(alpha)q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca(2+)(i)). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat beta(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta(3)-AR-induced coupling to G(alpha)q/s produced a rapid eightfold increase in Ca(2+)(i) followed by a slow decay to levels 25% above baseline. G(alpha)q/i also linked rat beta(3)-AR to mobilization of Ca(2+)(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca(2+)(i) was only 30% of the response obtained with G(alpha)q/s. Activation of the rat beta(3)-AR also increased GTP binding to endogenous G(alpha)i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse beta(3A)- and beta(3B)-AR to G(alpha)i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G(alpha)q/s and to increases in intracellular cAMP through endogenous G(alpha)s. The beta(3A)- and beta(3B)-AR coupled equivalently to G(alpha)q/i, but the temporal patterns of Ca(2+)(i) mobilization indicated that coupling was significantly less efficient than coupling to G(alpha)q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta(3A)- and beta(3B)-AR to G(alpha)i vs. G(alpha)s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta(3)-AR activation.
authors.affiliation: Laboratories of Adipocyte Signaling, Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA.
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Lenard
authors.forename: Veronica
authors.initials: V
authors.lastname: Prpic
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Adamson
authors.forename: Richard C
authors.initials: RC
authors.lastname: Rogers
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Gettys
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D005470:Fluorometry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D051381:Rats; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
chemical_list: D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists; D020033:Protein Isoforms; D022702:Receptors, Adrenergic, beta-3; D011993:Recombinant Fusion Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D002118:Calcium