RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein ␣ subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [ 3 H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for G i ␣. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both G iand G q -linked signaling pathways when expressed in HEK293T cells. These findings suggest that the aminoterminal region of RGS16 may affect the affinity of these proteins for G␣ subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to G␣ subunits on cellular membranes. RGS proteins1 enhance the GTPase activity of heterotrimeric G protein ␣ subunits to turn off signaling between cell-surface receptors and intracellular effectors (1). More than 20 mammalian RGS proteins have been identified by virtue of a common stretch of 125 amino acids termed the RGS box (2-3), which is a site of interaction of RGS proteins and G i ␣ subunits. RGS proteins bind and stabilize G i ␣ in its transition state (between GTP-and GDP-bound), thus lowering the energy barrier for GTP hydrolysis (4 -6). Recently a group of proteins with a divergent RGS domain that act exclusively on G 12 ␣ and G 13 ␣ to accelerate their GTPase activity has been identified (7). Studies of reconstituted signaling pathways in cell lines, consisting of overexpressed or endogenous receptors, RGS proteins, effectors (in some cases), and endogenous G proteins, have shown that transiently expressed RGS proteins inhibit G proteinmediated signaling in these systems (3, 8 -9).A perplexing question is the regulation of these 20 RGS proteins because almost all of them have powerful enzymatic activity toward the same substrates, and many may be expressed in the same cell type. Temporal or stimulus-specific transcriptional regulation, protein turnover, or post-translational modifications represent possible means of controlling the availability of a particular RGS protein to preside over a G protein-coupled signaling event at the plasma membrane. One such modification, addressed in this report, is palmitoylation.Palmitoylation, the reversible thioacylation of cysteine residues, occurs on several molecules involved in G protein-linked signaling pathways. G protein-coupled receptors incorporate [ 3 H]palmitate upon addition of...

Racemic albuterol has been one of the most widely used beta2-adrenoceptor agonists for the relief of the symptoms of asthma, yet the use of beta2 agonists has been known to induce bronchial hyperresponsiveness. To probe a possible role of the S-enantiomer for hyperresponsiveness, we determined the effects of (S)-albuterol on intracellular Ca2+ concentration ([Ca2+]i) in dissociated bovine tracheal smooth muscle cells. Both (S)-and (R,S)-albuterol increased [Ca2+]i at concentrations of >10 pM and 1 nM, respectively, with a maximal response by 150 and 100 nM, respectively. (S)-Albuterol (1 and 10 muM) induced Ca2+ oscillations, reaching 1-2 muM [Ca2+]i. This response is in a stark contrast to that of (R)-albuterol, which decreased [Ca2+]i. The increase in [Ca2+]i was blocked by 100 nM atropine or 500 nM 4-diphenylacetoxy-N-methylpiperidine but was insensitive to the beta2 antagonist ICI 118,551 (10 muM). (S)-Albuterol (10 muM) increased inositol-1,4,5-trisphosphate levels by 213 +/- 34.4% (p < 0.05, four experiments) in cells exposed for 30 sec. The sustained phase of the Ca2+ increase was absent in Ca2+-free solution, suggesting that Ca2+ influx was responsible for the sustained Ca2+ response. The results also suggest that (S)-albuterol may cross-react with muscarinic receptors. As a Ca2+ agonist in airway smooth muscle, (S)-albuterol may have profound clinical implications because 50% of prescribed racemic albuterol is composed of (S)-albuterol.

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of β-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from “system bias” with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.