Differential coupling of beta sub(3A)- and beta sub(3B)-adrenergic receptors to endogenous and chimeric G sub( alpha )s and G sub( alpha )i

• Published in: American journal of physiology: endocrinology and metabolism 2006-10, Vol.291 (4), p.E704-E715
• Main Authors: Lenard, Natalie R, Prpic, Veronica, Adamson, Aaron W, Rogers, Richard C, Gettys, Thomas W
• Format: Article
• Language: English
• ISSN: 0193-1849; 1522-1555

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G sub( alpha )q with the COOH-terminal heptapeptide of G sub( alpha )s or G sub( alpha )i were used to assess the relative coupling of beta sub(3)-adrenergic receptor ( beta sub(3)-AR) splice variants ( beta sub(3A) and beta sub(3B)) to G sub( alpha )s and G sub( alpha )i. The G sub( alpha )q/s and G sub( alpha )q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca super(2+) sub(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 sub(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta sub(3)-AR-induced coupling to G sub( alpha )q/s produced a rapid eightfold increase in Ca super(2+) sub(i) followed by a slow decay to levels 25% above baseline. G sub( alpha )q/i also linked rat beta sub(3)-AR to mobilization of Ca super(2+) sub(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca super(2+) sub(i) was only 30% of the response obtained with G sub( alpha )q/s. Activation of the rat beta sub(3)-AR also increased GTP binding to endogenous G sub( 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 sub(3A)- and beta sub(3B)-AR to G sub( alpha )i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G sub( alpha )q/s and to increases in intracellular cAMP through endogenous G sub( alpha )s. The beta sub(3A)- and beta sub(3B)-AR coupled equivalently to G sub( alpha )q/i, but the temporal patterns of Ca super(2+) sub(i) mobilization indicated that coupling was significantly less efficient than coupling to G sub( alpha )q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta sub(3A)- and beta sub(3B)-AR to G sub( alpha )i vs. G sub( alpha )s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta sub(3)-AR activation.