ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling.

ANG II type 1 (AT(1)) receptors respond to sustained exposure to ANG II by undergoing downregulation of absolute receptor numbers. It has been assumed previously that downregulation involves endocytosis. The present study hypothesized that AT(1) receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT(1) receptors with carboxy-terminal deletions internalized <5% of radioligand compared with 65% for wild-type AT(1) receptors. The truncated AT(1) receptors retained the ability to undergo downregulation. These data suggest the existence of an alternative pathway to AT(1) receptor degradation that does not require endocytosis, per se. Point mutations in either the second transmembrane region or second intracellular loop impaired G protein (G(q)) coupling. These receptors exhibited a biphasic pattern of downregulation. The earliest phase of downregulation (0-2 h) was independent of coupling to G(q), but no additional downregulation was observed after 2 h of ANG II exposure in the receptors with impaired coupling to G(q). These data suggest that coupling to G(q) is required for the later phase (2-24 h) of AT(1) receptor downregulation.

Molecular cloning and characterization of a novel regulator of G-protein signaling from mouse hematopoietic stem cells.

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha subunit of both G(i) and G(q) subfamilies. The results of in vitro GTPase single-turnover assays using Galpha(i) indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha(i). Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G(q)-mediated signaling pathways in vivo.

Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Galphai and Galphao deactivation. Galpha specificity of RGS4 AND RGS7.

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.

G(i) activator region of alpha(2A)-adrenergic receptors: distinct basic residues mediate G(i) versus G(s) activation.

The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrenergic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for G(i)-mediated inhibition of cAMP accumulation, whereas the G(s) response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as G(i) activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of G(i) but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal G(i) and G(s) responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the alpha(2a) adrenergic receptor.