RGS7 Is Palmitoylated and Exists as Biochemically Distinct Forms Jeremy J. Rose, Jackie B. Taylor, *Jing Shi, ²Mark I. Cockett, ²Philip G. Jones, and John R. Hepler Departments of Pharmacology and *Biochemistry, Emory University School of Medicine, Atlanta, Georgia; and ² Department of Neuroscience, Wyeth-Ayerst Research, Princeton, New Jersey, U.S.A. Abstract: R  egulator of G  protein s  ignaling (RGS) proteins are GTPase-activating proteins that modulate neuro- transmitter and G protein signaling. RGS7 and its binding partners G and G5 are enriched in brain, but biochem- ical mechanisms governing RGS7/G/G5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemi- cally distinct membrane-bound fractions (salt-extract- able, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and G5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane- bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. G5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane- derived) forms of RGS7, when complexed with G5, are equally effective stimulators of G o GTPase activity, sug- gesting that palmitoylation does not prevent RGS7/G o interactions. The isolated core RGS domain of RGS7 selectively binds activated G i/o in brain extracts and is an effective stimulator of both G o and G i1 GTPase activities in vitro. In contrast, the RGS7/G5 complex selectively interacts with G o only, suggesting that fea- tures outside the RGS domain and/or G5 association dictate RGS7–G interactions. These findings define pre- viously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoy- lated. Key Words: Regulator of G protein signaling pro- teins—G proteins—RGS7—G5—Palmitate. J. Neurochem. 75, 2103–2112 (2000). R  egulator of G  protein s ignaling (RGS) proteins are newly appreciated modulators of neurotransmitter ac- tions. Many neurotransmitters and hormones rely on G proteins to activate intracellular signaling pathways that, in turn, regulate nearly all aspects of cell and organ physiology (Hepler and Gilman, 1992; Hamm, 1998). Activated receptors stimulate GTP binding to G sub- units, and the strength and duration of a particular G protein-directed signaling event are limited by the life- time of the G–GTP species. G protein signaling is terminated by intrinsic G GTPase activity, and RGS proteins act as GTPase-activating proteins (GAPs) for G subunits to modulate hormone and G protein-di- rected signaling (Dohlman and Thorner, 1997; Berman and Gilman, 1998; Koelle, 1997; De Vries and Farquhar, 1999; Hepler, 1999). RGS proteins are defined by a conserved 120-amino acid domain (RGS domain) that is both necessary and sufficient for the stimulatory effects of RGS proteins on G GTPase activity in vitro (Berman et al., 1996; Hunt et al., 1996; N. Watson et al., 1996; Hepler et al., 1997; Tesmer et al., 1997). Apart from this shared domain, RGS proteins differ considerably and possess a remark- able variety of structural domains and motifs that interact with a growing list of protein binding partners (De Vries and Farquhar, 1999; Hepler, 1999). RGS7 belongs to a subfamily of RGS proteins (including RGS6, RGS9, and RGS11) that share a DEP (dishevelled, egl-10, pleck- strin) and a GGL (G protein -like) domain (Koelle and Horvitz, 1996; Cabrera et al., 1998; Snow et al., 1998). GGL domains selectively bind the 5 isoform of G subunits in vitro (Cabrera et al., 1998; Snow et al., 1998, 1999; Makino et al., 1999), and native RGS proteins that possess the GGL domain from a complex with G5 in vivo (Cabrera et al., 1998; Makino et al., 1999). G5 is enriched in brain (Watson et al., 1994, 1996; Jones et al., 1999), and its amino acid sequence shares only 53% amino acid identity with the other closely related G Received February 11, 2000; revised manuscript received June 15, 2000; accepted July 13, 2000. Address correspondence and reprint requests to Dr. J. R. Hepler at Department of Pharmacology, Emory University School of Medicine, 5009 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322- 3090, U.S.A. E-mail: jhepler@emory.edu Abbreviations used: cRGS7, cytosolic regulator of G protein signal- ing 7; DEP, dishevelled, egl-10, pleckstrin; GAIP, G interacting protein; GAP, GTPase-activating protein; GGL, G protein -like; His6, hexahistidine; mRGS7, membrane regulator of G protein signaling 7; Ni-NTA, nickel–nitrilotriacetic acid; PLC1, phospholipase C1; RGS, regulator of G protein signaling; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Sf9, Spodoptera frugi- perda; tRGS7, truncated form of regulator of G protein signaling 7; TX, Triton X. 2103 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry