Basal GTPase Activity Research Articles

Noradrenaline stimulation of high-affinity GTPase activity in membranes from rat aorta and caudal artery

The ability of noradrenaline (NA) to stimulate increases in high-affinity GTPase activity in sarcolemma-enriched rat aorta and caudal artery membranes was examined in the present study. In aortic membranes, NA significantly ( P < 0.05; N = 5) increased the V max from a basal value of 103 ± 29 to 156 ± 38 pmol P i/min/mg protein, but did not affect the K m which was 0.32 ± 0.08 μM in the absence and 0.58 ± 0.16 μM in the presence of NA. However, in caudal artery membranes, NA significantly ( P < 0.05; N = 6) increased both the V max and the K m from basal values of 69 ± 12 pmol P i/min/mg protein and 0.24 ± 0.05 μM, respectively, to 205 ± 54 pmol P i/min/mg protein and 1.01 ± 0.25 μM, respectively. Removing the endothelium from both artery preparations did not alter significantly basal GTPase activity or the magnitude of the increase stimulated by NA. Prazosin significantly inhibited NA-stimulated increases in GTPase activity in membranes from endothelium-denuded caudal artery and aorta, and in endothelium-intact caudal artery membranes. However, yohimbine significantly inhibited NA-stimulated increases in GTPase activity only in preparations from endothelium-intact caudal arteries. Therefore, in endothelium-intact caudal artery membranes, NA stimulated increases in GTPase activity that were apparently mediated by both α 1-adrenoceptors and α 2-adrenoceptors, while in endothelium-denuded aortic and caudal artery membranes this increase was mediated solely by α 1-adrenoceptors. Western blotting of these arteries confirmed the presence of both G i α2,3 and G q 11α , which are candidates for mediating the α 1-adrenoceptor-stimulated increases in GTPase activity.

ML-7 and W-7 facilitate thromboxane A 2-mediated Ca 2+ mobilization in rabbit platelets

The effects of 1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1, 4-diazepine (ML-7), a myosin light chain kinase inhibitor, and ( N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, on thromboxane A 2 receptor-mediated signal transduction were examined in rabbit washed platelets. ML-7 and W-7 at 10–30 μM slightly potentiated the aggregation induced by a thromboxane A 2 receptor agonist, 9, 11-dideoxy-9α, 11 α-epoxymethanoprostaglandin F 2 α (U46619), in spite of their known inhibitory actions. ML-7 and W-7 concentration-dependently enhanced U46619-induced phosphoinositide hydrolysis and the increase in internal free Ca 2+ concentration in the presence or absence of external Ca 2+. While ML-7 and W-7 inhibited basal GTPase activity, they augmented U46619-induced activation of GTPase in a concentration-dependent manner. The present results suggest that ML-7 and W-7 enhance thromboxane A 2 receptor-mediated signal transduction at the receptor/G protein coupling, leading to the enhancement of phosphoinositide hydrolysis and Ca 2+ mobilization, independently of the inhibition of myosin light chain kinase or calmodulin.

Molecular Cloning of Caveolin-3, a Novel Member of the Caveolin Gene Family Expressed Predominantly in Muscle

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene gamily, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are approximately 65% identical and approximately 85% similar. A single stretch of eight amino acids (FED-VIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a "caveolin signature sequence" that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue-types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.

Differential regulation of G protein α-subunit GTPase activity by peptides derived from the third cytoplasmic loop of the α2-adrenergic receptor

The effect of peptides homologous to segments of a G protein-coupled receptor on the GTPase activity of recombinant G 0α (rG oα) and G,a (rG sα) has been tested. These peptides contain overlapping sequences spanning from amino acid 212 of the putative fifth transmembrane domain to amino acid 229 of the third cytoplasmic loop of the a2 adrenergic receptor. Interestingly, two peptides (comprising residues 212–227 and 214–227) strongly inhibit the basal GTPase activity of both rG oα and rG sα. Instead, a C-terminally extended peptide (residues 216–229) stimulates rG oα but slightly inhibits rG sα. Circular dichroism spectroscopy of the peptides reveals that an a helical structure is more easily inducible in the inhibitory ones. These findings constitute an example of peptides representing cytoplasmic receptor sequences that differentially modulate the GTPase activity of recombinant G protein α-subunits.

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low Km and one with a high Km for GTP. The high-affinity GTPase activity, characteristic of the α subunits of the guanine nucleotide binding protein pool, was stimulated by the A1 adenosine receptor agonists N6(R-phenylisopropyl)adenosine and N6-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N6 (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 μmol·1-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent Km of GTP is decreased approximately 10% in each of the species, and the Vmax values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased Km of GTP and the increased Vmax ranged from-7.0 to-9.9 ml·mol-1. Increased pressure markedly decreased the efficacy of N6 (R-phenylisopropyl) adenosine, N6-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A1 adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.

Increased Platelet Reactivity to Prostaglandin E&lt;sub&gt;1&lt;/sub&gt; in Hypertension Is Linked With Altered Signal Transduction

Prostaglandin E1 has been shown to induce a greater accumulation of cAMP in platelets from spontaneously hypertensive (SHR) than in platelets from normotensive (Wistar-Kyoto, WKY) rats (Circ. Res. 1978;43:583-591. Thromb. Res. 1988;49:5-21). This study was conducted to determine the mechanisms of increased platelet reactivity to PGE1 in hypertension. The number of PGE1 binding sites/platelet (WKY 280 +/- 8, SHR 287 +/- 5) as well as the Kd for WKY (105 +/- 11 nmol/L) and SHR (120 +/- 14 nmol/L) were found to be similar in WKY and SHR rats. PGE1-induced GTPase activity was determined using WKY and SHR platelet membranes. The basal GTPase activity was similar in WKY and SHR platelets. Incubation of membranes with PGE1 (3 mumol/L) for 1 min increased GTPase activity by 46% in WKY and by 806% in SHR. Incubation of platelets with 1.0 mmol/L IBMX (3-isobutyl-1-methyl-xanthine) for 4 min resulted in a 327 +/- 57% and 320 +/- 11% increase in cAMP in WKY and SHR platelets, respectively. In other experiments, incubation of platelets with 3, 10 and 100 mumol/L forskolin, induced similar increases in cAMP levels in WKY (103 +/- 12%, 530 +/- 42%, 784 +/- 41%) and SHR (111 +/- 13%, 461 +/- 18%, 756 +/- 28%) platelets. These data lead us to suggest that the greater accumulation of cAMP induced by PGE1 in SHR than in WKY platelets is linked with altered PGE1-receptor mediated signal transduction at the G-protein level.

Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small G protein superfamily that is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A contains at least two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal C2 domain, which interacts with both Ca2+ and phospholipid. Because Rabphilin-3A interacts preferentially with GTP-Rab3A rather than with GDP-Rab3A, we have examined here whether Rabphilin-3A affects the GTPase activity of Rab3A. Rabphilin-3A and its N-terminal fragment, but not its C-terminal fragment, very weakly stimulated the basal GTPase activity of Rab3A. However, Rabphilin-3A and its N-terminal fragment strongly inhibited the Rab3A GAP-stimulated GTPase activity of Rab3A. Ca2+ and phospholipid showed no effect on these activities of Rabphilin-3A. The physiological significance of the GAP activity of Rabphilin-3A is obscure, but it is likely that Rabphilin-3A inhibits Rab3A GAP activity and keeps Rab3A in the GTP-bound active form during its action as a target molecule for Rab3A.

Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor

Rab3A is a Ras-like GTPase that is believed to function in regulated secretion. A GTPase activating protein (GAP) and a guanine nucleotide releasing factor (GRF) specific for Rab3A have recently been described. In this study we have described the biochemical activities of Rab3A mutants in codons 31, 81, 135, and 166, which correspond to codons 12, 61, 116, and 146 in Ras. The results demonstrate that simple extrapolations from the properties of Ras mutants are not valid for all small GTPases. S31V-Rab3A and Q81L-Rab3A had a reduced basal GTPase activity, but surprisingly were sensitive to the effects of Rab3A-GAP and insensitive to Rab3A-GRF. Q81L-Rab3A and wild-type Rab3A that were bound to a nonhydrolyzable GTP analog had similar affinities for Rab3A-GAP. In vivo, the percent of Q81L-Rab3A (46%) and wild-type Rab3A (43%) complexed to GTP was similar. These findings indicate that Rab3A-GRF and Rab3A-GAP interact with residues different from those of previously described GAPs and GRFs. Both A166V-Rab3A and N135I-Rab3A had increased intrinsic dissociation rates for GDP. However, the dissociation rate for N135I-Rab3A was > 100-fold higher than that for A166V-Rab3A suggesting that, in vivo, of the two, N135I-Rab3A would more likely be preferentially in the GTP-bound state.

GTP-binding proteins in polymorphonuclear granulocytes of severely burned patients

In order to study the biochemical mechanism underlying the cellular dysfunctions of polymorphonuclear granulocytes (PMNs) from severely burned patients, we analyzed the role of GTP-binding proteins (G-proteins) in PMNs from 11 burned patients. Our data demonstrate a significant enhancement of the basal GTPase activity within unstimulated neutrophils of severely burned patients compared to cells from healthy donors. This enhancement was significant within 4 weeks after the trauma, followed by a return to control levels. The increase in GTPase activity correlated with enhanced expression of the small G-protein Ras and the regulatory Ras-GTPase activating protein (Ras-GAP) compared to that in healthy donor cells. However, expression of the Ras-related protein Rap1, which is involved in initiation of the respiratory burst, was reduced. The observed changes in G-protein activity and expression impair the signal transduction cascade as well as bacterial killing and may lead to high susceptibility toward infections and finally to septic conditions.

Platelet activating factor stimulates a receptor-coupled membrane GTP ASE in guinea pig eosinophils

The platelet activating factor (PAF) receptor of the guinea pig peritoneal eosinophil was characterized by radioligand ([ 3H]WEB 2086) binding and measurement of PAF-stimulated membrane GTPase activity. Specific binding of [ 3H]WEB 2086 was rapid, reversible and saturable, with an equilibrium K D of 20.4 nM at 0°C, and was displaced competitively by unlabeled PAF with a K I of 3.26 nM and a pseudo-hill coefficient significantly less than unity (0.44). The affinity of PAF for these binding sites was reduced by the nonhydrolysable GTP analogue 5′-guanylylimidodiphosphate (GppNHp), suggesting the coupling of PAF receptors to intracellular effectors through a guanine nucleotide-binding protein (G protein). PAF stimulated a membrane-associated GTPase, indicating the formation of a G protein α subunit-GTP complex upon agonist occupation of the PAF receptor. The EC 50 for PAF stimulation was 25.5 nM and the Hill coefficient was significantly less than unity (0.56), while the response to 1 μM PAF was antagonised by WEB 2086 with an IC 50 of 128 nM and a slope factor not significantly different from unity (0.91), suggesting the coupling of multiple classes of PAF receptors to the G protein. The activation of GTPase by PAF was insensitive to inhibition by cholera toxin; basal GTPase activity was increased by pertussis toxin and no further stimulation was attainable with PAF.

The possible role of ADP ribosylation in physiological regulation of sporulation in Streptomyces griseus

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD +: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [α- 32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.

Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase A-catalyzed phosphorylation of smg p21.

We have previously shown that cyclic AMP-dependent protein kinase (protein kinase A) phosphorylates smg p21A and -B, ras p21-like small GTP-binding proteins. In the present study, we investigated the function(s) of this phosphorylation by use of the smg p21B purified from human platelets. smg p21B bound to plasma membranes and the protein kinase A-catalyzed phosphorylation of smg p21B reduced this binding. Moreover, the phosphorylation of smg p21B enhanced the two actions of its specific GDP/GTP exchange protein, named GDP dissociation stimulator, when tested in a cell-free system: one is the action to stimulate the GDP/GTP exchange reaction of smg p21B, and the other is the action to inhibit the binding of smg p21B to membranes. Consistently, smg p21B was translocated from the membranes to the cytoplasm when it was phosphorylated by protein kinase A in intact platelets in response to prostaglandin E1 or dibutyryl cyclic AMP. The protein kinase A-catalyzed phosphorylation of smg p21B affected neither its basal GDP/GTP exchange reaction, basal GTPase activity, nor the GTPase activity stimulated by its specific GTPase activating protein. On the other hand, we have recently clarified that the structure of the C-terminal region of the post-translationally processed human platelet smg p21B is Lys-Lys-Ser-Ser-all-trans-geranylgeranyl Cys181 methyl ester, and that this modification of the C-terminal region is essential for smg p21B to bind to membranes. We furthermore determined here that protein kinase A phosphorylated Ser179 in this C-terminal region of smg p21B. These results indicate that protein kinase A-catalyzed phosphorylation of smg p21B makes smg p21B sensitive to the actions of smg p21 GDP dissociation stimulator.

Binding of α‐ and βγ‐subunits of G0 to β1‐adrenoceptor in sealed unilamellar lipid vesicles

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150-200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30-70 nm (fraction II). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with alpha- and beta gamma-subunits of Go-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a beta 1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either alpha o or beta gamma alone with beta 1-adrenoceptors, some of these subunits appear together with beta 1-adrenoceptors in the small vesicle fraction II, but much more alpha o was bound to the receptor in the presence of beta gamma-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that beta gamma-subunits can bind to the non-activated beta 1-receptor where they may serve as an anchor for alpha-subunits. Secondly, the binding of alpha o- and beta gamma-subunits to the beta 1-adrenoceptors enhances the basal GTPase activity of alpha o. Thirdly, since the binding domains of the beta 1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the beta-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213-215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a beta 1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.

Activation of protein kinase C inhibits hormonal stimulation of the GTPase activity of Gi in human platelets

The effect of 12-tetradecanoyl phorbol 13-acetate on the GTPase activity of Gi was investigated. Treatment with TPA did not alter basal GTPase activity of membranes or the simulatory effect of prostaglandin E 1 (putatively via Gs). In contrast, the phorbol ester markedly diminished stimulation of GTPase by agents whose receptors are coupled to Gi such as epinephrine (α-adrenergic action), platelet activating factor or thrombin. Pertussis toxin catalyzed ADP-ribosylation was also decreased in membranes from TPA-treated platelets as compared to the controls. It is suggested that the Alteration in the hormonal activation of the GTPase activity of Gi is secondary to a perturbation in the receptor-Gi interaction.

Biochemical Characterization of Baculovirus-Expressed rap1A/ Krev-1 and Its Regulation by GTPase-Activating Proteins

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.

Na + regulation of formyl peptide receptor-mediated signal transduction in HL 60 cells. Evidence that the cation prevents activation of the G-protein by unoccupied receptors

In neutrophils and several other phagocytes, a pertussis and cholera toxin-sensitive guanine nucleotide-binding protein (G-protein) couples the receptors for formyl methionine-containing chemotactic peptides to stimulation of phospholipase C. We used membranes of myeloid-differentiated HL 60 cells to study the role of Na + in regulating both the interaction of the formyl peptide receptor with the chemotactic agonist, N-formyl-methionyl-leucyl-phenyl-alanine (FMLP), and the receptor-mediated activation of the G-protein. Monovalent cations (Na + > Li + > K + > choline +) markedly inhibited the binding of the radiolabeled oligopeptide [ 3H]FMLP by specifically reducing the number of receptors in the high-affinity state. Half-maximal and maximal inhibition of peptide binding were seen at cation concentrations of approximately 20 and 200 mM, respectively. Inhibition of peptide binding by Na + was observed in the presence and absence of divalent cations and was strictly additive to inhibition by the poorly hydrolyzable GTP analogue, guanosine-5′-O-(3-thiotriphosphate), or to ADP ribosylation of G-proteins by pertussis toxin. The inhibitory effect of Na + on peptide binding coincided with a marked reduction of the potency of FMLP to stimulate a high-affinity GTPase. In contrast, the degree of FMLP-stimulated GTPase activity was markedly enhanced in the presence of Na +. This was largely due to the fact that Na + reduced the agonist-independent basal GTPase activity in the same way but less so than pertussis toxin treatment. The results show that monovalent cations, Na + in particular, regulate the interaction of the formyl peptide receptor with both the chemotactic agonist and the G-protein by acting on a single site, possibly located on the receptor itself. The observation that basal GTPase activity is markedly reduced by both Na + and pertussis toxin treatment also suggests (a) that G-proteins interact with and are activated by receptors even in the absence of agonists and (b) that Na + uncouples unoccupied receptors from G-protein interaction and activation.

Studies on Nucleotide and Receptor Regulation of GiProteins: Effects of Pertussis Toxin

In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of Gi proteins are affected by PT. Experiments with the Gs-deficient Gi-regulated adenylyl cyclase of cyc- S49 cell membranes indicated that PT blocks Gi activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-Gi to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of Gi. Experiments in which Gi was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of Gi, but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes.

Secretion‐stimulating and secretion‐inhibiting hormones stimulate high‐affinity pertussis‐toxin‐sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.

Selective inactivation by endogenous protein kinase C of human platelet high-affinity GTPase coupled with PAF receptors

Incubation of human platelets with protein kinase C activator 4β-phorbol-12β-myristate-13α-acetate (PMA) abolished stimulation of membrane high-affinity GTPase by platelet-activating factor (PAF). GTPase stimulation by epinephrine decreased by 30%, while the prostaglandin E 1 (PGE 1) effect was unchanged. Basal GTPase activity (22.4±1.1 pmol P i/min per mg protein) was not affected by PMA. Therefore, a study was performed of the effect of endogenous protein kinase C activation on adenylate cyclase regulation by agonists. PMA pretreatment completely suppressed PAF inhibition of basal adenylate cyclase activity but hardly influenced the inhibition by PAF of forskolin-stimulated activity. Adenylate cyclase inhibition by epinephrine in the presence of propranolol was not suppressed completely after platelet incubation with PMA. Epinephrine effects on basal and forskolin-stimulated activities decreased equally. Platelet pretreatment with PMA increased PGE 1-stimulated activity by abolishing the inhibitory effect of high GTP concentrations. These studies indicate that protein kinase C selectively inhibits PAF effects, presumably by inactivating a GTP-binding protein coupled with PAF receptors.