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 αq contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of αq in signaling was addressed. For αq 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 αq 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 αq beyond simple plasma membrane localization. It is noteworthy that an αq4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although αq4Q 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 αq4Q showed a differing localization to plasma membrane nanodomains compared with wild-type αq. Thus, basic amino acids in the N terminus of αq can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of αq4Q. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.