Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

Observations of Golgi fragmentation upon introduction of G protein βγ (Gβγ) subunits into cells have implicated Gβγ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where Gβγ acts to provoke Golgi fragmentation is not known. Additionally, a role for Gβγ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of Gβγ to the Golgi, but not other subcellular locations, causes phospholipase C- and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted β₁γ₂ also activates protein kinase D. Moreover, the novel Gβγ inhibitor, gallein, and the Gβγ-sequestering protein, GRK2ct, reveal that Gβγ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized Gβγ, endogenous Gβ was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized Gβγ in regulating protein transport from the TGN to the cell surface.     

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.     

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).     

Gβγ regulates mitotic Golgi fragmentation and G2/M cell cycle progression

Heterotrimeric G proteins (αβγ) function at the cytoplasmic surface of a cell’s plasma membrane to transduce extracellular signals into cellular responses. However, numerous studies indicate that G proteins also play noncanonical roles at unique intracellular locations. Previous work has established that G protein βγ subunits (Gβγ) regulate a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the exit of select protein cargo. Now, we demonstrate a novel role for Gβγ in regulating mitotic Golgi fragmentation, a key checkpoint of the cell cycle that occurs in the late G2 phase. We show that small interfering RNA–mediated depletion of Gβ1 and Gβ2 in synchronized cells causes a decrease in the number of cells with fragmented Golgi in late G2 and a delay of entry into mitosis and progression through G2/M. We also demonstrate that during G2/M Gβγ acts upstream of protein kinase D and regulates the phosphorylation of the Golgi structural protein GRASP55. Expression of Golgi-targeted GRK2ct, a Gβγ-sequestering protein used to inhibit Gβγ signaling, also causes a decrease in Golgi fragmentation and a delay in mitotic progression. These results highlight a novel role for Gβγ in regulation of Golgi structure.     

G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein βγ subunits. Gβγ inhibition blocked ET-1–stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gβγ at the Golgi inhibited ET-1–dependent PI4P depletion and nuclear PKD activation. Translocation of Gβγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. Finally, blocking Gβγ at the Golgi or PM blocked ET-1–dependent cardiomyocyte hypertrophy. These data indicate that Gβγ regulation of the perinuclear Golgi PI4P pathway and a separate pathway at the PM is required for ET-1–stimulated hypertrophy, and the efficacy of Gβγ inhibition in preventing heart failure maybe due in part to its blocking both these pathways.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gα_o-, Gα_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Activation of Microtubule Dynamics Increases Neuronal Growth via the Nerve Growth Factor (NGF)- and Gαs-mediated Signaling Pathways

Signals that activate the G protein Gα_s and promote neuronal differentiation evoke Gα_s internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gα_s association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gα_s. To determine the function of Gα_s/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gα_s fusion protein. GFP-Gα_s concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gα_s translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gα_s binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gα_s. Together, these data suggest that activated Gα_s translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.     

Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.     

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα₁₃ downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα₁₃, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα₁₃. Rgnef co-immunoprecipitated with activated Gα₁₃Q226L but not Gα₁₂Q229L. The Rgnef C-terminal (CT, 1279–1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα₁₃-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα₁₃. Point mutations of Rgnef-CT residues disrupt association with active Gα₁₃ but not Gα_q. These results show that Rgnef functions as an effector of Gα₁₃ signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.     

Regulation of G-Protein Signaling by RKTG via Sequestration of the Gβγ Subunit to the Golgi Apparatus

Upon ligand binding, G-protein-coupled receptors (GPCRs) impart the signal to heterotrimeric G proteins composed of α, β, and γ subunits, leading to dissociation of the Gα subunit from the Gβγ subunit. While the Gα subunit is imperative for downstream signaling, the Gβγ subunit, in its own right, mediates a variety of cellular responses such as GPCR desensitization via recruiting GRK to the plasma membrane and AKT stimulation. Here we report a mode of spatial regulation of the Gβγ subunit through alteration in subcellular compartmentation. RKTG (Raf kinase trapping to Golgi apparatus) is a newly characterized membrane protein specifically localized at the Golgi apparatus. The N terminus of RKTG interacts with Gβ and tethers Gβγ to the Golgi apparatus. Overexpression of RKTG impedes the interaction of Gβγ with GRK2, abrogates the ligand-induced change of subcellular distribution of GRK2, reduces isoproterenol-stimulated phosphorylation of the β2-adrenergic receptor (β2AR), and alters β2AR desensitization. In addition, RKTG inhibits Gβγ- and ligand-mediated AKT phosphorylation that is enhanced in cells with downregulation of RKTG. Silencing of RKTG also alters GRK2 internalization and compromises ligand-induced Gβ translocation to the Golgi apparatus. Taken together, our results reveal that RKTG can modulate GPCR signaling through sequestering Gβγ to the Golgi apparatus and thereby attenuating the functions of Gβγ.Heterotrimeric G proteins are composed of distinct Gα, β, and γ subunits which relay extracellular signals from heptahelical G-protein-coupled receptors (GPCRs) to downstream effectors (16, 25, 30). Gα binds Gβγ when Gα is bound with GDP but dissociates from Gβγ after GDP is replaced with GTP upon activation of GPCRs by extracellular ligand (25). Under physiologic conditions, the Gβ and Gγ subunits form a dimer in which the two subunits are not separable (10, 30). Although Gα is the primary protein that transmits the signal of GPCRs to specific intracellular effectors, such as adenylyl cyclase and phospholipase C, emerging evidence has indicated that Gβγ is able to regulate GPCR signaling through interacting with GPCRs, the Gα subunit, and downstream effectors (30). Predominantly, Gβγ is able to directly interact with and affect the functions of a variety of membrane and intracellular effectors, such as ion channels, adenylyl cyclase, G-protein-coupled receptor kinases (GRKs), and phosphatidylinositol 3-kinase (PI3K) (30). The current model of Gβγ-mediated signaling restricts it mostly to the plasma membrane (PM) (30). In the case of membrane-bound effectors, such as adenylyl cyclases or GIRK channels, Gβγ regulates the activities of these transmembrane proteins through conformational alteration. In the case of cytosolic proteins such as PLCβ2 or GRK2, whose substrates are localized to PM, Gβγ regulates their activity by recruiting the proteins to PM. The activity of Gβγ is primarily regulated by GPCR and Gα, in which GPCR activation leads to conformational changes of Gα. Such change causes replacement of Gα-bound GDP with GTP and release of Gβγ from the heterotrimeric G proteins. The activity of Gβγ could also be regulated by interacting with cytosolic proteins such as RACK1 (7). However, how Gβγ-mediated signaling is regulated in a spatial manner via subcellular compartmentation is largely unknown.GRK2 is a member of a family of GRKs that can phosphorylate the agonist-occupied GPCRs (4). Specific phosphorylation of activated receptors is associated with a decreased responsiveness of GPCR to prolonged stimulation by the agonist, also known as desensitization (15, 26). Gβγ regulates the activities of GRK2 and GRK3 toward several GPCRs (9). In cooperation with phosphatidylinositol 4,5-bisphosphate, Gβγ binds to the pleckstrin homology (PH) domain of GRK2 and recruits GRK2 to PM, in which it phosphorylates activated GPCRs (18, 30). The crystallographic structure of GRK2 in complex with Gβ1γ2 has been solved (20, 32). On the other hand, AKT is an intracellular target of PI3K and plays a critical role in cell growth, proliferation, and survival. It has been reported that Gβγ could activate AKT in a PI3K-dependent fashion (5), and Gβγ could mediate AKT activation at endosomes (13). Recent data also indicate that the p110β subunit of PI3K signals downstream of GPCR, and the AKT activation mediated by p110β is G protein dependent (14, 17).PAQR3 is a member of the progestin and adipoQ receptor (PAQR) family, and the members of this family are predicted to have seven transmembrane domains similar to GPCRs (31). Recently, we demonstrated that PAQR3 is localized at the Golgi apparatus and is involved in the spatial regulation of Raf kinase, whereby this protein was named Raf kinase trapping to Golgi apparatus (RKTG) (12). Biochemical analysis of RKTG suggested that its N terminus is localized on the cytoplasmic side of the Golgi membrane (21). Using the N terminus of RKTG to screen a Saccharomyces cerevisiae two-hybrid library, we determined that RKTG is able to interact with Gβ, and detailed analyses indicate that RKTG is a spatial regulator of Gβγ signaling.     

A Chemical Biology Approach Demonstrates G Protein βγ Subunits Are Sufficient to Mediate Directional Neutrophil Chemotaxis

Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ “hot spot.” M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca²⁺ release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gα_i1β₁γ₂ heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.     

Integration of Fourier Transform Infrared Spectroscopy,Fluorescence Spectroscopy,Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gα_i1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg²⁺ binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s⁻¹ at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm⁻¹, respectively), α- and β-GDP (1214 and 1134/1103 cm⁻¹, respectively), and free phosphate (1078/991 cm⁻¹). In contrast to Ras·GAP catalysis, the bond breakage of the β-γ-phosphate but not the P_i release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gα_i1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gα_i1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gα_i1-D272N) and Ras-like/all-α interface coordination (Gα_i1-D229N/Gα_i1-D231N) on the nucleotide exchange reaction was furthermore elucidated.     

Biased Signaling at Chemokine Receptors

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as β-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gα_i subtypes (Gα_i1, Gα_i2, and Gα_i3) and the two Gα_o isoforms (Gα_oa and Gα_ob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα₁₂, but not Gα₁₃. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.     

Structure of the Regulator of G Protein Signaling 8 (RGS8)-Gαq Complex: MOLECULAR BASIS FOR Gα SELECTIVITY*

Regulator of G protein signaling (RGS) proteins interact with activated Gα subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Gα_i/o and Gα_q/11 subunits as substrates, the R7 and R12 subfamilies select against Gα_q/11. In contrast, only one RGS protein, RGS2, is known to be selective for Gα_q/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Gα_q revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Gα subunit, or both. To resolve this ambiguity, the 2.6 Å crystal structure of RGS8, an R4 subfamily member, was determined in complex with Gα_q. RGS8 adopts the same pose on Gα_q as it does when bound to Gα_i3, indicating that the non-canonical interaction of RGS2 with Gα_q is due to unique features of RGS2. Based on the RGS8-Gα_q structure, residues in RGS8 that contact a unique α-helical domain loop of Gα_q were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Gα_q seems more likely determined by whether strong contacts can be maintained between α6 of the RGS domain and Switch III of Gα_q, regions of high sequence and conformational diversity in both protein families.     

Identification of G Protein α Subunit-Palmitoylating Enzyme

The heterotrimeric G protein α subunit (Gα) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Gα palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Gα_q, Gα_s, and Gα_i2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Gα_q/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Gα_q rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Gα_q shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the α_1A-adrenergic receptor/Gα_q/11-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Gα localization to the plasma membrane.G-protein-coupled receptors (GPCRs) form the largest family of cell surface receptors, consisting of more than 700 members in humans. GPCRs respond to a variety of extracellular signals, including hormones and neurotransmitters, and are involved in various physiologic processes, such as smooth muscle contraction and synaptic transmission (20, 25). Heterotrimeric G proteins, composed of α, β, and γ subunits, transduce signals from GPCRs to their effectors and play a central role in the GPCR signaling pathway (13, 21, 24, 32). Although the Gα subunit seems to localize stably at the cytosolic face of the plasma membrane (PM), a recent report suggested that Gα_o, a Gα isoform, shuttles rapidly between the PM and intracellular membranes (2). The PM targeting of Gα requires both interaction with the Gβγ complex and subsequent lipid palmitoylation of Gα (22). Thus, palmitoylation of Gα is a critical determinant of membrane targeting of the heterotrimer Gαβγ.Protein palmitoylation is a common posttranslational modification with lipid palmitate and regulates protein trafficking and function (7, 18). Gα is a classic and representative palmitoyl substrate (19, 38), and recent studies revealed that protein palmitoylation modifies virtually almost all the components of G-protein signaling, including GPCRs, Gα subunits, several members of the RGS (regulators of G-protein signaling) family of GTPase-activating proteins, GPCR kinase GRK6, and some small GTPases (7, 33). This common lipid modification plays an important role in compartmentalizing G-protein signaling to the specific microdomain, such as membrane caveolae and lipid raft (26). The palmitoyl thioester bond is relatively labile, and palmitates on substrates turn over rapidly, allowing proteins to shuttle between the cytoplasm/intracellular organelles and the PM (2, 3, 27). For example, binding of isoproterenol to the β-adrenergic receptor markedly accelerates the depalmitoylation of the associated Gα_s, shifting Gα_s to the cytoplasm (37). This receptor activation-induced depalmitoylation was also observed in a major postsynaptic PSD-95 scaffold, which anchors the AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-type glutamate receptor at the excitatory postsynapse through stargazin (6). On glutamate receptor activation, accelerated depalmitoylation of PSD-95 dissociates PSD-95 from postsynaptic sites and causes AMPA receptor endocytosis (6). Thus, palmitate turnover on Gα_s and PSD-95 is accelerated by receptor activation, contributing to downregulation of the signaling pathway. However, the enzymes that add palmitate to proteins (palmitoyl-acyl transferases [PATs]) and those that cleave the thioester bond (palmitoyl-protein thioesterases) were long elusive.Recent genetic studies in Saccharomyces cerevisiae identified Erf2/Erf4 (1, 40) and Akr1 (29) as PATs for yeast Ras and yeast casein kinase 2, respectively. Erf2 and Akr1 have four- to six-pass transmembrane domains and share a common domain, referred to as a DHHC domain, a cysteine-rich domain with a conserved Asp-His-His-Cys signature motif. Because the DHHC domain is essential for the PAT activity, we isolated 23 mammalian DHHC domain-containing proteins (DHHC proteins) and developed a systematic screening method to identify the specific enzyme-substrate pairs (11, 12): DHHC2, -3, -7, and -15 for PSD-95 (11); DHHC21 for endothelial NO synthase (10); and DHHC3 and -7 for GABA_A receptor γ2 subunit (9). Several other groups also reported that DHHC9 with GCP16 mediates palmitoylation toward H- and N-Ras (36) and that DHHC17, also known as HIP14, palmitoylates several neuronal proteins: huntingtin (14), SNAP-25, and CSP (14, 23, 35). However, the existence of PATs for Gα has been controversial because spontaneous palmitoylation of Gα could occur in vitro (4).In this study, we screened the 23 DHHC clones to examine which DHHC proteins can palmitoylate Gα. We found that DHHC3 and -7 specifically and robustly palmitoylate Gα at the Golgi apparatus. Inhibition of DHHC3 and -7 reduces Gα_q/11 palmitoylation levels and delocalizes it from the PM to the cytoplasm in HeLa cells and primary hippocampal neurons. Also, DHHC3 and -7 are necessary for the continuous Gα_q shuttling between the Golgi apparatus and the PM. Finally, blocking DHHC3 and -7 inhibits the α_1A-adrenergic receptor [α_1A-AR]/Gα_q-mediated signaling pathway, indicating that DHHC3 and -7 play an essential role in GPCR signaling by regulating Gα localization.