The Membrane Association Domain of RGS16 Contains Unique Amphipathic Features That Are Conserved in RGS4 and RGS5

Abstract

Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for G i , G q , and G 12 ␣-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 NH 2 terminus of RGS16 is required for its function in vivo. In this study, we show that RGS16 lacking the NH 2 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 ␣-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.Regulators of G protein signaling (RGS 1 proteins) have emerged as major modulators of diverse aspects of biological activities (1-4). It has been well established that RGS proteins act as GTPase-activating factors for many of the heterotrimeric G protein ␣-subunits (5-7). The core RGS domain is responsible for GTPase-activating protein activity, although it is not sufficient for biological function in vivo (8 -12).Many RGS proteins have been shown to be membrane-associated. GAIP behaves as an integral membrane protein as judged by its resistance to the stripping effect of Na 2 CO 3 treatment (13). Plasma membrane association has also been shown to be required for RGS4 function (12). RGSZ1, a G z -selective RGS protein in the brain, is also tightly membrane-bound and requires membrane association for its GTPase-activating protein activity toward G z (14, 15). However, it is unclear how they are targeted to the membrane. RET-RGS1, another potentially membrane-bound RGS member, contains a putative transmembrane domain and multiple cysteine residues for palmitoylation (9). GAIP and RGS4 also possess cysteine string motifs in their NH 2 terminus and have been shown to be palmitoylated (12, 13). Although palmitoylated GAIP is found only in pellet fractions, the functional significance of palmitoylation has yet to be established. Surprisingly, palmitoylation of RGS4 is shown not to be required for...

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.…”

The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signalling 16 through physical interaction

“…1). The N-terminal 33 aa, which are required for the association with the plasma membrane and RGS protein activity, are conserved within RGS4, RGS5 and RGS16 (Chen et al, 1999). Interestingly, our results indicate that an interaction between PCV2 ORF3 and RGS16 facilitates degradation of RGS16 through a ubiquitin-mediated proteasome-dependent pathway (Fig.…”

The BNIP-2 and Cdc42GAP Homology Domain of BNIP-2 Mediates Its Homophilic Association and Heterophilic Interaction with Cdc42GAP

“…Molecular Modeling and Sequence Alignments-The methods for modeling the BNIP-2 BCH domain were as described previously (25,31). One of the templates used in comparative modeling was the crystal structure of the breakpoint cluster region homology (BH) domain from the phosphatidylinositol 3-kinase p85␣ regulatory subunit (Protein Data Bank code: 1PBW).…”