Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

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 alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(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 alpha(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 betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.     

N-terminal polybasic motifs are required for plasma membrane localization of Galpha(s) and Galpha(q)

Heterotrimeric G proteins typically localize at the cytoplasmic face of the plasma membrane where they interact with heptahelical receptors. For G protein alpha subunits, multiple membrane targeting signals, including myristoylation, palmitoylation, and interaction with betagamma 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 alpha subunits. Mutations of N-terminal basic residues in alpha(s) and alpha(q) caused defects in plasma membrane localization, as assessed through immunofluorescence microscopy and biochemical fractionations. In alpha(s), mutation of four basic residues to glutamine was sufficient to cause a defect, whereas in alpha(q) 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. betagamma 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 alpha(s) and alpha(q) 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 alpha(s) and alpha(q) 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 alpha subunits.     

N-Myristoylation and betagamma play roles beyond anchorage in the palmitoylation of the G protein alpha(o) subunit

Many of the alpha subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of alpha subunits from the G(i) family is preceded by N-myristoylation, which alone or together with betagamma probably supports a reversible interaction of the alpha subunit with membrane as a prerequisite to the eventual incorporation of palmitate. Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with betagamma, or other events, is sufficient for palmitoylation. We report here for alpha(o) that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of alpha(o) designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [(3)H]palmitate. Mutants of alpha(o) unable to interact normally with betagamma yet still attach to membrane demonstrate that betagamma, in contrast, is not required for palmitoylation. betagamma becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and betagamma, while almost certainly relevant to the reversible interaction of alpha(o) with membrane, also play at least partly overlapping, post-anchorage roles in palmitoylation.     

The importance of N-terminal polycysteine and polybasic sequences for G14alpha and G16alpha palmitoylation, plasma membrane localization, and signaling function

Plasma membrane targeting of G protein alpha (Galpha) subunits is essential for competent receptor-to-G protein signaling. Many Galpha are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqalpha,G11alpha,G14alpha, and G16alpha) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14alpha and G16alpha. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14alpha and G16alpha. We find that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G16alpha-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14alpha and G16alpha expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16alpha, more so than G14alpha, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Galpha palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.     

Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-mediated signaling.

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein alpha subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Gialpha. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Galpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Galpha subunits on cellular membranes.     

Yeast One-Hybrid Gγ Recruitment System for Identification of Protein Lipidation Motifs

Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs.     

Sequence landmark patterns identify and characterize protein families

The three-dimensional structures of homologous proteins are usually conserved during evolution, as are critical residues in a few short sequence motifs that often constitute the active site in enzymes. The precise spatial organization of such sites depends on the lengths and positions of the secondary structural elements connecting the motifs. We show how members of protein superfamilies, such as kinesins, myosins, and G(alpha) subunits of trimeric G proteins, are identified and classed by simply counting the number of amino acid residues between important sequence motifs in their nucleotide triphosphate-hydrolyzing domains. Subfamily-specific landmark patterns (motif to motif scores) are principally due to inserts and gaps in surface loops. Unusual protein sequences and possible sequence prediction errors are detected.     

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.     

A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.     

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol- anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.     

Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN- enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).     

Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling

The G protein subunit, betagamma, plays an important role in targeting alpha subunits to the plasma membrane and is essential for binding and activation of the heterotrimer by heptahelical receptors. Mutation of residues in the N-terminal alpha-helix of alpha s and alpha q that contact betagamma in the crystal structure of alpha i reduces binding between alpha and betagamma, inhibits plasma membrane targeting and palmitoylation of the alpha subunit, and results in G proteins that fail to couple receptor activation to stimulation of effector. Overexpression of betagamma can recover this loss of signaling through Gs but not Gq. In fact, a single mutation (I25A) in alpha q can block alpha q-mediated generation of inositol phosphates. Function is not recovered by betagamma overexpression nor myristoylation directed plasma membrane localization. Introduction of a Q209L activating mutation with I25A results in a constitutively active alpha q as expected, but surprisingly a R183C activating mutation does not result in constitutive activity when present with I25A. Examination of binding between alpha and betagamma via a pull down assay shows that the N-terminal betagamma-binding mutations inhibit alpha-betagamma binding significantly more than the R183C or Q209L activating mutations do. Moreover, introduction of the I25A mutation into alpha q RC disrupts co-immunoprecipitation with PLCbeta1. Taken together, results presented here suggest that alpha-betagamma binding is necessary at a point downstream from receptor activation of the heterotrimeric G protein for signal transduction by alpha q.     

Interaction of alpha1-syntrophin with multiple isoforms of heterotrimeric G protein alpha subunits

Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.     

G protein selectivity is a determinant of RGS2 function

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Galpha subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward G(q) versus G(i) family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of G(q)-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of G(i)-mediated signaling. RGS2 mutants were identified that display increased potency toward G(i) family members without affecting potency toward G(q). These mutations and the structure of RGS4-G(i)alpha(1) complexes suggest that RGS2-G(i)alpha interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the alpha8-alpha9 loop of RGS2 and alphaA of G(i) class alpha subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Galpha subunits.     

Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins

Many receptors for neurotransmitters and hormones rely upon members of the Gqalpha family of heterotrimeric G proteins to exert their actions on target cells. Galpha subunits of the Gq class of G proteins (Gqalpha, G11alpha, G14alpha and G15/16alpha) directly link receptors to activation of PLC-beta isoforms which, in turn, stimulate inositol lipid (i.e. calcium/PKC) signalling. Although Gqalpha family members share a capacity to activate PLC-beta, they also differ markedly in their biochemical properties and tissue distribution which predicts functional diversity. Nevertheless, established models suggest that Gqalpha family members are functionally redundant and that their cellular responses are a result of PLC-beta activation and downstream calcium/PKC signalling. Growing evidence, however, indicates that Gqalpha, G11alpha, G14alpha and G15/16alpha are functionally diverse and that many of their cellular actions are independent of inositol lipid signalling. Recent findings show that Gqalpha family members differ with regard to their linked receptors and downstream binding partners. Reported binding partners distinct from PLC-beta include novel candidate effector proteins, various regulatory proteins, and a growing list of scaffolding/adaptor proteins. Downstream of these signalling proteins, Gqalpha family members exhibit unexpected differences in the signalling pathways and the gene expression profiles they regulate. Finally, genetic studies using whole animal models demonstrate the importance of certain Gqalpha family members in cardiac, lung, brain and platelet functions among other physiological processes. Taken together, these findings demonstrate that Gqalpha, G11alpha, G14alpha and G15/16alpha regulate both overlapping and distinct signalling pathways, indicating that they are more functionally diverse than previously thought.     

Whither goest the RGS proteins?

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits - the "regulator of G protein signaling" or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.     

Whither Goest the RGS Proteins?

Studies of the desensitization of G protein-coupled signal transduction have led to the discovery of a family of guanosine triphosphatase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits — the “regulator of G protein signaling” or RGS proteins. In considering both documented and potential functions of several RGS protein family members with demonstrable multidomain compositions (p115RhoGEF, PDZRhoGEF, Axin, Axil/Conductin, D-AKAP2, the G protein-coupled receptor kinases [GRKs], the DEP/GGL/RGS subfamily [RGS6, RGS7, RGS9, RGS11], and RGS12), this review explores the shift in our appreciation of the RGS proteins from unidimensional desensitizing agents to multifocal signal transduction regulators.     

Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi

Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane.