The β3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G; proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual G(s)/G(i) protein coupling of the beta(3)-adrenergic receptor (beta(3)AR) in 3T3-F442A adipocytes. The beta(3)AR selective agonist disodium (R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further a-fold increase in cAMP production by CL316,243, CL316,243 (5 mu M) stimulated the incorporation of 8-azido-[P-32]GTP into G alpha(s) (1.57 +/- 0.12; n = 3) and G alpha(i) (1.68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta(3)AR stimulation results in G(i)-GTP exchange. The beta(3)AR-stimulated increase in 8-azido-[P-32]GTP labeling of G alpha(i) was equivalent to that obtained with the A(1)-adenosine receptor agonist N-6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 mu M) eliminated all binding. Stimulation of the beta(3)AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta(3)AR, demonstrating that this response required receptor coupling to Gi, Expression of the human beta(3)AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta(3)AR, ERK1/2 activation by the beta(3)AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta(3)AR coupling to G(i) proteins serves both to restrain G(s)-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.