Members of the regulators of G protein signaling (RGS) family modulate Ga-directed signals as a result of the GTPaseactivating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To de®ne the cellular and biochemical properties of RGS14 we utilized two different af®nity puri®ed antisera that speci®cally recognize recombinant and native RGS14. In brain, we observed two RGS14-like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent-extractable and the other detergent-insensitive. Recombinant RGS14 binds speci®cally to activated Ga i/o , but not Ga q/11 , Ga 12/13 , or Ga s in brain membranes. In reconstitution studies, we found that RGS14 is a non-selective GAP for Ga i1 and Ga o and that full-length RGS14 is an approximately 10-fold more potent stimulator of Ga GTPase activity than the RGS domain alone. In contrast, neither fulllength RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Ga i but not Ga o , and this activity is restricted to the C-terminus containing the GoLoco domain. These ®ndings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the ®rst examples of an RGS protein that is a bifunctional regulator of Ga actions.

R គ egulator of G គ protein s គignaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners G␣ and G␤5 are enriched in brain, but biochemical mechanisms governing RGS7/G␣/G␤5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, 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 membranebound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. G␤5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membranederived) forms of RGS7, when complexed with G␤5, are equally effective stimulators of G␣ o GTPase activity, suggesting 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 features outside the RGS domain and/or G␤5 association dictate RGS7-G␣ interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated. Key Words: Regulator of G protein signaling proteins-G proteins-RGS7-G␤5-Palmitate.

No abstract