ABSTRACT
Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Membrane Proteins/metabolism , Nitric Oxide Synthase/metabolism , Receptors, Estrogen/metabolism , Animals , Cell Line, Transformed , Cell Membrane/metabolism , Enzyme Activation , Nitric Oxide Synthase Type III , Precipitin Tests , Signal TransductionABSTRACT
Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.
Subject(s)
GTP-Binding Proteins/metabolism , Pancreas/metabolism , RGS Proteins , Receptors, Cell Surface/metabolism , Submandibular Gland/metabolism , Animals , Antibodies/pharmacology , Calcium/metabolism , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/immunology , Enzyme Inhibitors/pharmacology , Isoproterenol/pharmacology , Mice , Mice, Knockout , Pertussis Toxin , Phosphatidylinositol 4,5-Diphosphate/immunology , Proteins/pharmacology , Receptors, Adrenergic, beta/metabolism , Receptors, Vasoactive Intestinal Peptide/metabolism , Signal Transduction , Vasoactive Intestinal Peptide/pharmacology , Virulence Factors, Bordetella/pharmacology , beta-Adrenergic Receptor KinasesABSTRACT
Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors. G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP. However, the topological fate of activated G protein alpha subunits is disputed. One model declares that depalmitoylation of alpha, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm. Our data suggest that activation of G protein alpha subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, alpha subunits remained bound to the membrane when they were activated with guanosine 5'-(3-O-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of alpha subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.
Subject(s)
GTP-Binding Proteins/metabolism , Palmitic Acid/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Immunohistochemistry , Macaca mulatta , Membrane Proteins/metabolism , Palmitoyl-CoA Hydrolase/metabolism , Signal Transduction , Transfection/geneticsABSTRACT
There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein alpha and beta subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein alpha subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.
Subject(s)
Adenylyl Cyclases/metabolism , Caveolins , Cell Membrane/enzymology , Heterotrimeric GTP-Binding Proteins/metabolism , Animals , Antibody Specificity , Caveolin 1 , Cell Fractionation , Cell Line , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dogs , Fibroblasts , Fluorescent Antibody Technique , Humans , Membrane Proteins/metabolism , Mice , Microscopy, Immunoelectron , Protein Binding , Subcellular Fractions/enzymology , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructureABSTRACT
Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo was similar to that seen in vitro. RGS4 and GAIP both attenuated Gi-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta.
Subject(s)
Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , RGS Proteins , Signal Transduction , Animals , Base Sequence , Bradykinin/pharmacology , Cell Line , Clone Cells , DNA Primers , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Isoproterenol/pharmacology , Kinetics , Mammals , Molecular Sequence Data , Oligodeoxyribonucleotides , Phosphoproteins/biosynthesis , Polymerase Chain Reaction , Protein Biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Sequence Tagged Sites , Transfection , Virulence Factors, Bordetella/pharmacologyABSTRACT
Palmitoylation is unique among lipid modifications of proteins in that it is reversible and regulable. Recent advances in the study of palmitoylation include the following: the correlation of this modification with the localization of a signaling protein to specific membrane subdomains; the demonstration of a specific protein-protein interaction that is promoted by palmitoylation; and the identification, characterization, and purification of enzymes catalyzing this modification.
Subject(s)
Palmitic Acid/metabolism , Signal Transduction , Cell Membrane/metabolism , GTP-Binding Proteins/metabolism , Receptors, Cell Surface/metabolismABSTRACT
Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein alpha-subunits present in mammalian brain--alpha o, alpha i1, and alpha i2--using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant alpha i2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein alpha-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of alpha i1 but not alpha i2 increased during nerve growth factor-induced differentiation. In contrast, alpha i2 but not alpha i1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of alpha o increased in both cell lines during differentiation. Electrophoretic resolution of alpha o subtypes revealed that although alpha o2 was the major subtype in undifferentiated cells, only the concentration of alpha o1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with alpha o, increased similarly to that of alpha o1. ADP-ribosylation of alpha o, alpha i1, and alpha i2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of alpha q, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of alpha i and alpha o associated with neuronal differentiation.
Subject(s)
Antibodies, Monoclonal/immunology , GTP-Binding Proteins/immunology , Pertussis Toxin , Virulence Factors, Bordetella/pharmacology , Adenosine Diphosphate Ribose/metabolism , Animals , Antibody Specificity , Blotting, Western , Brain/metabolism , Cattle , Cell Differentiation , GAP-43 Protein , GTP-Binding Proteins/metabolism , Humans , In Vitro Techniques , Membrane Glycoproteins/metabolism , Nerve Growth Factors/pharmacology , Nerve Tissue Proteins/metabolism , Neuroblastoma , PC12 Cells , Rats , Synaptosomes/metabolism , Tumor Cells, CulturedSubject(s)
GTP-Binding Proteins/metabolism , Palmitic Acids/metabolism , Protein Processing, Post-Translational , Receptors, Adrenergic, alpha-2/physiology , Receptors, Cell Surface/physiology , Animals , Cell Line , Chlorocebus aethiops , Homeostasis , Mutagenesis, Site-Directed , Palmitic Acid , Point Mutation , Receptors, Adrenergic, alpha-2/biosynthesis , Recombinant Proteins/metabolism , Signal TransductionSubject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Palmitic Acids/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Carbon Radioisotopes , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Chromatography, Thin Layer/methods , Cytosol/metabolism , Indicators and Reagents , Kinetics , Macromolecular Substances , Mammals , Molecular Sequence Data , Myristic Acid , Myristic Acids/metabolism , Palmitic Acid , Radioisotope Dilution Technique , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Spodoptera , Transfection/methods , TritiumABSTRACT
Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.
Subject(s)
Caveolins , Cell Compartmentation , Cell Membrane/chemistry , Membrane Proteins/chemistry , Muscle, Smooth/chemistry , Animals , Caveolin 1 , Cell Fractionation/methods , Cell Membrane/ultrastructure , Chickens , GTP-Binding Proteins/isolation & purification , Gizzard, Avian/cytology , Glycosylphosphatidylinositols , Immunohistochemistry , Microscopy, Immunoelectron , Muscle, Smooth/ultrastructure , Subcellular Fractions/chemistry , Subcellular Fractions/ultrastructureABSTRACT
Many alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are palmitoylated. Exposure of cells to the beta-adrenergic agonist isoproterenol increased incorporation of [3H]palmitate specifically into alpha s, the alpha subunit that mediates stimulation of adenylyl cyclase. Pulse-chase experiments suggested that isoproterenol increased turnover of alpha s-bound palmitate. Mutagenesis of Cys-3 in alpha s or alpha o (a homologous alpha subunit) prevented palmitoylation of these proteins. Differing results were obtained when mutations of Cys-3 in alpha s or alpha o were expressed in cells and assayed for their distribution between soluble and membrane fractions. Some alpha subunits, including alpha o, are myristoylated at the amino-terminal glycine residue. Mutation of this glycine prevented both myristoylation and palmitoylation of alpha o, indicating that myristoylation precedes palmitoylation of dually acylated alpha subunits. The amino-terminal sequences and fatty acylation properties of dually acylated alpha subunits are strikingly similar to those of some members of the Src family of protein-tyrosine kinases. The amino-terminal sequence Met-Gly-Cys-Xaa-Xaa-Ser/Cys shared by these proteins may represent a motif for cotranslational and posttranslational processing that includes myristoylation of the glycine residue and reversible palmitoylation of the cysteine residue.
Subject(s)
GTP-Binding Proteins/metabolism , Palmitic Acids/metabolism , Protein Processing, Post-Translational , Receptors, Adrenergic, beta/metabolism , Acylation , Amino Acid Sequence , Animals , Base Sequence , DNA Mutational Analysis , GTP-Binding Proteins/genetics , Isoproterenol/pharmacology , Molecular Sequence Data , Palmitic Acid , Rats , Receptors, Adrenergic, beta/drug effects , Recombinant Proteins/metabolismSubject(s)
GTP-Binding Proteins/metabolism , Myristic Acids/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Cell Line , Chlorocebus aethiops , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/isolation & purification , Gene Expression , Macromolecular Substances , Mass Spectrometry/methods , Molecular Sequence Data , Mutagenesis, Site-Directed , Myristic Acid , Plasmids , Radioisotope Dilution Technique , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Transfection/methods , TritiumABSTRACT
A small number of membrane-associated proteins are reversibly and covalently modified with palmitic acid. Palmitoylation of G-protein alpha and beta subunits was assessed by metabolic labeling of subunits expressed in simian COS cells or insect Sf9 cells. The fatty acid was incorporated into all of the alpha subunits examined (alpha s, alpha o, alpha i1, alpha i2, alpha i3, alpha z, and alpha q), including those that are also myristoylated (alpha o, alpha i, and alpha z). Palmitate was released by treatment with base, suggesting attachment to the protein through a thioester or ester bond. Limited tryptic digestion of activated alpha o and alpha s resulted in release of the amino-terminal portions of the proteins and radioactive palmitate. These data are consistent with palmitoylation of the proteins near their amino termini, most likely on Cys-3. Reversible acylation of G-protein alpha subunits may provide an additional mechanism for regulation of signal transduction.
Subject(s)
GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Palmitic Acids/metabolism , Protein Processing, Post-Translational , Animals , Autoradiography , Base Sequence , Cell Line , Cell Membrane/metabolism , Chlorocebus aethiops , Cytosol/metabolism , GTP-Binding Proteins/isolation & purification , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Macromolecular Substances , Methionine/metabolism , Molecular Sequence Data , Moths , Myristic Acid , Myristic Acids/metabolism , Oligodeoxyribonucleotides , Palmitic Acid , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , TritiumABSTRACT
Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma.
Subject(s)
Cell Membrane/metabolism , GTP-Binding Proteins/metabolism , Animals , Base Sequence , Cell Line , Chemical Fractionation , Electrophoresis, Polyacrylamide Gel , GTP-Binding Proteins/genetics , Humans , Immunoblotting , Immunohistochemistry , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , PlasmidsABSTRACT
A cDNA clone encoding a stimulatory G-protein alpha subunit (Gs alpha) was isolated from a cDNA library derived from cultured rat astrocytes. The nucleotide sequence of the cDNA indicated that it corresponds to the Gs alpha-2 form of Gs alpha mRNA, one of four Gs alpha mRNAs known to be derived by alternative splicing from the human Gs alpha gene. A ribonuclease protection assay using cRNA from this clone allowed distinction between the Gs alpha-1 and Gs alpha-2 mRNAs, which encode the 52-kDa (Gs-L) forms of Gs alpha. Astrocytes express relatively high amounts of Gs alpha-1 mRNA, much lower amounts of the Gs alpha-2 mRNA, and no detectable amounts of the mRNAs (Gs alpha-3 and Gs alpha-4) encoding the two 45-kDa forms of Gs alpha (Gs alpha-S). Similar results were obtained with RNA samples isolated from whole brain. The 45-kDa form of Gs alpha protein was not detectable by immunoblot analysis of a membrane preparation from rat cerebral cortex (the source of the astrocyte cultures). These results indicate that the expression of Gs alpha forms in astrocytes is similar to that found in whole brain.
Subject(s)
Astrocytes/metabolism , Cerebral Cortex/cytology , GTP-Binding Proteins/genetics , RNA, Messenger/analysis , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , DNA/genetics , GTP-Binding Proteins/biosynthesis , Molecular Sequence Data , Rats , Rats, Inbred StrainsSubject(s)
Antibody Specificity , GTP-Binding Proteins/analysis , Immune Sera , Peptides/chemical synthesis , Amino Acid Sequence , Animals , Blotting, Western/methods , Chromatography, Gel/methods , DNA/genetics , Enzyme-Linked Immunosorbent Assay , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Indicators and Reagents , Macromolecular Substances , Molecular Sequence Data , Rabbits/immunologyABSTRACT
A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the alpha and gamma subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The beta and gamma subunits were isolated by specific association with an affinity column of immobilized alpha subunits. The gamma subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein alpha or beta subunits. Isoprenylation of gamma subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.
Subject(s)
Diterpenes/analysis , GTP-Binding Proteins/isolation & purification , Adrenal Gland Neoplasms , Animals , Brain Chemistry , Cattle , Cell Line , Chromatography, Affinity , Chromatography, High Pressure Liquid , Macromolecular Substances , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/isolation & purification , Pheochromocytoma , RatsABSTRACT
Myristoylation of seven different alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [3H]myristate was incorporated into alpha i1, alpha i2, alpha i3, alpha 0, alpha t, and alpha z but not alpha s subunits. The role of myristoylation in the association of alpha subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of alpha 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated alpha subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein alpha subunits dissociate from beta gamma.
Subject(s)
Cell Membrane/metabolism , GTP-Binding Proteins/genetics , Myristic Acids/metabolism , Protein Processing, Post-Translational , Transfection , Animals , Cell Line , Cytomegalovirus/genetics , DNA/genetics , GTP-Binding Proteins/metabolism , Genetic Vectors , Kinetics , Macromolecular Substances , Mutation , Myristic Acid , PlasmidsABSTRACT
We studied G proteins and regulation of adenylate cyclase in nervous tissue and muscle of Aplysia using bacterial toxin-catalyzed ADP-ribosylation. We identified Gs alpha, a Mr 45,000 cholera toxin substrate, Go alpha, a Mr 40,000 pertussis toxin substrate, and G beta (Mr 37,000) by Western blot analysis with antisera specific for bovine brain G protein subunits. Partial proteolysis suggests that the neuronal pertussis toxin substrates are heterogeneous. The concentration of these substrates in membranes from Aplysia ganglia is similar to that of rat, squid and Helix; in Aplysia nervous tissue, G protein subunits are most enriched in synaptosomes and neuropil. The stimulation of adenylate cyclase by serotonin (5-HT), low concentrations of GTP-gamma-S, and cholera toxin, and the inhibition by high concentrations of GTP-gamma-S that is blocked by pertussis toxin indicate that both a Gs and a Gi protein regulate the Aplysia enzyme. These results support the idea that G proteins in Aplysia are important in regulating synaptic function.
Subject(s)
Adenylyl Cyclases/metabolism , Aplysia/metabolism , GTP-Binding Proteins/metabolism , Muscles/metabolism , Nervous System/metabolism , Adenosine Diphosphate/metabolism , Adenylate Cyclase Toxin , Animals , GTP-Binding Proteins/immunology , Ganglia/metabolism , Immune Sera , Pertussis Toxin , Subcellular Fractions/metabolism , Virulence Factors, BordetellaABSTRACT
GTP-binding proteins were purified from human neutrophils, including a 40,000-Da pertussis toxin substrate (Gn) and 22,000-, 24,000-, and 26,000-Da proteins, termed G22K, G24K, and G26K, respectively. The latter proteins were shown to be immunologically unrelated to Gn. G22K cross-reacted with anti-ras monoclonal antibody 142-24EO5, but not with monoclonal antibody Y13-259. A single 22,000-Da substrate for botulinum toxin-catalyzed ADP-ribosylation present in neutrophil membranes co-migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with G22K. In the presence of a cytosolic factor, G22K could serve as a specific botulinum toxin substrate. The 22,000-Da botulinum toxin substrate in neutrophil membranes could be immunoprecipitated by antibody 142-24EO5, but not by antibody Y13-259. G22K appears to be a unique GTP-binding protein which serves as a substrate for ADP-ribosylation by a component of botulinum toxin and which may be involved in exocytotic secretion or cellular differentiation.