N-terminal polybasic motifs are required for plasma membrane localization of Gαs and Gαq

Abstract

Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein α subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with βγ subunits, facilitate membrane localization. Here we show that an additional membrane targeting signal, an N-terminal polybasic region, plays a key role in plasma membrane localization of non-myristoylated α subunits. Mutations of N-terminal basic residues in αs and αq caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In αs, mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in αq a defect in membrane localization was not observed unless nine basic residues were mutated to glutamine or if three basic residues were mutated to glutamic acid. βγ co-expression only partially rescued the membrane localization defects; thus, the polybasic region is also important in the context of the heterotrimer. Introduction of a site for myristoylation into the polybasic mutants of αs and αq recovered strong plasma membrane localization, indicating that myristoylation and polybasic motifs may have complementary roles as membrane targeting signals. Loss of plasma membrane localization coincided with defects in palmitoylation. The polybasic mutants of αs and αq were still capable of assuming activated conformations and stimulating second messenger production, as demonstrated through GST-RGS4 interaction assays, cAMP assays, and inositol phosphate assays. Electrostatic interactions with membrane lipids have been found to be important in plasma membrane targeting of many proteins, and these results provide evidence that basic residues play a role in localization of G protein α subunits.

An N-Terminal Polybasic Motif of GαqIs Required for Signaling and Influences Membrane Nanodomain Distribution

“…Consistent with this variability, mutation of basic residues in other ␣ q family members, ␣ 14 and ␣ 16 , had minimal effects on their PM targeting or palmitoylation (Pedone and Hepler, 2007). Defects in PM localization of polybasic mutants of ␣ s and ␣ q could be overcome by myristoylation of these subunits, supporting the proposal that these targeting signals, myristoylation and polybasic motifs, have overlapping roles in promoting PM localization of G␣ (Kosloff et al, 2002;Crouthamel et al, 2008).…”

“…It is noteworthy that these polybasic motifs lie on the opposite face of the N-terminal helix from the G␤␥-contacting residues, and therefore allow G␣, even when bound to G␤␥, to make electrostatic interactions with a negatively charged interface of the PM. Mutation of residues in the polybasic motifs of ␣ s and ␣ q caused defects in PM localization and palmitoylation (Crouthamel et al, 2008). Substitution of four basic residues with neutral glutamines in the N terminus of ␣ s was sufficient to decrease its PM localization, as observed by immunofluorescence microscopy and cell fractionation.…”

“…However, when ␣ 2A AR was coexpressed with wild-type ␣ q , agonist-dependent inositol phosphate stimulation was observed. First, we examined four polybasic mutants that properly localized at the PM (Crouthamel et al, 2008). ␣ q 3Q (mutations K33Q, R37Q, R38Q), ␣ q 2Q (mutations R19Q, R20Q), ␣ q 3Qn (mutations K16Q, R19Q, R20Q), and ␣ q 2E (mutations R19E, R20E) were able to promote ␣ 2A AR agonist-dependent increases in inositol phosphate levels (Fig.…”

“…Our recent work examined the importance of N-terminal basic amino acids of ␣ q for its PM localization (Crouthamel et al, 2008). Extensive mutagenesis of the 10 basic amino acids, either arginines or lysines, located between amino acids 16 to 34 (Fig.…”

“…Recent work has demonstrated that clusters of basic amino acid side chains also contribute to PM localization of G proteins (Pedone and Hepler, 2007;Crouthamel et al, 2008). Polybasic motifs are present in the N termini of nonmyristoylated G␣ family members, namely ␣ s , ␣ q , and ␣ 12/13 (Kosloff et al, 2002).…”