title: An N-terminal polybasic motif of Gαq is required for signaling and influences membrane nanodomain distribution.
abstract: Regions of basic amino acids in proteins can promote membrane localization through electrostatic interactions with negatively charged membrane lipid head groups. Previous work showed that the heterotrimeric G protein subunit α(q) contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of α(q) in signaling was addressed. For α(q) mutants, loss of plasma membrane localization correlated with loss of signaling function, as measured by the ability to couple activated G protein-coupled receptors (GPCRs) to stimulation of inositol phosphate production. However, recovery of plasma membrane localization of α(q) polybasic mutants by introduction of a site for myristoylation or by coexpression of βγ failed to recover signaling, suggesting a role for N-terminal basic amino acids of α(q) beyond simple plasma membrane localization. It is noteworthy that an α(q)4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although α(q)4Q failed to couple activated receptors to inositol phosphate production, it was able to bind βγ, bind RGS4 in an activation-dependent manner, stimulate inositol phosphate production in a receptor-independent manner, and productively interact with a GPCR in isolated membranes. It is noteworthy that α(q)4Q showed a differing localization to plasma membrane nanodomains compared with wild-type α(q). Thus, basic amino acids in the N terminus of α(q) can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of α(q)4Q.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 S. 10th Street, 839 BLSB, Philadelphia, PA 19107, USA.
authors.forename: Marykate
authors.initials: M
authors.lastname: Crouthamel
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Cherisse
authors.initials: C
authors.lastname: DiLizio
authors.forename: David R
authors.initials: DR
authors.lastname: Manning
authors.forename: John F
authors.initials: JF
authors.lastname: Hancock
authors.forename: Philip B
authors.initials: PB
authors.lastname: Wedegaertner
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D049329:Nanostructures / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D018411:Spodoptera / Q000166:cytology / Q000235:genetics
chemical_list: D024361:Amino Acids, Basic; D010446:Peptide Fragments; D043802:GTP-Binding Protein alpha Subunits, Gq-G11