Interaction with Gβγ is required for membrane targeting and palmitoylation of Gα(s) and Gα(q)

Abstract

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein α subunits. For α(s) and α(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein βγ dimer for their proper plasma membrane localization and palmitoylation. Conserved α subunit N- terminal amino acids predicted to mediate binding to βγ were mutated to create a series of βγ binding region mutants expressed in HEK293 cells. These α(s) and α(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional α(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to βγ as a critical membrane attachment signal for α(s) and α(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of βγ binding-deficient α(q) subunits.