Philip B. Wedegaertner

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_q4Q 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_q4Q 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_q4Q 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_q4Q.

PDZ-RhoGEF is a member of the regulator family of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein α subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561 and 585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as being necessary for binding to actin and for colocalization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate an LIxxFE motif, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin-binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure in a manner independent of its ability to activate RhoA.

G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

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 current topic 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.

The G-protein α subunit, α₁₃, regulates cell growth and differentiation through the monomeric Rho GTPase. α₁₃ activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for Gα₁₃. The mechanism for the Gα₁₃/p115RhoGEF interaction is not well understood. Here, we determined specific α₁₃ residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating α₁₃ residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated α₁₃ with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated α₁₃ to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an α₁₃ mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other α disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical α/RGS interaction are preserved in the α₁₃/p115RhoGEF interaction, there are also significant differences.

Regulator of G protein signaling (RGS) proteins modulate G protein signaling by acting as GTPase-activating proteins for G protein α-subunits. RGS7 belongs to a subfamily of RGS proteins that exist as dimers with the G protein β₅-subunit. In this report, we addressed the mechanisms of plasma membrane localization of β₅RGS7. When expressed in human embryonic kidney 293 cells, β₅RGS7 was found to be cytoplasmic and soluble. Expression of α_o promoted a strong redistribution of β₅RGS7 to the plasma membrane. Expression of α_q, however, failed to affect the subcellular localization of β₅RGS7. The constitutively active mutant α_oR179C, like wild-type α_o, strongly recruited β₅RGS7 to plasma membranes; however, inactive α_oG204A, RGS-insensitive α_oG184S, and lipidation-deficient α_oG2A were all defective in the ability to promote plasma membrane localization of β₅RGS7. In addition, palmitoylation of RGS7 was demonstrated, and palmitoylation required expression of α_o or α_oR179C. To examine potential palmitoylation sites of RGS7, several cysteines were substituted with serines. β₅RGS7C133S failed to localize to plasma membranes when coexpressed with α_o, suggesting cysteine 133 of RGS7 as a putative palmitoylation site. Finally, deletion of amino acids 76 to 128 of RGS7, which includes part of the disheveled, EGL-10, pleckstrin (DEP) domain, prevented α_o-mediated plasma membrane recruitment of β₅RGS7. These findings are the first to demonstrate Gα-regulated plasma membrane localization and palmitoylation of β₅RGS7 and suggest that membrane targeting of β₅RGS7 is a complex process requiring at least RGS domain-mediated interaction with α_o and RGS7 palmitoylation.