Protein-mediated Pathway Research Articles

Purification of G protein subunits from Sf9 insect cells using hexahistidine-tagged alpha and beta gamma subunits.

G protein-mediated pathways are the most fundamental mechanisms of cell signaling. In order to analyze these pathways, the availability of purified recombinant G proteins are critically important. Using Sf9-Baculovirus expression system, a general and simplified method to purify various G protein subunits is described in this chapter. This method is useful for purification of most of G protein subunits.

Mechanisms of Guanine Nucleotide Exchange and Rac-mediated Signaling Revealed by a Dominant Negative Trio Mutant

Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis. Dominant negative mutants of Rho GTPases, such as T17NRac1, that block the endogenous Rho protein activation by sequestering upstream guanine nucleotide exchange factors (GEFs) have been widely used to implicate specific members of the Rho family in various signaling pathways. We show here that such an approach could produce potentially misleading results since many Rho GEFs can interact with multiple Rho proteins promiscuously, and overexpression of one dominant negative Rho protein mutant may affect the activity of other members of the Rho family. Based on the available structural information, we have identified the highly conserved amino acid pairing of Asn(1406)Trio-Asp(65)Rac1 of the GEF-Rho GTPase interaction as the critical catalytic machinery required for the Rac1 GDP/GTP exchange reaction. The N1406A/D1407A mutant of Trio acted dominant negatively in vitro by retaining Rac1 binding activity but losing GEF catalytic activity and competitively inhibited Rac1 activation by wild type Trio. It readily blocked the platelet-derived growth factor (PDGF)-induced lamellipodia formation and inhibited the wild type Trio-induced serum response factor activation. Moreover the mutant was able to selectively inhibit Dbl-induced Rac1 activation without affecting RhoA activity in cells. In contrast to the non-discriminative inhibitory effect displayed by T17NRac1, the Trio mutant was ineffective in inhibiting PDGF-stimulated DNA synthesis and Dbl-induced transformation, revealing the Rac-independent functions of PDGF and Dbl. These studies identify a conserved pair of amino acid residues of the Trio-Rac interaction that is likely to be essential to the GEF catalysis of Rho family GTPases and demonstrate that a dominant negative mutant derived from a Rho GTPase regulator constitutes a new generation of specific inhibitors of Rho GTPase signaling pathways.

Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.

Independent β-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

GluR5 Goes Both Ways

The neurotoxin kainate activates a particular class of ionophoric glutamate receptor, the kainate receptor. Kainate also inhibits neurotransmitter release through a pathway that involves a pertussis toxin-sensitive G protein (heterotrimeric guanine nucleotide-binding protein) and protein kinase C (PKC). Rozas et al . investigated the relation between these effects in cultured rat dorsal root ganglion cells (DRGs) and discovered that kainate's activation of the G protein-coupled pathway depended on expression of GluR5, an ion channel-forming subunit of the kainate receptor, but was independent of functional ion channel activity. The authors used calcium imaging to show that kainate stimulated a rise in intracellular calcium ([Ca 2+ ] i ); a fraction that was sensitive to pertussis toxin persisted in the absence of extracellular calcium, which suggests that it represented release from intracellular stores. Calcium imaging in combination with electrophysiological analysis indicated that pretreatment with kainate inhibited Ca 2+ influx in response to depolarization with potassium; this was sensitive to pertussis toxin and inhibitors of PKC. There was no correlation between kainate-stimulated Ca 2+ influx (a marker of ionophoric activity) and kainate inhibition of depolarization-dependent Ca 2+ influx. Moreover, inhibition of depolarization-dependent Ca 2+ influx occurred at kainate concentrations that were too low to affect [Ca 2+ ] i directly and in the absence of extracellular sodium (which affects receptor gating). Both kainate-stimulated Ca 2+ influx and kainate's inhibition of depolarization-dependent Ca 2+ influx were abolished in DRGs from mice lacking GluR5. Pharmacological analysis suggested that kainate-dependent release of glutamate from primary sensory afferents involved a G protein-mediated pathway. Thus GluR5 appears to mediate distinct kainate signaling pathways; one involves formation of an ionophoric receptor and one involves G protein activation. J. L. Rozas, A. V. Paternain, J. Lerma, Noncanonical signaling by ionotropic kainate receptors. Neuron 39 , 543-553 (2003). [Online Journal]

Specificity of G Protein-RGS Protein Recognition Is Regulated by Affinity Adapters

RGS proteins regulate the duration of cell signaling by modulating the lifetime of activated G proteins. The specificity of RGS-G protein mutual recognition is critical for meeting unique timing requirements of numerous G protein-mediated pathways. Our study of two splice isoforms of RGS9 expressed in different types of neurons revealed a novel mechanism whereby this specificity is determined by specialized protein domains or subunits acting as affinity adapters. The long RGS9 isoform contains a C-terminal domain that provides high-affinity interaction with its target G protein. The lack of this domain in the short RGS9 isoform is compensated by the action of a G protein effector subunit that is structurally similar to this C-terminal domain. This allows the short isoform to specifically target the complex between the G protein and its effector. Thus, the specific timing needs of different signaling pathways can be accommodated by affinity adapters positioned at various pathway components.

Ozone-induced disruptions of lung transcriptomes

We have analyzed changes in ∼4000 lung mRNAs, with GeneChips, in mice exposed to 1 ppm O 3 for three consecutive nights (8 h per night). Differential gene expression analysis identified ∼260 O 3 sensitive genes; ∼80% of these were repressed and ∼20% were induced in O 3-exposed mice compared to the air-exposed controls. A 20-fold induction of serum amyloid A3 mRNA by O 3 suggested activation of NF-κB and CCAAT/enhancer binding protein-mediated pathways by inflammatory cytokines. Induction (up to 14-fold) of 12 genes that increase DNA synthesis and cell cycle progression, and increase (∼7-fold) in CD44 mRNA and macrophage metalloelastase suggested a state of O 3-induced hyperplasia and lung remodeling. Several mRNAs encoding enzymes of xenobiotic metabolism and cytoskeletal functions were repressed and may suggest cytokine mediated suppression of cytochrome P450 expression and cachexia-like inflammatory state in ozone-exposed lungs. The expressions of ∼30 genes of immune response were also repressed. Collectively this genome-wide analysis of lungs identified ozone-induced disruption of gene transcriptional profile indicative of increased cellular proliferation under suppressed immune surveillance and xenobiotic metabolism.

Signal-Dependent Translocation of Transducin, RGS9-1-Gβ5L Complex, and Arrestin to Detergent-Resistant Membrane Rafts in Photoreceptors

Many lines of evidence show that membranes contain microdomains, “lipid rafts”, that are different from the rest of the membrane in specific lipid and protein composition [1]. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gβ5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPγS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF−4 causes both proteins to move to the raft in the dark. This shows that the Gαt-RGS9-1-Gβ5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPγS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.

VIP and PACAP inhibit L-, N- and P/Q-type Ca2+ channels of parasympathetic neurons in a voltage independent manner.

In this study, we investigated the effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP) on the voltage-gated calcium currents in hamster submandibular ganglion neurons. VIP and PACAP inhibited the high threshold voltage-gated calcium current in a voltage-independent and a concentration-dependent manner via the G protein-mediated pathway. L-, N- and P/Q-type components of the total maximum voltage-gated calcium current accounted for 48.0 +/- 3.1% (n = 4), 35.1 +/- 4.7% (n = 4), and 13.5 +/- 2.3% (n = 3) of the total peak amplitude, respectively. VIP at a concentration of 1 microM inhibited the L-type calcium current by 33.2% +/- 1.4% (n = 4), the N-type current by 31.0 +/- 3.6%, and the P/Q-type current by 3.2 +/- 1.1% (n = 3). PACAP at a concentration of 1 microM inhibited the L-type current by 35.6 +/- 5.7%, the N-type current by 34.4 +/- 3.1% (n = 4), and the P/Q-type current by 6.4 +/- 2.1% (n = 2). However, VIP and PACAP did not inhibit the low threshold voltage-gated (T-type) calcium current. The rank order of potency was PACAP > VIP. In experiments replacing GTP with GDP-beta-S, the inhibitory effects of VIP and PACAP were prevented. In experiments of double-pulse protocol, depolarizing conditioning pulses could not relieve the inhibition of total high threshold voltage-gated calcium currents produced by VIP and PACAP. Therefore, the inhibition of the high threshold voltage-gated calcium channels produced by VIP and PACAP in hamster parasympathetic neurons differed in its mechanisms from that of N-type calcium channels in rat sympathetic neurons.

Trout ovulatory proteins: site of synthesis, regulation, and possible biological function.

The mRNA transcripts for trout ovulatory proteins (TOPs) are dramatically up-regulated at the time of ovulation. Previous studies indicated that TOPs were produced by the ovaries and were also present in the coelomic fluid that bathes ovulated eggs. In the present study, Western analysis indicated that TOPs were not present in the coelomic fluid prior to ovulation and therefore must be secreted into the coelomic fluid in large quantities during and after ovulation. Using in situ hybridization and immunocytochemistry, TOP mRNA and proteins were localized to the granulosa cell layer of the postovulatory follicle. A whole-follicle in vitro incubation system was used to look at the effects of various mediators on TOP mRNA and protein levels. Results of several different secondary messenger agonists suggest that TOPs are regulated through a G protein-mediated pathway that does not involve cAMP but may involve the activation of protein kinase C. Other agonists that had significant effects on TOP RNA and/or protein included transforming growth factor alpha (TGF-alpha), serine proteases, corticosteroids, bacterial lipopolysaccharide, and the nitric oxide generator SNAP ([+/-]-S-nitroso-N-acetylpenicillamine). Overall, while several compounds caused significant effects, none were able to reproduce the increase in TOP RNA and protein that occurs in vivo, suggesting that the natural mediator of TOPs may still be untested, or that a combination of mediators may be involved. Finally, coelomic fluid inhibited the growth of the Gram negative bacterium, P. aeruginosa, and this inhibition was lost following immunoprecipitation of TOPs. This suggests that one function of TOPs may be to protect ovulated eggs from bacterial infection.

High-density lipoprotein increases intracellular calcium levels by releasing calcium from internal stores in human endothelial cells

Elevated levels of high-density lipoproteins (HDL) appear to delay or prevent the development of atherosclerosis. The intracellular signaling mechanisms activated by HDL in vascular cells are currently under active investigation. In this study the effects of HDL on endothelial intracellular Ca levels (EC Ca i) are investigated. We show that HDL, like low density lipoproteins (LDL), increases EC Ca i in a dose-dependent fashion by releasing Ca from internal stores. Neither apolipoprotein A-I (apo A-I) nor apolipoprotein A-II (apo A-II) was responsible for the increase in EC Ca i. HDL appeared to release Ca from the same internal stores as did LDL, since preincubation of EC with LDL prevented subsequent responses to HDL but not to the vasodilator ATP. In addition, preincubation of EC with pertussis toxin, an inhibitor of specific G proteins, as well as U73122, an inhibitor of phospholipase C, prevented a rise in EC Ca i in response to HDL. These findings suggest that HDL, like LDL, can modulate EC Ca i and that this occurs via a pertussis toxin-sensitive G protein-mediated pathway which involves phospholipase C.

Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca 2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1–100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca 2+ (≤10 −6M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca 2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5′-O-(2-thiodiphosphate) (GDP βs). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca 2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.

Ethanol inhibits G-protein-mediated glucose uptake by C6 glioma cells.

The mechanism of ethanol inhibition of glucose uptake was investigated using C6 glioma cells. Basal [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was inhibited by ethanol in a concentration-dependent manner. Fifty, 75 and 100 mM ethanol significantly inhibited basal 2DG uptake by 12, 20 and 23%, respectively (p < 0.05). Carbachol (an agonist acting via G protein-coupled receptors) stimulated the uptake by 26% (p < 0.05). In the presence of 100 mM ethanol, the ability of carbachol to stimulate 2DG uptake was abolished. In contrast, ethanol did not inhibit the ability of insulin to stimulate 2DG uptake. These results suggest that ethanol inhibits 2DG uptake by selectively interfering with G protein-mediated signal transduction pathway.

Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12-amino acid sequence located in the NH2-terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+-independent and not disassociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The association of prosaposin-GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22-mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide-induced neurite outgrowth, as well as prosaptide-enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein-mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

The role of G proteins in insulin signalling.

Insulin modulates many intracellular processes including cellular metabolism, cell proliferation and cell differentiation. Some of these processes involve significant changes in the traffic of intracellular vesicles or in the structural organization of the cell. These phenomena have been linked to the activity of regulatory GTP-binding proteins. Most, if not all functions, of the insulin receptor are associated with its tyrosine kinase activity. Thus, over the past few years, a significant effort has been dedicated to elucidate the cross-talk between the tyrosine kinase activity of the receptor and the regulation of G protein-mediated pathways. Recent progress indicates that G proteins may mediate the control of several of insulin's intracellular functions. These include the regulation of the MAP kinase pathway, the activation of phospholipase D and the regulation of glucose uptake. This article discusses some recent advances in this area.

Monitoring of hematopoietic recovery after autologous stem cell transplantation by analysis of G alpha 16 mRNA and CD34 surface glycoprotein.

The hematopoiesis-specific G protein alpha subunit G alpha16 was shown to be expressed in early normal and malignant hematopoietic cell lines and has been suggested to play an important role in signal transduction of hematopoiesis. We previously demonstrated a strict correlation of G alpha16 mRNA and CD34 antigen expression in peripheral blood stem cells (PBSC). In PBSC mobilization, both markers are detectable at the time of hematopoietic recovery and progenitor cell release. In this study the possible use of G alpha16 determination in peripheral blood samples for monitoring patients undergoing stem cell transplantation was investigated. Normal peripheral blood is negative for G alpha16 expression. In all five patients G alpha16 mRNA expression appeared shortly before the time of blood cell recovery. When tested together with CD34 (three cases) a pattern different from CD34 antigen expression was found, reflecting a different mechanism of action. In two cases with different time points of leukocyte and platelet recovery G alpha16 mRNA was detected at both time points but not in the interval, thus suggesting a role of G alpha16 in multipotent precursor cells. CD34 mRNA tested in three patients was not detected at any time; this argues for different regulation of CD34 and G alpha16 mRNA. G alpha16 may be used as an indicator of hematopoietic recovery after autologous stem cell transplantation, suggesting that there are cell type-specific G protein-mediated signal transduction pathways of early hematopoiesis.

Molecular diversity and double regulatory mechanism of activation of phospholipase C in rat brain

Whereas evidence for a G protein-dependent stimulation of phospholipase C (PLC) is abundant, reports on the inhibition of PLC through a G protein-mediated pathway have only recently begun to appear. In the present study, cerebral cortex membranes were chosen since they have a readily measurable Gpp[NH]p and Ca 2+-stimulated PLC activity. Nanomolar concentrations of Gpp[NH]p, a hydrolysis-resistant GTP analogue, inhibited basal inositol 1,4,5-trisphosphate (IP 3) production, with a maximum inhibition of 25% at 10nM. Increasing the concentrations of Gpp[NH]p to over 10nM resulted in a reversal of the inhibitory effect and onset of stimulation of IP 3 production. GDPβS as a G protein inhibitor and U-73122 as a putative PLC-β inhibitor had little effect on basal IP 3 production at 100μM and 1μM, respectively. However, GDPβS and U-73122 completely antagonized both the inhibition and the stimulation of IP 3 production produced by lower and higher concentrations, respectively, of Gpp[NH]p. Rat cortical membranes expressed a greater amount of PLC- β 1. These data suggest that PLC- β 1 isozymes may be regulated by both inhibitory and stimulatory G protein-mediated mechanisms.

Cl- currents activated via purinergic receptors in Xenopus follicles.

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course (F(in)). In Ringer solution, reversal potential (Erev) of F(in) was -22 mV, which did not change with external substitutions of Na- or K+, whereas solutions containing 50 or 5% of normal Cl- concentration shifted Erev to about +4 and +60 mV, respectively, and decreased F(in) amplitude, indicating that F(in) was carried by Cl-.F(in) had an onset delay of approximately 400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 microns). F(in) was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. F(in) was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl- currents and displayed cross-inhibition on the others. F(in) activation by acetylcholine also cross-inhibited F(in)-ATP responses, suggesting that all act on a common channel-activation pathway.

Role of Gqα in insulin-stimulated glucose uptake by C6 glioma cells

To investigate the possibility that insulin-stimulated glucose uptake in C6 cells is due to transactivation of a G protein-mediated pathway, the role of Gq alpha in insulin signaling was studied. Insulin stimulation of [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was time- and concentration-dependent: at a concentration of 1 microM, insulin stimulated 2DG uptake by C6 cells by about 30% (p < 0.05). Pertussis toxin treatment of C6 cells did not alter the ability of insulin (1 microM) to promote 2DG uptake, ruling out the involvement of Gion in insulin-stimulated hexose uptake. Next, C6 cells were transfected with Gq alpha cDNA for 48 h, challenged with 1 microM insulin, and 2DG uptake by the cells was determined. Insulin-stimulated 2DG uptake was 1.14 +/- 0.03 and 1.75 +/- 0.19 nmol/min/mg protein in mock- and Gq alpha-transfected cells, respectively (p < 0.05); insulin stimulated 2DG uptake in Gq alpha-transfected cells by 54%. These results suggest an involvement of Gq alpha in the transactivation of the G protein signal transduction pathway by insulin.

Activation of porcine oocytes via an exogenously introduced rat muscarinic M1 receptor.

Thirty hours after the beginning of in vitro maturation, porcine oocytes were microinjected with mRNA coding for the rat muscarinic M1 receptor. They were then incubated for 15 h to allow sufficient time for completing maturation, translation of the mRNA, and insertion of the receptor into the plasma membrane. They were then treated with acetylcholine, the receptor's agonist, and its effect on inducing various activation-related changes was examined. Acetylcholine treatment triggered the release of Ca2+ from internal stores that could be blocked by atropine, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertussis toxin (PT) had no effect on the Ca2+ transients induced by acetylcholine, suggesting that the signal transduction pathway involved a PT-insensitive G protein. Electron microscopy revealed that in the injected oocytes, acetylcholine induced cortical granule exocytosis. The oocytes were released from meiotic arrest as evidenced by the decrease in H1 kinase activity measured in the oocytes during the histone H1 kinase assay. After resuming meiosis they entered interphase: 58.8% of the injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholine incubation without prior injection with the receptor mRNA, did not cause these changes. The results provide further evidence that the components of a G protein-mediated signal transduction pathway exist in porcine oocytes and that the activation of this pathway via an exogenously supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activating signal at sperm-induced oocyte activation requires further examination.