Mechanisms Governing Subcellular Localization and Function of Human RGS2

Abstract

RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells. Expression of activated G q increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain is an amphipathic ␣-helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G q . Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear accumulation of RGS2 does not limit its ability to attenuate G q signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes in the nucleus.Many hormones, neurotransmitters, and sensory stimuli elicit specific physiological responses in target tissues by activating receptors that are coupled to heterotrimeric G proteins 1 (1, 2). Activated receptors promote exchange of GTP for GDP on G␣ subunits leading to dissociation of GTP-bound G␣ subunits from G␤␥ heterodimers and activation of downstream effector pathways. Signals are terminated following G␣-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins act as molecular switches to coordinate the wide range of responses elicited by extracellular stimuli.The regulators of G protein signaling (RGS) proteins are a large family that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein ␣ subunits (3-6). The GAP activity of RGS proteins shortens the half-life of active, GTP-bound G␣ subunits and leads to attenuation of a response during prolonged stimulation or accelerated termination of the signal following removal of agonist (7-11). Binding of RGS proteins to active G␣ subunits can also interfere with effector binding, thereby blocking activation and downstream signaling (12). These activities are mediated by the ϳ120-amino acid RGS domain, a conserved feature of this protein family.Genetic studies in budding yeast (13) and Caenorhabditis elegans (14) indicate that eukaryotes express several types of RGS proteins to provide selective regulation of distinct G protein signaling pathways, and a means of differentially regulating a single pathway in response to various physiologic conditions. Similarly, certain mammalian RGS proteins have b...

A Predicted Amphipathic Helix Mediates Plasma Membrane Localization of GRK5

“…Amphipathic helix membrane-targeting motifs have been identified in several other signaling proteins, including the regulator of G protein signaling (RGS) proteins RGS2, RGS4, and RGS16 (17)(18)(19) and the small GTPase ARF1 (20). For the RGS proteins, helical models predict a hydrophobic face and a hydrophilic face rich in positive charged amino acids, and, similar to the results described here for GRK5, both the hydrophobic and basic residues contribute to membrane binding.…”

“…The other side of the helix consists mostly of hydrophilic residues, and a number of the basic residues are predicted to surround the hydrophobic patch. Thus, an amphipathic helical membrane binding motif, similar to that proposed recently for several other proteins (17)(18)(19)(20), is predicted to exist within amino acids 546 -565 in GRK5. The hydrophobic residues can directly insert into membrane lipids, while the surrounding positively charged amino acids would further enhance membrane binding by interacting with acidic phospholipids.…”

“…1A). Analysis of the predicted helical region by a helical wheel or helical net projection (17) (Fig. 1, B and C) reveals an amphipathicity for this membrane-targeting region.…”

RGS2 Binds Directly and Selectively to the M1 Muscarinic Acetylcholine Receptor Third Intracellular Loop to Modulate Gq/11α Signaling

“…ous reports (13,27). These data suggest that membrane-targeting factors intrinsic to RGS2 are sufficient for its localization at the plasma membrane, as is the case for RGS4 (28).…”

“…Thus, it is likely that the relative ratios of G protein subunits and receptor could influence which interaction dictates the response. Consistent with this idea, overexpression of G␣ subunits is sufficient to recruit RGS2 and RGS4 to the plasma membrane (13,27,38). Alternatively, the mechanism for RGS recruitment among G i -linked receptors may differ from that of G q -linked receptors such that different regions of the receptor bind to the RGS.…”