Guanine Nucleotide-binding Regulatory Protein Research Articles

Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.

Direct regulation of vascular smooth muscle contraction by mastoparan-7

Mastoparan-7 (mas-7) is a basic tetradecapeptide isolated from wasp venom, which activates guanine nucleotide-binding regulatory proteins (G-proteins) and stimulates apoptosis. In smooth muscle cells, mas-7 leads to an increase in the perfusion pressure. The main aim of this study was to evaluate the physiological effect of the direct stimulation of G-proteins in comparison to the typical stimulation of receptors in vascular smooth muscle cells (VSMCs). Experiments were performed on the isolated and perfused tail artery of Wistar rats. The contraction force in our model was measured by an increased level of perfusion pressure with a constant flow. The concentration response curves (CRCs) obtained for mas-7 were sigmoidal. In comparison to the curves for phenylephrine and vasopressin, the mas-7 curve was significantly shifted to the right with a significant reduction in maximal response. Mas-7 significantly increased the perfusion pressure for the intra- and extracellular calcium (Ca2+) influx to the cytoplasm. The presence of the pertussis toxin (PT) did not affect the mas-7-induced contraction. In comparison to phenylephrine and vasopressin, all the values of perfusion pressure following stimulation of the G-proteins by mas-7 were significantly lower. The results of our experiments suggested that mas-7 significantly induces the contraction of VSMCs. The binding site for mas-7 is different from that for PT; thus, PT does not affect VSMC contraction. The tissue effect of this stimulation is comparable to the stimulatory effect of partial agonists. Current knowledge regarding the apoptosis pathway reveals the significance of Ca2+ ions involved in this process. Therefore, mas-7 may induce apoptosis through an increase in the cytoplasmic Ca2+ concentration; however, the use of this mechanism in anticancer therapy must be preceded by a molecule modification that eliminates the vasoconstrictive effect.

Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes.

The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5–13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.

Regulation of G protein signaling by the 70 kDa heat shock protein

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha2A adrenergic receptor (α2A-R) by the ubiquitous stress-inducible 70kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α2A-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α2A-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α2A-R-catalyzed [35S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine1A receptor (5-HT1A-R). In heat-stressed CHO cells, the α2A-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α2A-R compared to the 5-HT1A-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.

Contribution of G inhibitory protein alpha subunits in paradoxical hyperalgesia elicited by exceedingly low doses of morphine in mice

AimsAlthough morphine, at higher doses, induces analgesia, it may also enhance sensitivity to pain at extremely low doses as shown in studies for testing an animal's sensitivity to pain. We used an antisense approach capable of selectively down-regulating in vivo Gi(G inhibitory protein),Go and Gs members of the Gα sub-family protein subunits in order to establish if these proteins might be implicated in the effects induced by extremely low morphine doses on acute thermonociception. Main methodsMice pretreated with a morphine hyperalgesic dose (1μg/kg) were submitted to hot plate test after pre-treatment with antisense oligodeoxynucleotides (aODNs) targeting Giα, Goα and Gsα regulatory proteins. The association of G-protein (guanine nucleotide-binding regulatory protein) coupled receptors with G protein was investigated using co-immunoprecipitation procedure. Key findingsThe downregulation of the Giα1–3 and Goα1 proteins reversed the licking latency responses induced by 1μg/kg morphine administration toward the basal value whereas downregulation of the Goα2 and Gsα proteins did not significantly modify the hyperalgesic response. SignificanceThese results suggest that G inhibitory proteins play a role in the production of low dose evoked morphine hyperalgesia in mouse. Immunoprecipitation studies revealed that both μ opioid receptor (μOR) and α2 adrenoreceptor (α2 AR) are bound to G inhibitory proteins in hyperalgesic response to morphine extremely low dose. Both μOR and α2 AR appear to be necessary for low morphine dose induced hyperalgesic response through G inhibitory proteins.

Gα13 is closely related to hematopoiesis in zebrafish

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) function as signal transducers and control many different physiologic processes. G proteins can be grouped into four families: Gs, Gi, Gq and G12. Gα13 belongs to the G12 family. In zebrafish, there are two isoforms of Gα13: Gα13a and Gα13b. We show here that knockdown of Gα13b in zebrafish results in hematopoietic and angiogenic defects. The Gα13b morphants don't show complete loss of expression of gata1, pu.1 or flk until 35 hpf suggests that Gα13b is closely related to the development of hematopoietic cells. Further studies reveal that blood cells and vascular endothelial cells have undergone apoptosis through a p53-dependent pathway in Gα13b-depleted embryos. Injection of p53 morpholino could partially rescue the phenotype of Gα13b morphants. These data possibly demonstrate a new role for Gα13 in cell survival.

Developmental sperm contributions: fertilization and beyond

The objective of this review was to examine the role of the various spermatozoal components suspected of actively participating in early human development. The contributions of the fertilizing spermatozoon to the oocyte include, as a minimum, the delivery of the DNA/chromatin, a putative oocyte-activating factor (OAF), and a centriole. Recent data indicate that spermatozoa may also provide the zygote with a unique suite of paternal mRNAs; some transcripts might be crucial for early and late embryonic development and deficient delivery, or aberrant transcription might contribute to abnormal development and arrest. Clinical evidence from assisted reproduction suggests that failure to complete the fertilization process, syngamy, or early cleavage might be the result of an early paternal effect. It is speculated that an abnormal release of a putative OAF and/or dysfunctions of the centrosome and cytoskeletal apparatus may mediate these effects. On the other hand, a later paternal effect resulting in embryonic failure to achieve implantation, pregnancy loss, and/or developmental abnormalities resulting from "carried over" sublethal effects may be associated with sperm nuclear/chromatin defects, including the presence of aneuploidy, genetic anomalies, DNA damage, and possibly other causes. These findings highlight the need for continuous monitoring of clinical results.

Segment-Dependent Activation of Muscarinic Acetylcholine Receptor-Mediated [35S]Guanosine-5′-O-(γ-Thiotriphosphate) Binding in Airway Tissue Membranes

Muscarinic acetylcholine receptor (mAChR)-mediated guanine nucleotide-binding regulatory protein (G protein) activation and the functional interaction between receptors and the respective G proteins were investigated using an agonist-induced [³⁵S]guanosine-5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS)-binding approach in membranes of 3 native equine airway segments (trachea, bronchus and lung), which differ tremendously in mAChR density and subtype distribution; especially subtypes that couple negatively to adenylyl cyclase through G_i/0 proteins, i.e. M₂ receptors. The assay was initially optimized by determining the influence of incubation time, guanosine 5′-diphosphate (GDP), MgCl₂ and NaCl on basal and agonist-stimulated [³⁵S]GTPγS binding. In standard assays, the presence of 10 μmol/l GDP, 10 mmol/l MgCl₂ and 200 mmol/l NaCl increased carbachol-induced specific [³⁵S]GTPγS binding in a segment- and receptor-density-dependent manner. Hereby, mAChR agonists stimulated [³⁵S]GTPγS binding with a rank order of potency (oxotremorine M > carbachol > acetylcholine), and in a specific segment- and receptor-density-dependent manner (trachea > bronchus > lung). The increase in the specific [³⁵S]GTPγS binding was potently inhibited by the mAChR antagonist atropine. Pertussis toxin and N-ethylmaleimide as G_i/0 protein ADP-ribosylating and alkylating agents reduced basal and agonist-stimulated [³⁵S]GTPγS binding. The mAChR-stimulated [³⁵S]GTPγS binding serves as a useful method for investigating the functional interaction between mAChRs and their respective G proteins in native airway tissue membranes of equines.

Intracellular Sodium Regulates Proteolytic Activation of the Epithelial Sodium Channel

Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).

Subcellular redistribution of trimeric G-proteins – potential mechanism of desensitization of hormone response; internalisation, solubilization, down-regulation

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Somatostatin modulates voltage-dependent Ca2+ channels in GH3 cells via a specific G(o) splice variant.

In rat pituitary GH3 cells Ca2+ current through L-type channels is reduced by somatostatin. This modulation of channel activity by somatostatin receptors is mediated by a guanine nucleotide-binding regulatory protein (G protein). It is sensitive to pertussis toxin, indicating the involvement of a G(o)- or Gi-type G protein in this pathway. The identity of this G protein was determined by suppressing the expression of endogenous G proteins individually via intranuclear injection of antisense oligonucleotides. This method was applied to GH3 cells to screen several G protein alpha, beta and gamma subunits for their roles in the defined signal transduction pathway. The loss of somatostatin's modulating activity on the voltage-dependent Ca2+ channel after oligonucleotide injection revealed the involvement of G(o) alpha 2 beta 1 gamma 3 to the exclusion of other closely related subtypes.

G proteins in signal transduction: the regulation of phospholipase C.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate by specific phospholipase C (PLC) enzymes produces two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) of the Gq subfamily activate the PLC beta 1 isoform of PLC. We have purified three isozymes of PLC beta: PLC beta 1 and PLC beta 3 from rat brain and PLC beta 2 from HL-60 cells. Whereas the beta 1 and beta 2 isozymes appear restricted to a few cell types, beta 3 is broadly distributed. Gq alpha (the alpha subunit of the Gq subfamily) can activate all three isoforms but PLC beta 2 is much less sensitive. Thus all three enzymes are potential effectors for pertussis toxin-insensitive regulation by hormones. The three beta isozymes can also be activated by purified beta gamma subunits. The PLC beta 3 isoform gives the greatest activation with beta gamma; PLC beta 1 is least responsive. The results indicate that all the known isoforms of mammalian PLC beta can be regulated at unique sites by both Gq alpha and beta gamma subunits. The effect of beta gamma subunits may provide a pathway for the regulation of PLC beta isozymes by pertussis toxin-sensitive G proteins or may indicate that the alpha subunit of Gq and its associated beta gamma both participate in regulation of the same phospholipase molecule.

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

BackgroundHeterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain.ResultsWe show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain.ConclusionA fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed Gβ5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the Gβ5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the Gβ5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex.

The Role of Internal Water Molecules in the Structure and Function of the Rhodopsin Family of G Protein‐Coupled Receptors

Membrane receptors coupled to guanine nucleotide-binding regulatory proteins (commonly known as G protein-coupled ACHTUNGTRENNUNGreceptors, GPCRs) constitute one of the most attractive pharmaceutical targets, as around 40% of clinically prescribed drugs and 25% of the top-selling drugs act at these receptors. GPCRs are receptors for sensory signals of external origin such as odors, pheromones, or tastes; and for endogenous signals such as neurotransmitters, (neuro)peptides, divalent cations, proteases, glycoprotein hormones, and purine ligands. Phylogenetic analyses of the human genome have permitted GPCR sequences to be classified into five main families: rhodopsin (class A or family 1), secretin (class B or family 2), glutamate (class C or family 3), adhesion, and frizzled/ taste2. Specialized databases of GPCRs can be found at http://www.gpcr.org/7tm, http://gris.ulb.ac.be/, and http:// www.iuphar-db.org. Due to the low natural abundance of GPCRs and the difficulty in producing and purifying recombinant protein, only one member of this family, rhodopsin, the photoreceptor protein of rod cells, has been crystallized so far. Five structural models of inactive rhodopsin are available at the Protein Data Bank, at resolutions of 2.8 A (PDB IDs: 1F88 and 1HZX), 2.65 A (1GZM), 2.6 A (1L9H), and 2.2 A (1U19). Structural models of rhodopsin photointermediates such as bathorhodopsin (2G87), lumirhodopsin (2HPY), metarhodopsin I, and a photoactivated deprotonated intermediate reminiscent of metarhodopsin II (2I37) are also available. Rhodopsin is formed by an extracellular N terminus of four b-strands, seven transmembrane helices (TM1 to TM7) connected by alternating intracellular (I1 to I3) and extracellular (E1 to E3) hydrophilic loops, a disulfide bridge between E2 and TM3, and a cytoplasmic C terminus containing an a-helix (Hx8) parallel to the cell membrane. Statistical analysis of the residues forming the TM helices of the rhodopsin family of GPCRs shows a large number of conserved sequence patterns; this suggests a common TM structure. Thus, the availability of the rhodopsin structure allows the use of homology modeling techniques to build three-dimensional models of other homologous GPCRs. The putative structural homology between rhodopsin and other GPCRs probably does not extend to the extracellular domain, since the extracellular N terminus and loop fragments are highly variable in length and amino acid content. The class A family of GPCRs contains highly conserved Pro residues in the middle of TMs 5 (P5.50, conserved in 77% of the sequences), 6 (P6.50, 100%), and 7 (P7.50, 96%; residues are identified by the generic numbering scheme of Ballesteros and Weinstein, which allows easy comparison among residues in the 7TM segments of different receptors). Pro residues are normally observed in the TM helices of membrane proteins where they usually induce a significant distortion named a “Pro-kink”. This break arises in order to avoid a steric clash between the pyrrolidine ring of the Pro side chain (at position i) and the carbonyl oxygen of the residue in the preceding turn (position i 4) and leads to a distortion of the helical structure. However, TM segments of rhodopsin, either with or without Pro residues in their sequence are far from being standard Pro-kinked or ideal helices, respectively. Their distortions are energetically stabilized through complementary intraand interhelical interactions involving polar side chains, backbone carbonyls, and, in some cases, specific structural and functional water molecules embedded in the TM bundle. Here we review the role of these water molecules in the structure and function of GPCRs and in building computergenerated homology models of class A GPCRs. We propose that water molecules present in the vicinity of highly conserved motifs are most likely present in the rhodopsin family of GPCRs, being another conserved structural element in the family.

Influence of Differential Stability of G Protein βγ Dimers Containing the γ11 Subunit on Functional Activity at the M1 Muscarinic Receptor, A1 Adenosine Receptor, and Phospholipase C-β

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.

Dopamine D 2 receptor-mediated G protein activation assessed by agonist-stimulated [ 35S]guanosine 5′- O-(γ-thiotriphosphate) binding in rat striatal membranes

In order to investigate the functional interaction between the native dopamine receptors and their coupled guanine nucleotide-binding regulatory (G) proteins, dopamine-stimulated [ 35S]guanosine 5′- O-(γ-thiotriphosphate) ([ 35S]GTPγS) binding was pharmacologically characterized in rat striatal membranes. Following optimizing the experimental conditions as to the concentrations of GDP, MgCl 2 and NaCl in the assay medium, the agonist and antagonist properties for a series of dopamine receptor ligands were determined mainly under the standard assay condition. The pharmacological profile of this response clearly indicated the involvement of dopamine D 2-like receptors, but not of dopamine D 1-like receptors. Among the types of dopamine D 2-like receptors, dopamine D 2 receptors most likely appeared to be involved in dopamine-stimulated [ 35S]GTPγS binding in rat striatal membranes, because the affinities of agonists and antagonists determined in the present study were significantly correlated with those reported in the previous literature only for dopamine D 2 receptors, but not for dopamine D 3 or D 4 types. Though the concentration-dependent inhibition curves of dopamine-stimulated [ 35S]GTPγS binding by spiperone and S(−)-raclopride were apparently biphasic, the origin of the low-affinity minor components was not fully determined. The antiparkinsonian drugs with the properties of dopamine receptor agonism were shown to behave as stimulants with varied affinities and relative efficacies in the current assay system. On the other hand, neither phencyclidine (PCP) nor ketamine stimulated the specific [ 35S]GTPγS binding, in contrast with the previous report demonstrating that these two N-methyl- d-aspartic acid (NMDA) receptor antagonists behaved as agonists at human dopamine D 2 receptors expressed in Chinese hamster ovary (CHO) cells. These results provide important information about the functional activation of G proteins coupled with dopamine D 2 receptors as well as agonist actions of various compounds at native dopamine D 2 receptors, which are potentially involved in pathophysiology and pharmacotherapy of neuropsychiatric diseases such as Parkinson's disease, schizophrenia and depression.

5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through Gi type G proteins. In the present study, 5-HT-stimulated [35S]GTPgammaS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 microM GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [35S]GTPgammaS binding was stimulated by several compounds that had been shown to be agonists at 5-HT(1B/1D) receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC50) for these agonists were significantly correlated with their pKi's reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [35S]GTPgammaS binding in response to 1 microM 5-HT was potently inhibited by several 5-HT(1B/1D) receptor antagonists as well as beta-adrenoceptor antagonists such as S(-)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 microM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(-)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [35S]GTPgammaS binding was reduced by 1'-methyl-5-([2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4'-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(-)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [35S]GTPgammaS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.

Differential Sensitivity of P-Rex1 to Isoforms of G Protein βγ Dimers

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.

IL-15 induces mast cell migration via a pertussis toxin-sensitive receptor

IL-15 induces proliferation, inhibits apoptosis and increases IL-4 production in murine mast cells. There is evidence that these activities are mediated via the uncharacterised receptor, IL-15R-X, rather than the classical three-chain IL-15 receptor. Effects of IL-15 on important aspects of mast cell biology, such as migration and degranulation, are unknown. We report that IL-15 induces migration of murine and human mast cells in a dose-dependent and biphasic manner, with peaks of migration occurring at approximately 10(-15) and approximately 10(-9) M. The potency of the response was similar to that induced by other well-established mast cell chemoattractants. Competition assays performed with murine and human mast cells indicate that both peaks of migration are due to chemotaxis. Pre-treatment of cells with pertussis toxin (PTX), a guanine nucleotide-binding regulatory protein (G-protein) inhibitor, resulted in complete inhibition of murine mast cell migration at approximately 10(-15) M IL-15, and human mast cell migration at approximately 10(-15) and approximately 10(-9) M. This demonstrates that murine and human mast cells express a PTX-sensitive receptor, activated in response to IL-15. Additionally, IL-15 did not induce degranulation in murine mast cells. Locally-produced IL-15 may contribute to mast cell recruitment during inflammatory responses, thereby acting as a linking cytokine between innate and adaptive arms of the immune system.

Loss-of-function mutations identified in the Helical domain of the G protein α-subunit, Gα2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of alpha-, beta-, and gamma-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the alpha-subunit. The alpha-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known alpha-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of alpha-subunit function, we performed a screen for loss-of-function mutations, using the G alpha2-subunit of Dictyostelium. G alpha2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of G alpha2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to G alpha2 function. A library of random point mutations in the g alpha2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a g alpha2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the g alpha2 insert. To date, sixteen single amino acids changes have been identified in G alpha2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the alpha-subunit. These loss-of-function mutations in the alpha-subunit Helical domain may provide important insight into its function.