The beta3-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 Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR 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 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR 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 beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR 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 beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.
Soeder
K J
KJ
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Snedden
S K
SK
Cao
W
W
Della Rocca
G J
GJ
Daniel
K W
KW
Luttrell
L M
LM
Collins
S
S
D016475:3T3 Cells; D017667:Adipocytes / Q000201:enzymology*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D008066:Lipolysis; D051379:Mice; D011485:Protein Binding; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D022702:Receptors, Adrenergic, beta-3
D000318:Adrenergic beta-Agonists; D004149:Dioxoles; D011943:Receptors, Adrenergic, beta; D022702:Receptors, Adrenergic, beta-3; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go