Activation of mu-opioid receptors transfers control of Galpha subunits to the regulator of G-protein signaling RGS9-2: role in receptor desensitization

In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of Galpha subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their co-precipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5'-O-(3-[35S]thiotriphosphate) binding as well as low Km GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.     

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.     

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

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

Peptide interactions with G-protein coupled receptors.

Peptide recognition by G-protein coupled receptors (GPCRs) is reviewed with an emphasis on the indirect approach used to determine the receptor-bound conformation of peptide ligands. This approach was developed in response to the lack of detailed structural information available for these receptors. Recent advances in the structural determination of rhodopsin (the GPCR of the visual system) by crystallography have provided a scaffold for homology modeling of the inactive state of a wide variety of GPCRs that interact with peptide messages. Additionally, the ability to mutate GPCRs and assay compounds of similar chemical structure to test a common binding site on the receptor provides a firm experimental basis for structure-activity studies. Recognition motifs, common in other well-studied systems such as proteolytic enzymes and major histocompatibility class receptors (MHC) are reviewed briefly to provide a basis of comparison. Finally, the development of true peptidomimetics is contrasted with nonpeptide ligands, discovered through combinatorial chemistry. In many systems, the evidence suggests that the peptide ligands bind at the interface between the transmembrane segments and the extracellular loops, while nonpeptide antagonists bind within the transmembrane segments. Plausible models of GPCRs and the mechanism by which they activate G-proteins on binding peptides are beginning to emerge.     

Potent activation of RhoA by Galpha q and Gq-coupled receptors

Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.     

Decreased ADP-ribosylation of the Galpha(olf) and Galpha(s) subunits by high glucose in pancreatic B-cells

In HIT-T15 insulinoma B-cells incubated in presence of [(32)P]NAD, we identified by autoradiography and immunoblotting ADP-ribosylation (ADP-R) of the trimeric G-protein Galpha(s) and Galpha(olf) subunits (45 kDa) induced by cholera toxin in M1 (120,000g) and M2 (70,000g) subcellular fractions containing plasma membranes, insulin granules, and mitochondria. This ADP-R indicates that these two fractions contain functionally competent Galpha subunits for adenylyl cyclase activation. Prolonged exposure of HIT-T15 cells to high glucose (25 mM instead of 6 mM) specifically reduced the ADP-R in Galpha(s) and Galpha(olf) subunits in the M1 fraction only, despite the clear increase of their accumulation in this compartment. A similar alteration in the ADP-R of the M1-associated Galpha(s) and Galpha(olf) subunits was observed in pancreatic islets isolated from fasted and fed rats. These results may explain, at least in part, the undesirable effects of sustained hyperglycemia on the cAMP-dependent process of insulin secretion in diabetes.     

The importance of G-protein beta lambda subunits

As the properties of more and more isoforms of the molecules involved in G-protein-mediated signal transduction pathways are unravelled, surprising diversity and versatility are being revealed. The path from receptor to effector is not dictated exclusively by the alpha subunits of heterotrimetric G proteins. The nature of the beta lambda subunit complex probably controls interactions of G(alpha) with receptors. In addition, dissociation of G(alpha)-GTP from G(beta lambda)provides two signalling complexes, and these proteins regulate effectors independently or synergistically. Synergistic or conditional regulation of effectors by G(alpha) and G(beta lambda)can provide a molecular signal that records the association of independent events.     

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.     

Regulatory interactions between the amino terminus of G-protein betagamma subunits and the catalytic domain of phospholipase Cbeta2

We previously identified a 10-amino acid region from the Y domain of phospholipase Cbeta2 (PLCbeta2) that associates with G-protein betagamma subunits (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154). We mapped the site for cross-linking of a synthetic peptide (N20K) corresponding to this Y domain region to Cys(25) within the amino-terminal coiled-coil domain of Gbetagamma (Yoshikawa, D. M., Bresciano, K., Hatwar, M., and Smrcka, A. V. (2001) J. Biol. Chem. 276, 11246-11251). Here, further experiments with a series of variable length cross-linking agents refined the site of N20K binding to within 4.4-6.7 angstroms of Cys(25). A mutant within the amino terminus of the Gbeta subunit, Gbeta(1)(23-27)gamma(2), activated PLCbeta2 more effectively than wild type, with no significant change in the EC(50), indicating that this region is directly involved in the catalytic regulation of PLCbeta2. This mutant was deficient in cross-linking to N20K, suggesting that a binding site for the peptide had been eliminated. Surprisingly, N20K could still inhibit Gbeta(1)(23-27)gamma(2)-dependent activation of PLC, suggesting a second N20K binding site. Competition analysis with a peptide that binds to the Galpha subunit switch II binding surface of Gbetagamma indicates a second N20K binding site at this surface. Furthermore, mutations to the N20K region within the Y-domain of full-length PLCbeta2 inhibited Gbetagamma-dependent regulation of the enzyme, providing further evidence for aGbetagamma binding site within the catalytic domain of PLCbeta2. The data support a model with two modes of PLC binding to Gbetagamma through the catalytic domain, where interactions with the amino-terminal coiled-coil domain are inhibitory, and interactions with the Galpha subunit switch II binding surface are stimulatory.     

Activation of RhoA by association of Galpha(13) with Dbl

RhoA is a small G protein that is implicated in the regulation of the actin cytoskeleton, gene expression, and cell cycle progression. It is activated by many agonists whose receptors are linked to heterotrimeric G proteins, but the mechanisms are incompletely understood. In this study, we show that the constitutively active alpha-subunit of the heterotrimeric G protein G(13) associated with the Rho family guanine nucleotide exchange factor Dbl in NIH 3T3 cells and that this resulted in activation of RhoA. This activation was not seen with wild-type Galpha(13) or if Dbl and active Galpha(13) were expressed separately and mixed. In contrast, coexpression of constitutively active Galpha(q) with Dbl did not lead to their association and caused a weak activation of RhoA that was no greater than that observed with wild-type Galpha(q). These findings illustrate that activated Galpha(13) and Dbl can associate in vivo and that this leads to Rho activation.     

Stabilization of C5a receptor–G-protein interactions through ligand binding

Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor–G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor–effector interactions. When biotin-C5a–ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both G_ialpha₂ and G_ialpha₃ subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems. © 1994 Wiley-Liss, Inc.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mechanism of membrane-translocation of regulator of G-protein signaling (RGS) 8 induced by Galpha expression

RGS (regulators of G-protein signaling) proteins comprise a large family that modulates heterotrimeric G-protein signaling. This protein family has a common RGS domain and functions as GTPase-activating proteins for the alpha-subunits of heterotrimeric G-proteins located at the plasma membrane. RGS8 was identified as a neuron-specific RGS protein, which belongs to the B/R4 subfamily. We previously showed that RGS8 protein was translocated to the plasma membrane from the nucleus on coexpression of GTPase-deficient Galphao (GalphaoQL). Here, we first examined which subtypes of Galpha can induce the translocation of RGS8. When the Galphai family was expressed, the translocation of RGS8 did occur. To investigate the mechanism of this translocation, we generated a mutant RGS8 with reduced affinity to Galphao and an RGS-insensitive (RGS-i) mutant of GalphaoQL. Co-expression experiments with both mutants revealed that disruption of the Galpha-RGS8 interaction abolished the membrane-translocation of RGS8 despite the apparent membrane localization of RGS-i GalphaoQL. These results demonstrated that RGS8 is recruited to the plasma membrane where G-proteins are activated mainly by direct association with Galpha.     

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

Signaling and regulation of G-protein coupled receptors encoded by cytomegaloviruses.

Cytomegaloviruses (CMVs) are species-specific beta-herpesviruses whose replicative success is largely due to establishment of novel mechanisms for altering the host immune response. CMV encodes 3 families of putative G-protein coupled receptors (GPCRs) likely pirated from the host cell. While the functions of these virally encoded GPCRs remain unclear, the receptors possess potent signaling abilities. Understanding the molecular regulation of these GPCRs will provide important insight into CMV pathogenesis.     

Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.     

Immunolocalization of multiple Galpha subunits in mammalian spermatozoa and additional evidence for Galphas

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.     

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.