Sar1 GTPase Activity Is Regulated by Membrane Curvature.

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5'-[(ß,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

Regulation of the Melanocortin-Sensitive Adenylate Cyclase System by N-Acylated Peptide 71-82 of Type 4 Melanocortin Receptor.

The peptides structurally corresponding in to cytoplasmic loops of G protein-coupled receptors (GPCR) are able to control functional activity of homologous receptors and the corresponding signaling pathways. Modification of these peptides with hydrophobic radicals enhances their biological activity due to penetration of lipophilic derivatives through the membrane and anchoring near their targets, GPCR. We synthesized an N-palmitoylated peptide Palm-Val-[Lys-Asn-Lys-Asn-Leu-His-Ser-Pro-(Nle)-Tyr-Phe-Phe71-82]-amide-Palm-Val-(71-82) structurally corresponding to cytoplasmic loop 1 of melanocortin 4 receptor (M4R). We found that in micromolar concentrations it very effectively suppresses stimulation of basal adenylate cyclase activity and basal level of GppNHp binding of heterotrimeric G proteins produced by THIQ and α-melanocyte stimulating hormone (α-MSH), agonists of M4R homologous to the peptide, in synaptosomal membranes of rat brain. The peptide Palm-Val-(71-82) also reduced, albeit to a significantly less extent, stimulation of adenylate cyclase and G-proteins by M3R agonist of γ-MSH, due to high homology of the peptide primary structure to M3R cytoplasmic loop 1. The synthesized peptide with activity of M4R/M3R antagonist can be used for the development of regulators of M4R and M3R and the corresponding biochemical and physiological processes.

[The influence of two-month treatment with bromocryptine on activity of the adenylyl cyclase signaling system in the myocardium and testes of rats with type 2 diabetes mellitus].

One of the common complications of type 2 diabetes mellitus (DM2) are cardiovascular diseases and dysfunctions of the reproductive system, indicating the urgency of developing new approaches to their correction. Last years for the treatment of DM2 began to use bromocryptine (BC), the agonist of type 2 dopamine receptors, which not only restores the energy metabolism, but also prevents the development of cardiovascular diseases. However, the mechanisms and targets of BC action are poorly understood. The purpose of this study was to investigate the effect of BC treatment on functional activity of adenylyl cyclase signaling system (ACSS) in the myocardium and testes of male rats with DM2, which is caused by high-fat diet and treatment with streptozotocin (25 mg/kg). The treatment with BC (60 days, orally at a dose of 0.6 mg/kg once every two days) was started 90 days after the beginning of high-fat diet. Diabetic rats had an increased body weight, elevated triglycerides level, impaired glucose tolerance, and insulin resistance. The treatment with BC resulted in the restoration of glycometabolic indicators and in the improvement of insulin sensitivity. Adenylyl cyclase (AC) stimulating effects of guanylylimidodiphosphate (GppNHp), relaxin, and agonists of ß-adrenergic receptors (ß3-AR)--isoproterenol and norepinephrine were decreased in the miocardium of the diabetic rats. The corresponding effects of the ß-agonists BRL-37344 and CL-316243 was preserved. The inhibitory effect of somatostatin on forskolin-stimulated AC activity was attenuated, while the inhibitory effect of noradrenaline mediated through α2-AR increased. The treatment with BC resulted in the normalization of the adrenergic signaling in the myocardium and partially restoration of AC effects of relaxin and somatostatin. In the testes of diabetic rats, the basal and stimulated by GppNHp, forskolin, human chorionic gonadotropin and pituitary AC-activating polypeptide AC activity were decreased, and the inhibitory effect of somatostatin was attenuated. The changes in testicular ACSS in the case of BC treatment were weakly expressed. Thus, long-term BC treatment restores the functional activity of ACSS in the myocardium and testes of diabetic rats that underlies the therapeutic effect of BC on functions of the cardiovascular and reproductive systems disturbed in DM2 and should be considered when developing strategies for treatment type 2 diabetes and its complications.

Agonist binding fraction of dopamine D2/3 receptors in rat brain: a quantitative autoradiographic study.

There has arisen considerable interest in the study of dopamine D(2/3) agonist binding sites by positron emission tomography (PET), based on the claim that agonist sites represent a functional subset of the total number of sites labeled by more conventional antagonist ligands. To test the basis of this claim, we used quantitative autoradiography to measure the abundance of binding sites of a dopamine D(2/3) agonist ([(3)H]NPA) and an antagonist ([(3)H]raclopride) in cryosections of rat brain. Saturation binding studies revealed that the B(max) for [(3)H]NPA was nearly identical to that of [(3)H]raclopride in dorsal brain regions, but was 25% less in the ventral striatum and 56% less in the olfactory tubercle. We also tested the displacement of the two ligands by the hallucinogen LSD, which is known to have dopamine agonist properties. Whereas displacement of [(3)H]raclopride by increasing LSD concentrations was monophasic, displacement of [(3)H]NPA was biphasic, suggesting an action of LSD via a subset of dopamine D(2/3) agonist binding sites. Addition of the stable GTP analogue Gpp(NH)p to the medium abolished 90% of the [(3)H]NPA binding, and increased [(3)H]raclopride binding by 10%, with a shift to the right in the LSD competition curve, suggesting retention of endogenous dopamine in washed cryostat sections. Thus [(3)H]NPA and [(3)H]raclopride binding sites have nearly identical abundances in rat dorsal striatum, but are distinct in the ventral striatum, and with respect to their displacement by LSD.

The site for the allosteric activator GTP of Escherichia coli UMP kinase.

UMP kinase (UMPK), a key bacterial pyrimidine nucleotide biosynthesis enzyme, is UTP-inhibited and GTP-activated. We delineate the GTP site of Escherichia coli UMPK by alanine mutagenesis of R92, H96, R103, W119 or R130, abolishing GTP activation; of S124 and R127, decreasing affinity for GTP; and of N111 and D115, with little detrimental effect. We exclude the correspondence with the modulatory ATP site of Bacillus anthracis UMPK, confirming the functionality of the GTP site found by Evrin. Mutants R92A, H96A and R127A are constitutively activated, suggesting key roles of these residues in allosteric signal transduction and of positive charge neutralization in triggering activation. No mutation hampered UTP inhibition, excluding overlapping of the UTP and GTP sites.

Antagonistic cannabinoid CB1/dopamine D2 receptor interactions in striatal CB1/D2 heteromers. A combined neurochemical and behavioral analysis.

In vitro results show the ability of the CB(1) receptor agonist CP 55,940 to reduce the affinity of D(2) receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D(2) receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB(1)-D(2) receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB(1)/D(2) receptor-receptor interactions may occur in CB(1)/D(2) formed heteromers. Antagonistic CB(1)/D(2) interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB(1) receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB(1) receptor antagonist rimonobant but also the specific A(2A) receptor antagonist MSX-3 blocked the inhibitory effect of CB(1) receptor agonist on D(2)-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB(1)/D(2) receptor-receptor interactions within CB(1)/D(2) heteromers in which A(2A) receptors may also participate.

Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway.

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids.

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles.

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.

Gender-dependent regulation of G-protein-gated inwardly rectifying potassium current in dorsal raphe neurons in knock-out mice devoid of the 5-hydroxytryptamine transporter.

Agonists at G-protein-coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock-out mice devoid of the serotonin transporter (5-HTT(-/-)) exhibit lower efficacy to inhibit cellular discharge than in wild-type counterparts. Using patch-clamp whole-cell recordings, we found that a G-protein-gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5-HT1A agonists (5-carboxamidotryptamine (5-CT) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT); 50 nM-30 microM) in both wild-type and 5-HTT(-/-) female and male mice. These effects were mimicked by 5'-guanylyl-imido-diphosphate (Gpp(NH)p; 400 microM) dialysis into cells with differences between genders. The 5-HTT(-/-) knock-out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5-HT1A receptors to agonists in 5-HTT(-/-) mutants reflects notably alteration in the coupling between G-proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5-HT neurotransmission appear to depend-at least in part-on sex-related variations in corresponding receptor-G protein signaling mechanisms.

The first luminal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake.

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites.

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

A role for ethylene in low-oxygen signaling in rice roots.

Inhibitors of action and synthesis of ethylene (Ag(+), norbornadien, Co(2+)) were able to reduce the level of gamma-aminobutyric acid (Gaba) in rice roots during the development of an anaerobic environment. The inhibitory effect was reversed by the addition of the G protein activator 5'-guanylylimidodiphosphate. Gaba accumulation was modulated by the presence of CO(2) (inhibitor of ethylene action and synthesis) and stimulated by 2-chloroethylphosphonic acid (ethefon). These findings are consistent with a role of ethylene during a low-oxygen stress.

Use of probes with fluorescence indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor.

Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.

Effects of urea pretreatment on the binding properties of adenosine A1 receptors.

The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.

Paracrine calcitonin in prostate cancer is linked to CD44 variant expression and invasion.

BACKGROUND: Calcitonin (CT) exerts an autocrine/paracrine influence on prostatic tumor invasion through coupling to transduction protein Gsalpha. Cell adhesion glycoprotein CD44 variant v7-v10 also faciliates invasion, but its modulation by the CT-Gsalpha system was unexplored. MATERIALS AND METHODS: LnCaP, PC-3 and metastasis-derived PC-3M cell lines were studied, including cells modified therefrom: Gsalpha-QL, expressing mutant constitutively active Gsalpha protein, and CT+, overexpressing CT. CD44 variant expression was evaluated in vivo after orthotopic implantion into nude mice, and in vitro by real-time RT-PCR and Western blotting. RESULTS: Both mRNA and protein levels of the CD44 variant were minimal in PC-3M tumor implants, but elevated in Gsalpha-QL. Exogenous CT stimulated invasion into Matrigel strongly in LnCaP and CT+, and less in PC-3 and Gsalpha-QL. By Western blot analysis, untreated Gsalpha-QL and CT+ cells overexpressed CD44 variant compared with LnCaP or PC-3. By quantitative RT-PCR, exogenous CT dose-dependently increased CD44 variant mRNA to seven-fold. Pharmacologic agents that stimulated or inhibited Gsalpha activity or stimulated adenylyl cyclase produced proportionate dose-dependent effects on both CD44 variant expression and Matrigel invasion. CONCLUSION: This paracrine factor, acting though cyclic AMP, regulates the expression of CD44v7-10, which modulates the tumor phenotype.

Characterization of [3H]-forskolin binding sites in young and adult rat brain cortex: identification of suramin as a competitive inhibitor of [3H]-forskolin binding.

Little is know about forskolin binding in the rat brain during ontogenetic development. For this paper, we have characterized specific binding sites for [3H]-forskolin in cerebrocortical membranes from young (12-day-old) and adult (90-day-old) rats. High-affinity, as well as super-high-affinity, [3H]-forskolin binding sites were detected in samples from both age groups tested, and the binding parameters of these sites differed significantly. Whereas the number of high-affinity [3H]-forskolin binding sites was higher by about 50% in adult than in young rats, their affinity was markedly (about 4 times) lower. In the presence of AlF4-, the number high-affinity [3H]-forskolin binding sites in samples from young rats rose to the level determined in samples from adult animals, and the number of super-high-affinity sites considerably increased in both age groups. The different characteristics of [3H]-forskolin binding found in cerebrocortical membranes from young and adult rats may be closely related to markedly diminished adenyl cyclase activity in preparations from adult animals. Results of our experiments with suramin indicated that this drug may act as a competitive inhibitor of [3H]-forskolin binding.

Galphao2 regulates vesicular glutamate transporter activity by changing its chloride dependence.

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Effects of the C-terminal peptide of the alphaS subunit of the G protein on the regulation of adenylyl cyclase and protein kinase A activities by biogenic amines and glucagon in mollusk and rat muscles.

The C-terminal parts of the a subunits of heteromeric G proteins play an important role in the functional linkage of G proteins with receptors of the serpentine type. The present report describes studies of the effects of the C-terminal octapeptide 387-394 of the alphaS subunit of the mammalian G protein on the transmission of the hormonal signal via the hormone-sensitive adenylyl cyclase signal system, whose major components are receptors of the serpentine type, G proteins, and the enzymes adenylyl cyclase and protein kinase A. The peptide synthesized here, 387-394 amide (10(-7) - 10(-4) M), dose-dependently decreased adenylyl cyclase and protein kinase A activities stimulated by serotonin and glucagon in smooth muscle from the freshwater bivalve mollusk Anodonta cygnea and by the beta agonist isoproterenol in rat skeletal muscle. At a concentration as low as 10(-7) M, the peptide released potentiation of the stimulatory effects of hormones on adenylyl cyclase activity due to the non-hydrolyzable guanine nucleotide analog Gpp[NH]p. At the same time, it had almost no effect on the stimulation of adenylyl cyclase activity by non-hormonal agents (NaF, Gpp[NH]p, and forskolin). The inhibitory effects of hormones on adenylyl cyclase and protein kinase A activities persisted in the presence of the peptide. Our data demonstrate the importance of the C-terminal part of the alphaS subunit of the stimulatory G protein for its functional linkage with receptors of the serpentine type and throw light on the molecular mechanisms of the interactions between G proteins and receptors.