Characterization of a phospholipase C beta 2-binding site near the amino-terminal coiled-coil of G protein beta gamma subunits

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 that does not overlap with the alpha(i1)-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta(1) which is located in the amino-terminal coiled-coil region of beta(1) and identifies a PLC-binding site distinct from the alpha subunit interaction site.     

Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.     

Modification of AlF-4- and receptor-stimulated phospholipase C activity by G-protein beta gamma subunits

Turkey erythrocyte membranes possess a phospholipase C that is markedly activated by P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of [3H]inositol-labeled turkey erythrocyte membranes with guanine nucleotide regulatory protein (G-protein) beta gamma subunits resulted in inhibition of both AlF-4-stimulated adenylate cyclase and AlF-4-stimulated phospholipase C activities. The apparent potency (K0.5 approximately 1 microgram or 20 pmol of beta gamma/mg of membrane protein) of beta gamma subunits for inhibition of each enzyme activity was similar and occurred with beta gamma purified by different methodologies from turkey erythrocyte, bovine brain, or human placenta membranes. In contrast to the effect on AlF-4-stimulated activity, the stimulatory effect on phospholipase C of the P2Y-purinergic receptor agonist 2-methylthioadenosine 5'-triphosphate in the presence of guanine nucleotides was potentiated by 50-100% in a concentration-dependent manner by reconstitution of beta gamma subunits. beta gamma subunits did not affect the K0.5 value of 2-methylthioadenosine 5'-triphosphate for the stimulation of phospholipase C activity. These results indicate that beta gamma subunits influence phospholipase C activity in a concentration range similar to that necessary for regulation of adenylate cyclase activity and suggest the involvement of a G-protein possessing an alpha beta gamma heterotrimeric structure in coupling hormone receptors to phospholipase C.     

Activation of cytosolic phosphoinositide phospholipase C by G-protein beta gamma subunits.

Bovine liver cytosol contains a phosphoinositide phospholipase C (PLCcyt) that is activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S)-activated G-proteins from liver plasma membranes. Heparin-Sepharose chromatography indicated that PLCcyt was immunologically distinct from PLC-beta 1, PLC-gamma 1, or PLC-delta 1 from brain. Initial purification of the GTP gamma S-activated G-proteins that stimulated PLCcyt indicated that the beta gamma complex was responsible. G-proteins were subsequently extracted from liver membranes as heterotrimers and purified in the presence of AlCl3, MgCl2, and NaF to allow reversible activation. Immunoblot analysis with an antiserum selective for the beta subunit showed that the stimulatory activity corresponded with the presence of this protein at every chromatographic step. When liver beta gamma complex was purified and separated from all detectable alpha subunits, as shown by immunoblotting and silver staining, it strongly stimulated PLCcyt after removal of the activating ligand [AlF4]- by gel filtration. beta gamma prepared from brain was approximately equipotent with that from liver. beta gamma was half-maximally effective at 33 nM and produced a maximal 50-fold activation of the PLC. Under identical conditions, beta gamma had no effect on brain PLC-gamma 1 or PLC-delta 1 and produced a 2-fold stimulation of PLC-beta 1 activity. Addition of purified GDP-bound alpha o, which had no effect by itself, completely reversed the beta gamma activation of PLCcyt, confirming that beta gamma was the active species. These data provide evidence for a novel mechanism by which beta gamma subunits of pertussis toxin-sensitive or -insensitive G-proteins activate phospholipase C.     

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.     

G蛋白βγ亚单位介导的信号转导途径

跨膜信息传递有关的Ｇ蛋白由α、β和γ亚单位所组成,受体激动后,引起ＧＴＰ与α亚单位结合,导致Ｇα与Ｇβγ分离。近年来发现Ｇα、受体本射和许多效应分子如Ｋ＾＋通道、Ｇａ＾２＋通道、磷脂酶Ｃ－β、腺苷酸环化酶、酷氨酸、ＭＡＰＫ和受体激酶等都受Ｇβγ的调节,Ｇβγ同Ｇα一样均可引起效应蛋白的激活,在细胞信号转导中起同样重要作用,共同介导一系列的生物学效应。     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.     

Role of the G protein gamma subunit in beta gamma complex modulation of phospholipase Cbeta function

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase C isozymes (PLCbetas). Different domains on the beta subunit are known to contact phospholipase Cbeta and affect its regulation. In contrast, the role of the gamma subunit in Gbetagamma modulation of PLCbeta function is not known. Results here show that the gamma subunit C-terminal domain is involved in mediating Gbetagamma interactions with phospholipase Cbeta. Mutations were introduced to alter the position of the post-translational prenyl modification at the C terminus of the gamma subunit with reference to the beta subunit. These mutants were appropriately post-translationally modified with the geranylgeranyl moiety. A deletion that shortened the C-terminal domain, insertions that extended this domain, and a point mutation, F59A, that disrupted the interaction of this domain with the beta subunit were all affected in their ability to activate PLCbeta to varying degrees. All mutants, however, interacted equally effectively with the G(o)alpha subunit. The results indicate that the G protein gamma subunit plays a direct role in the modulation of effector function by the betagamma complex.     

Distinct sites on G protein beta gamma subunits regulate different effector functions

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents. Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K(+) channels. With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors. These data show that distinct domains within Gbetagamma subserve specific functional roles.     

Role of the gamma subunit prenyl moiety in G protein beta gamma complex interaction with phospholipase Cbeta

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase Cbeta isozymes (PLCbetas). Prenyl modification of the gamma subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein gamma subunit prenyl group and PLCbeta isozymes. A geranylgeranylated peptide corresponding to the C-terminal region of the gamma subunit type, gamma2, strongly inhibits stimulation of PLCbeta2 and PLCbeta3 activity by the betagamma complex. This effect is specific because the same peptide has no effect on stimulation of PLCbeta by an alpha subunit type, alphaq. Prenylation of the gamma peptide is required for its inhibitory effect. When interaction of prenylated gamma subunit peptide to fluorophore-tagged PLCbeta2 was examined by fluorescence spectroscopy, prenylated but not unprenylated peptide increased PLCbeta2 fluorescence emission energy, indicating direct binding of the prenyl moiety to PLCbeta. In addition, fluorescence resonance energy transfer was detected between fluorophore tagged PLCbeta and wild type betagamma complex but not an unprenylated mutant betagamma complex. We conclude that a major function of the gamma subunit prenyl group is to facilitate direct protein-protein interaction between the betagamma complex and an effector, phospholipase Cbeta.     

Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.     

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits

Novel G protein alpha subunits were purified from rat brain by an affinity matrix containing immobilized beta gamma subunits (Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These alpha subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known alpha subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other alpha subunits. Sequences are identical with one or more members of a new family of alpha subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an alpha subunit labeled alpha q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP gamma S only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new alpha subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.     

G-protein beta gamma forms: identity of beta and diversity of gamma subunits

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of critical residues controlling G protein-gated inwardly rectifying K(+) channel activity through interactions with the beta gamma subunits of G proteins.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are activated through direct interactions of their cytoplasmic N- and C-terminal domains with the beta gamma subunits of G proteins. By using a combination of biochemical and electrophysiological approaches, we identified minimal N- and C-terminal G beta gamma -binding domains responsible for stimulation of GIRK4 channel activity. Within these domains one N-terminal residue, His-64, and one C-terminal residue, Leu-268, proved critical for G beta gamma-mediated GIRK4 activity. Moreover, mutations at these GIRK4 sites reduced significantly binding of the channel domains to G beta gamma . The corresponding residues in GIRK1 also showed a critical involvement in G beta gamma sensitivity. In GIRK4/GIRK1 heteromers the GIRK4 His-64 and Leu-268 residues showed greater contributions to G beta zeta sensitivity than did the corresponding GIRK1 His-57 and Leu-262 residues. These results identify functionally important channel interaction sites with the beta gamma subunits of G proteins, critical for channel activity.     

Specificity and structural requirements of phospholipase C-beta stimulation by Rho GTPases versus G protein beta gamma dimers

Phospholipase C-beta(2) (PLC beta(2)) is activated both by heterotrimeric G protein alpha- and beta gamma- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC beta isozymes with the rank order of PLC beta(2) > PLC beta(3) > or = PLC beta(1). The sensitivity of PLC beta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLC beta(3) > PLC beta(2) > PLC beta(1) for beta(1)gamma(1/2) and PLC beta(2) > PLC beta(1) > PLC beta(3) for beta(5)gamma(2). Rac1 and Rac2 were found to be more potent and efficacious activators of PLC beta(2) than was Cdc42Hs. The stimulation of PLC beta(2) by Rho GTPases and G protein beta gamma dimers was additive, suggesting that PLC beta(2) activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC beta(1)-PLC beta(2) enzymes, beta gamma dimers, and Rho GTPases are shown to require different regions of PLC beta(2) to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC beta(2) were sufficient for efficient stimulation by beta gamma, the presence of the putative pleckstrin homology domain of PLC beta(2) was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC beta regulators, which mediate PLC beta isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC beta(2) by extracellular mediators in intact cells.     

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).     

Regulation of the lateral association of phospholipase Cbeta2 and G protein subunits by lipid rafts

One function of membrane domains of liquid-ordered lipids or "rafts" may be to stabilize complexes of signaling proteins, thereby playing a role in the transduction of cellular signals. Here, we have used fluorescence methods to directly test this idea by assessing the ability of phospholipase Cbeta2 (PLCbeta2) to associate with G protein subunits on model membranes in the fluid phase and on membranes that contain domains of lipids in the liquid-ordered phase (rafts). We find that the apparent dissociation constant for the equilibrium between PLCbeta2 and Galpha(q)(GTPgammaS) was identical on both types of membrane surfaces. However, the degree of association between PLCbeta2 and Gbetagamma subunits was significantly reduced on the surfaces containing rafts. Time studies indicate that this phenomenon is a dynamic process. Incorporating the lipid substrate of PLCbeta2 into membranes that forms rafts, we find that its basal activity is unaffected. However, its activation by Gbetagamma subunits is inhibited, supporting a reduced degree of interaction between these two proteins when rafts are present. Since lipid rafts affected PLCbeta2-Gbetagamma association and not PLCbeta2-Galpha(q)(GTPgammaS) association, we explored the possibility that the membrane interaction of Gbetagamma differed when rafts are present. We find that although the membrane partition coefficient of Gbetagamma is not significantly changed in the presence of rafts, proteolysis of Gbetagamma by trypsin increases and the ability of Gbetagamma Tyr/Trp fluorescence to be quenched by iodide ions decreases when rafts are present. These results suggest a model in which lipid rafts occlude the PLCbeta2 interaction site on Gbetagamma subunits by localizing these subunits at the domain interface.     

Evidence that v-Src-induced phospholipase D activity is mediated by a G protein.

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.