RGS7 is palmitoylated and exists as biochemically distinct forms.
Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Galpha and Gbeta5 are enriched in brain, but biochemical mechanisms governing RGS7/Galpha/Gbeta5 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 Gbeta5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gbeta5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with Gbeta5, are equally effective stimulators of Galpha(o) GTPase activity, suggesting that palmitoylation does not prevent RGS7/Galpha(o) interactions. The isolated core RGS domain of RGS7 selectively binds activated Galpha(i/o) in brain extracts and is an effective stimulator of both Galpha(o) and Galpha(i1) GTPase activities in vitro. In contrast, the RGS7/Gbeta5 complex selectively interacts with Galpha(o) only, suggesting that features outside the RGS domain and/or Gbeta5 association dictate RGS7-Galpha interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.
Rose
J J
JJ
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090, USA
Taylor
J B
JB
Shi
J
J
Cockett
M I
MI
Jones
P G
PG
Hepler
J R
JR
D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry; D019308:Palmitic Acid / Q000737:chemistry*; D054799:Phospholipase C beta; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera; D010738:Type C Phospholipases / Q000737:chemistry
D003902:Detergents; D044387:GTP-Binding Protein beta Subunits; C490610:Gnb5 protein, rat; D007527:Isoenzymes; D020710:RGS Proteins; D011994:Recombinant Proteins; C492038:Rgs7 protein, mouse; D019308:Palmitic Acid; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins