Mechanisms governing subcellular localization and function of human RGS2.
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 alpha-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.
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. sheximer@cellbio.wustl.edu
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002942:Circular Dichroism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D006868:Hydrolysis; D008081:Liposomes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000096:biosynthesis* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D018076:DNA, Complementary; D008081:Liposomes; D008164:Luminescent Proteins; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; C105345:Rgs2 protein, mouse; D049452:Green Fluorescent Proteins