Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via G q . Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10 4 -fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates G q class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by G␣ q and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled G q signaling.Heterotrimeric G proteins of the G q class are mediators of Ca 2ϩ responses in animal cells. Signaling is initiated by agonist binding to heptahelical transmembrane receptors complexed with G q ␣␤␥ and phospholipase C-␤ (PLC␤) 1 (1), which generates IP 3 to trigger Ca 2ϩ release from internal stores (2).Many cells express several G q -coupled receptors that regulate the location, intensity, and propagation of intracellular Ca 2ϩ waves. For example, pancreatic acini respond to acetylcholine, bombesin, and cholecystokinin by activating the same set of G q class proteins and mobilizing the same Ca 2ϩ pool, but each receptor evokes distinct patterns of Ca 2ϩ waves (3). Ca 2ϩ release may be regulated by intracellular proteins that interact with guanine nucleotide binding proteins, such as regulators of G protein signaling (RGS) proteins.2 RGS proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling (5-8). Mammals express over 20 different RGS proteins, of which RGS4 has received the most extensive biochemical characterization (5, 7-12). RGS4 is composed of a central domain of 120 amino acids that is homologous to other RGS proteins, termed the RGS box, flanked by less well conserved N-and C-terminal sequences (13). In rat pancreatic acinar cells, RGS4 preferentially inhibited G q/11 -mediated signaling evoked by carbachol relative to bombesin and cholecystokinin regardless of the identity of the G q class ␣ subunit. 2Regulatory specificity was apparently conferred by direct or indirect interaction between RGS4 and the receptor.In the present study, we used deletion mutations to identify two domains in RGS4 that regulate agonist-dependent Ca 2ϩ signaling. The RGS box accelerates GTP hydrolysis by G␣ q whereas the N terminus conveys high affinity and receptorselective inhibition. These combined activities are required for receptor selectivity and high potency i...

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A series of analogues of the satiety-inducing peptide cholecystokinin (CCK-8) was prepared in which the sulfated tyrosine required for activation of peripheral receptors was replaced with a carboxy(alkyl)- or tetrazolyl(alkyl)-phenylalanine to investigate whether an organic acid could serve the role of the sulfate group at the receptor. The necessary intermediates were prepared by previously reported procedures or by alkylation of carboxy(alkyl)- or tetrazolyl(alkyl)phenylmethyl bromides with a glycine-derived anion followed by protecting-group manipulations, and these were incorporated into derivatives of acetyl-CCK-7 using solid-phase synthesis. Peptide analogues were evaluated in a CCK-binding assay for affinity for either peripheral (CCK-A) receptors using homogenated rat pancreatic membranes as the receptor source or for central (CCK-B) receptors using bovine striatum as the receptor source. They were further evaluated for effects on food intake in rats after intraperitoneal (ip) injection. A number of the compounds reported are active in the CCK-A receptor binding assay although less potent than acetyl-CCK-7 and decrease food intake with comparable potency to acetyl-CCK-7. In a meal feeding model designed to assess appetite suppressant activity, acetyl-CCK-7 has an ED50 of 7 nmol/kg ip, while the ED50s of Ac-Phe(4-CH2CO2H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (28) and Ac-Phe[4-(tetrazol-5-yl)]-Met-Gly-Trp-Met-Asp-Phe-NH2 (34) were 9 and 11 nmol/kg ip, respectively. An analogue of 28 lacking the N-terminal acetamido group, 3-[4-(carboxymethyl)-phenyl]propanoyl-Met-Gly-Trp-Met-Asp-Phe-NH2 (50), was also active in the meal feeding assay with an ED50 of 3 nmol/kg ip. Its anorexic effect was blocked by simultaneous administration of the CCK-A receptor antagonist MK 329, indicating that the observed anorexic activity is mediated by CCK-A receptors. We conclude from this work that the requirement for a negative charge at the CCK-A receptor provided in the natural substrate by a sulfate group can be satisfied by organic acids.