Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus.
The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.
Feldman
David S
DS
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Zamah
A Musa
AM
Pierce
Kristen L
KL
Miller
William E
WE
Kelly
Francine
F
Rapacciuolo
Antonio
A
Rockman
Howard A
HA
Koch
Walter J
WJ
Luttrell
Louis M
LM
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007545:Isoproterenol / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D010455:Peptides; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D007545:Isoproterenol