The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein G␣ 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 G␣ s (GsCT). In membranes prepared from GsCT-expressing cells, the peptide

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... locked high affinity agonist binding to ␤ 2 adrenergic receptors (AR) and inhibited ␤ 2 AR-induced [ 35 S]GTP␥S loading of G␣ s . GsCT expression inhibited ␤ 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. [ 35 S]GTP␥S loading of G␣ q/11 by ␣ 1B ARs and G␣ i by ␣ 2A ARs and G q/11or 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 ␤ 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, ␣ 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 ␣ 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. Heptahelical, or G protein-coupled, receptors represent the single most diverse class of cell surface receptors, both evolutionarily and within the human genome. The basic unit of G protein-coupled receptor signaling is composed of three parts as follows: a heptahelical receptor, a heterotrimeric G protein, 1 and an effector, such as a G protein-regulated enzyme or ion channel. The binding of an extracellular agonist ligand to the receptor changes its conformation so as to permit productive coupling with the G protein, thereby catalyzing the exchange of GTP for GDP on the G␣ subunit, and dissociation of G␣-GTP from G␤␥ subunits. Regulation of effectors is achieved through their interaction with free GTP-bound G␣ or G␤␥ subunits. Based upon data from crystallographic, biochemical, and mutagenesis studies, physical coupling of receptor and G protein is thought to involve primarily the second and third intracellular domains of the receptor, which make physical contact with the carboxyl terminus of the G␣ subunit (1-6). In particular, the last ϳ50 amino acids of the G␣ subunit are important for discriminating between different receptor subtypes and between different functional states of the receptor (3, 4, 6 -9). Pharmacologic agents that act as agonists or antagonists of heptahelical receptors represent the most common type of drug in clinical use today. Irrespective of chemical composition, these agents share a common mechanism of action in that they act extracellularly either to mimic, or to preclude, agonist binding at its receptor. By interacting with the molecular determinants of ligand binding in the extracellular or transmembrane domains of the receptor, often remarkable receptor subtypespecific agonist or antagonist effects can be obtained. An alternative approach to antagonism of heptahelical receptor signaling is to target the receptor-G protein interface with agents that block coupling between the receptor and G protein intracellularly. Such an approach differs fundamentally from classical heptahelical receptor pharmacology in that the blockade of receptor-G protein coupling might be expected to produce G protein-specific, rather than receptor-specific, antagonism. Several successful applications of this strategy, using polypeptides derived from the putative contact surfaces on