Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Gαs) and its inhibition (mediated by Gαi) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gαq (<50%) but not Gαi, Gαz, Gα12, and Gα13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gαq in thrombin-induced responses.

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.

Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABAB) receptors, activates G protein-gated inwardly rectifying K+ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABAB receptors in wild-type granule cells induced an inwardly rectifying K+ current, which was sensitive to pertussis toxin and inhibited by external Ba2+ ions. The amplitude of the GABAB receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K+ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABAB receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABAB receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gαi/Gαq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gαi3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl− currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5–11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl− current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

Activation of protein kinase A-independent pathways by Gsα in Drosophila

One of the best-described transmembrane signal transduction mechanisms is based on receptor activation of the α subunit of the heterotrimeric G protein Gs, leading to stimulation of adenylyl cyclase and the production of cAMP. Intracellular cAMP is then thought to mediate its effects largely, if not entirely, by activation of protein kinase A and the subsequent phosphorylation of substrates which in turn control diverse cellular phenomena. In this report we demonstrate, by two different methods, that reduction or elimination of protein kinase A activity had no effect on phenotypes generated by activation of Gsα pathways in Drosophila wing epithelial cells. These genetic studies show that the Gsα pathway mediates its primary effects by a novel pathway in differentiating wing epithelial cells. This novel pathway may in part be responsible for some of the complex, cell-specific responses observed following activation of this pathway in different cell types.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Gαq C terminus antibody; and (iv) was associated with a net increase in Gαq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Gα subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Gαq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.

Regulation of the p21-activated kinase (PAK) by a human Gβ-like WD-repeat protein, hPIP1

The family of p21-activated protein kinases (PAKs) is composed of serine–threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein β-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor κ B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.