The N-terminal Domain of RGS4 Confers Receptor-selective Inhibition of G Protein Signaling

Abstract

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...

Regulators of G protein signaling 12 promotes osteoclastogenesis in bone remodeling and pathological bone loss

“…These results further confirmed that osteoclastogenesis was impaired in the CD11b;Rgs12 fl/fl group. Accumulating studies showed that Rgs proteins have a critical role in regulating Ca 2+ oscillations in various cells, including pancreatic acinar cells and T lymphocytes, [30][31][32][33] and that Ca 2+ oscillations and NFAT2 activation are essential for RANKL-induced OC differentiation. 13 Thus we hypothesized that Rgs12 is a specific regulator of Ca 2+ oscillations in response to RANKL during OC differentiation.…”

A regulator of G protein signaling interaction surface linked to effector specificity

“…Since most of the RGS residues in direct contact with G ␣ are well conserved across the entire family, these residues are unlikely to determine RGS-G protein specificity alone. Rather, additional regulatory proteins, such as effectors (11), G␤ proteins (9,(35)(36)(37)(38), or G proteincoupled receptors (7,39) may be involved. Since five classspecific residues (r77, r117, r121, r122, r124, and r125) cluster above the RGS-G ␣ interface to form an active site common to all members of the family, a reasonable hypothesis is that this is the binding site for additional proteins that mediate specificity (Fig.…”

“…Growing evidence suggests these domains do play an important role in the function of the RGS domain itself. For example, the amino terminus of RGS4 has been shown to provide agonist-dependent regulation of PLCmediated Ca 2ϩ release in rat pancreatic acinar cells (39), and the amino terminus of GAIP provides specificity for the G omediated desensitization of presynaptic Ca 2ϩ channels in chick sensory neurons (40). Furthermore, truncation of the amino terminus from RGS16 (41) results in improper cellular localization and a corresponding loss of intracellular GAP activity.…”

Homer proteins in Ca2+ signaling by excitable and non-excitable cells

“…The RGS proteins are Gα GTPase activating proteins (GAP) composed of a highly conserved RGS box of about 110 amino acids and divergent C-and N-termini. The GAP activity of the RGS proteins is mediated by the RGS box [39], whereas the N-terminus domain mediates membrane targeting and confers receptor recognition [40]. Receptor recognition is mediated by binding of the Nterminus of RGS proteins and the third intracellular loop of GPCRs to the scaffolding protein spinophilin [41].…”