To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This review will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.
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.
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 ␣ 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 ␣ q in signaling was addressed. For ␣ 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 ␣ q polybasic mutants by introduction of a site for myristoylation or by coexpression of ␥ failed to recover signaling, suggesting a role for N-terminal basic amino acids of ␣ q beyond simple plasma membrane localization. It is noteworthy that an ␣ 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 ␣ q 4Q failed to couple activated receptors to inositol phosphate production, it was able to bind ␥, 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 ␣ q 4Q showed a differing localization to plasma membrane nanodomains compared with wild-type ␣ q . Thus, basic amino acids in the N terminus of ␣ q can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of ␣ q 4Q.
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