The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5.
Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for Gi, Gq, and G12 alpha-subunits of heterotrimeric G proteins. Although it is known that membrane association is critical for the biological activities of many RGS proteins, the mechanism underlying this requirement remains unclear. We reported recently that the NH2 terminus of RGS16 is required for its function in vivo. In this study, we show that RGS16 lacking the NH2 terminus is no longer localized to the plasma membrane as is the wild type protein, suggesting that membrane association is important for biological function. The region of amino acids 7-32 is sufficient to confer the membrane-targeting activity, of which amino acids 12-30 are predicted to adopt an amphipathic alpha-helix. Site-directed mutagenesis experiments showed that the hydrophobic residues of the nonpolar face of the helix and the strips of positively charged side chains positioned along the polar/nonpolar interface of the helix are crucial for membrane association. Subcellular fractionation by differential centrifugation followed by conditions that distinguish peripheral membrane proteins from integral ones indicate that RGS16 is a peripheral membrane protein. We show further that RGS16 membrane association does not require palmitoylation. Our results, together with other recent findings, have defined a unique membrane association domain with amphipathic features. We believe that these structural features and the mechanism of membrane association of RGS16 are likely to apply to the homologous domains in RGS4 and RGS5.
Regulatory Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore.
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Chen
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Seow
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Guo
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Yaw
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Lin
D000595:Amino Acid Sequence; D019204:GTP-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D015003:Yeasts
D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C098799:RGS4 protein; D019308:Palmitic Acid; D019204:GTP-Binding Proteins