title: Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members.
abstract: RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated Galpha(q). Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or Galpha(q). Together, these data indicate that novel features within the RGS2 amphipathic alpha helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.
authors.affiliation: Department of Physiology, Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada.
authors.forename: Steven
authors.initials: S
authors.lastname: Gu
authors.forename: Janet
authors.initials: J
authors.lastname: He
authors.forename: Wing-Ting
authors.initials: WT
authors.lastname: Ho
authors.forename: Suneela
authors.initials: S
authors.lastname: Ramineni
authors.forename: David M
authors.initials: DM
authors.lastname: Thal
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Natesh
authors.forename: John J G
authors.initials: JJ
authors.lastname: Tesmer
authors.forename: John R
authors.initials: JR
authors.lastname: Hepler
authors.forename: Scott P
authors.initials: SP
authors.lastname: Heximer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D020710:RGS Proteins / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D010716:Phosphatidylinositols; D020710:RGS Proteins; D043643:Receptor, Muscarinic M1; D007930:Leucine; D002118:Calcium