©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The G Protein Subunit Transduces the Muscarinic Receptor Signal for Ca Release in Xenopus Oocytes (*)

(Received for publication, June 22, 1995; and in revised form, September 19, 1995)

Lisa Stehno-Bittel (§) Grigory Krapivinsky Lyubov Krapivinsky Carmen Perez-Terzic David E. Clapham (¶)

From the Department of Pharmacology, Mayo Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

At least 30 G protein-linked receptors stimulate phosphatidylinositol 4,5-bisphosphate phosphodiesterase (phospholipase Cbeta, PLCbeta) through G protein subunits to release intracellular calcium from the endoplasmic reticulum (Clapham, D. E.(1995) Cell 80, 259-268). Although both G [Medline] alpha and Gbeta G protein subunits have been shown to activate purified PLCbeta in vitro, Galphaq has been presumed to mediate the pertussis toxin-insensitive response in vivo. In this study, we show that Gbeta plays a dominant role in muscarinic-mediated activation of PLCbeta by employing the Xenopus oocyte expression system. Antisense nucleotides and antibodies to Galphaq/11 blocked the m3-mediated signal transduction by inhibiting interaction of the muscarinic receptor with the G protein. Agents that specifically bound free Gbeta subunits (Galpha-GDP and a beta-adrenergic receptor kinase fragment) inhibited acetylcholine-induced signal transduction to PLCbeta, and injection of Gbeta subunits into oocytes directly induced release of intracellular Ca. We conclude that receptor coupling specificity of the Galphaq/Gbeta heterotrimer is determined by Galphaq; Gbeta is the predominant signaling molecule activating oocyte PLCbeta.


INTRODUCTION

Muscarinic acetylcholine (ACh) (^1)receptors are heptahelical G protein-linked receptors widely dispersed in a variety of tissues including neurons of the central and peripheral nervous system, heart, smooth muscle, and exocrine glands(1, 2) . The five muscarinic receptor subtypes (referred to as m1-m5) can be grouped into two broad categories of signal transduction. Stimulation of m2 and m4 subtypes inhibits adenylyl cyclase activity (3) and only weakly activates phosphoinositide turnover(4) ; activation of m1, m3, and m5 receptors strongly induces phosphoinositide hydrolysis through a pertussis toxin (PTX)-insensitive G protein(4, 5) .

Convincing biochemical and functional evidence established that members of the Galphaq/11 family of G proteins activate PLCbeta(6, 7, 8, 9, 10, 11) , but not PLC or PLC isoforms(7) . Equally strong evidence demonstrated that PLCbeta enzymes are activated by free G protein beta complexes (12, 13, 14, 15, 16) . Thus muscarinic receptor activation releases Galpha-GTP and Gbeta, both of which can stimulate PLCbeta(17) .

The goal of this project was to identify which G protein subunits activate PLCbeta following stimulation of the m3 receptor in Xenopus laevis oocytes. The Xenopus oocyte PLCbeta has been cloned and is unique, containing 33-64% amino acid identity to mammalian PLCbeta isoforms(18) . Experiments with antisense oligonucleotides designed to block synthesis of members of the Galphaq-11 family of G proteins in Xenopus oocytes decreased the peak m3 receptor-mediated calcium (Ca) release as measured by the Ca-sensitive chloride current (I). Specific Galphaq function-blocking antibodies also abrogated the m3 receptor-mediated response. Direct injection of Gbeta into oocytes increased intracellular Ca [Ca] and injection of specific Gbeta-binding agents, Galpha-GDP, and a beta-adrenergic receptor kinase (betaARK) fragment (19) attenuated the muscarinic receptor-mediated response in a dose-dependent manner. We conclude that the m3 muscarinic signal requires Galphaq for specificity, but the majority of the signal is transduced by the Gbeta dimer.


EXPERIMENTAL PROCEDURES

Oocyte Preparation

Cell preparation has been described in detail previously(20) . Briefly, stage V and VI oocytes were removed from X. laevis frogs (Nasco, Fort Atkinson, WI) and manually defolliculated. Stage III-IV oocytes used for imaging were obtained following enzymatic dispersion(21) . Oocytes were stored in L-15 supplemented medium (Life Technologies, Inc.) containing 5% horse serum at 19 °C. Medium was replaced daily, and 24 h prior to voltage-clamp cells were placed in medium without horse serum(20) . Injection electrodes were pulled from capillary tubes and broken to a tip diameter of approximately 15 µm and baked at 300 °C. Cells were placed in a Ca-free solution (10 mM EGTA) during injections using a Drummond microinjector. Pertussis toxin (PTX, stock 100 µg/ml) was activated by incubating in 100 mM dithiothreitol (2:1 volume PTX to dithiothreitol) at 37 °C for 30 min. Cells injected with PTX (30 µg/ml) were also stored in PTX-containing medium (2 µg/ml) for 18-20 h prior to voltage clamp.

Antisense Design

Oligonucleotides were manufactured by the Mayo Molecular Biology Core Facility and purified in one step by high performance liquid chromatography on a reverse phase column. Oligonucleotide concentrations were determined by measuring the optical density at 260 nm. Antisense oligonucleotides specific to each Galpha subunit were designed by choosing regions with the least homology between the oocyte Galpha genes (see Table 1for oligonucleotide sequences). The specific Galpha subtype oligonucleotides contained phosphorothioate modifications that increased nuclease resistance of the oligonucleotide, but still allowed RNase H to degrade the target mRNA(22) .



For experiments determining the time course of the antisense oligonucleotide effects, all oocytes were injected with antisense common1 or sense oligonucleotides (0.8 mg/ml) on day 0 and groups of cells assayed for the m3-mediated response during the subsequent 7 days. In order to rule out an effect of varying levels of m3 receptor expression, the m3 mRNA transcript was always injected 2 days prior to voltage clamp. Constituitively active Galphaq (GalphaqQ209L DNA) was subcloned into Bluescript SK+ (Promega) and translated into capped cRNA using the Megacript kit (Ambion). Thus, for cells measured on day 0, mRNA for the muscarinic receptor had been injected 2 days prior to day 0 and cells were voltage clamped the same day as antisense or sense oligonucleotides were injected. In contrast, oocytes measured on day 7 were injected with oligonucleotides on day 0 and mRNA for m3 receptors on day 5.

We found no appearance of nonspecific effects of oligonucleotide injection on oocytes at concentrations below 1.0 mg/ml (see Fig. 1C). However, at antisense and sense oligonucleotide concentrations greater than 1.5 mg/ml, oocytes showed visible signs of deterioration (loss of pigmentation of animal pole, loss of intracellular contents, and deterioration of the resting membrane potential, n = 26), illustrating that, with higher oligonucleotide concentrations, global effects occurred that were not specific to G protein function.


Figure 1: Galpha antisense oligonucleotides decrease the m3-mediated response. A, oocytes were injected with 40 ng/oocyte of common1 Galpha oligonucleotides 4 days prior to voltage clamp (holding potential, -70 mV) and 5 µM ACh added to the bath (arrow). The endogenous (no m3 expression) ACh-induced response was less than 0.3 µA. The m3-expressing, sense-injected oocyte responded to ACh with a 5.2-µA increase in current (exogenous, sense). The m3-expressing, antisense nucleotides (common1)-injected ACh-induced current increased to 0.6 µA (exogenous, antisense). The delay in the response in the antisense nucleotide-injected oocyte was likely due to diffusion of ACh in the bath. B, oocytes injected with antisense nucleotides common to the Galphaq family (q/11-com) displayed 70% smaller ACh-stimulated I than sense controls (2.7 ± 0.3 and 0.8 ± 0.2 µA in Galphaq sense- and antisense-injected cells, respectively). In Galpha11 antisense nucleotide-injected oocytes, peak ACh-induced I decreased 76% compared to sense-injected cells (0.7 ± 0.2 and 2.9 ± 0.5 µA, respectively). I in oocytes injected with antisense specific to Galphaq alone were reduced by 81% from sense controls (2.6 ± 0.5 and 0.5 ± 0.2 µA in sense- and antisense-injected cells, respectively). The ACh-induced responses in oocytes injected with sense and antisense nucleotides to other G protein subunits were not statistically different then water-injected oocytes (HO). Average n = 33 oocytes/group. C, the optimal dose for inhibition of the ACh-induced current with antisense common1 was 0.8 mg/ml (pipette concentration). The antisense nucleotides targeted to a common region of Galphaq and Galpha11 maximally inhibited the ACh current at 0.6 mg/ml. Sense oligonucleotides (0.6 mg/ml) to the same region did not significantly alter the m3-mediated response. Average n = 15 oocytes/group.



Electrophysiology

A two-electrode voltage clamp (Turbo TEC 01, npi Instruments, Germany) was used to measure ACh-induced currents. Electrodes were pulled using a horizontal puller (Sutter Instruments) to resistances of 1 and 4-6 megaohm for current and potential recording electrodes, respectively. Both electrodes were filled with 2 M KCl. The bath contained Barth's solution (in mM): 88 NaCl, 1 KCl, 2.4 NaHCO(3), 0.8 MgSO(4), 10 HEPES, 0.3 Ca(NO(3))(2), 0.4 CaCl(2), plus 1% of each of the following antibiotics (vol/vol): Pen/Strep (Sigma), Fungizone (Life Technologies, Inc.), and gentamycin (Life Technologies, Inc.). The membrane potential was held at -70 mV for all experiments; near the estimated reversal potential for K ensuring that the measured whole-cell current was mainly carried by I(20) . All currents were filtered at 1 kHz, and data were stored and analyzed using Axobasic software (Axon Instrs., Inc.). Statistical differences were defined at the level p leq 0.05 using hierarchal analysis of variance(23) . ACh was added directly to the bath for a final concentration of 5 µM, except for dose-response experiments. All experiments were conducted at room temperature (22 ± 2 °C).

Immunoblots

Membrane proteins for immunoblotting were obtained using the following procedure. Ten oocytes were homogenized in 1 ml of buffer A (in mM): 10 HEPES, 20 KCl, 2.5 Mg(2)Cl, 1 EGTA, 1 dithiotreitol (pH 7.5) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride and 2 µg/ml of each leupeptin, aprotinin, and pepstatin), and the samples were centrifuged for 10 min at 10,000 times g. Membrane proteins were extracted from the pellet with 0.3 ml of buffer A supplemented with 0.5% Lubrol-PX for 30 min on ice. Insoluble material was precipitated using the same conditions described above. Solubilized proteins were precipitated by trichloroacetic acid in the presence of 30% EtOH and solubilized in SDS sample buffer (3 µl/oocyte). This method resulted in 80-90% extraction of the total p35 as measured by comparison with direct SDS solubilization of total oocyte proteins.

After SDS electrophoresis (24) and transfer onto a poly(vinylidene fluoride) membrane, proteins were immunoblotted according to published procedures(25) . Visualization was accomplished with I-labeled goat anti-rabbit IgG-F(ab`)(2) fragments and autoradiography. For Galphaq staining, three different antibodies to Galphaq were used. Two were raised to the C-terminal amino acids (CILQLNLKEYNLV) of Galphaq from two different sources, Z811 (26) and CQ2(27) , and the third antibody was raised to the internal portion of Galphaq, W082 (EVDVEKVSAFENPYVDAIK) (28) . Block of Z811 antibody by the epitope peptide was performed by preincubation of antibodies with 50 µM C-terminal peptide of Galphaq followed by 10-fold dilution for immunoblotting. Bovine brain G protein subunits were isolated as described previously(29) . They were shown to be active by their ability to bind GTPS(29) , and Galpha subunits were able to bind immobilized Gbeta and vice versa(30) .

Confocal Microscopy

Images were taken on a Bio-Rad MRC 600 confocal microscope adapted as described previously(31) . Oocytes were injected with 1.2 mM (pipette concentration) Indo-1 1 h prior to experiments. Light sources were argon lasers tuned to either 488 (green) or 350 nm (ultraviolet). Simultaneous images of G protein subunit diffusion and intracellular Ca distribution were obtained by switching the filter settings in the Bio-Rad scanning box from 520 nm for fluorescein to 485 and 405 nm for Indo-1 emission during the experiments (approximately 5 s between images)(32) . Images were processed on a Silicon Graphics Personal IRIS system using ANALYZE (Mayo Foundation), and displayed using Adobe Photoshop software on a MacIntosh Quadra 700 computer. Purified G protein subunits were labeled with fluorescein using methods described previously(33) .


RESULTS

m3 Muscarinic-dependent Ca^2 Release in Xenopus Oocytes

Two days following injection of mRNA encoding m3 muscarinic receptors, Xenopus oocytes responded to ACh application with rapid and dramatic increases in [Ca](i). The [Ca](i) transient was monitored by recording changes in endogenous I, a current that has been closely correlated to submembranous increases in [Ca](i)(4, 20) . The mean peak I in response to 5 µM ACh in cells expressing m3 receptors was 3.6 ± 0.7 µA (n = 120), in contrast to a peak of 0.1 µA in control water-injected oocytes (Fig. 1A, n = 16). Injection of the Ca chelator, EGTA (100 µM) prior to voltage clamp, abolished the ACh-induced response (mean peak = 0.3 ± 0.1 µA), indicating the dependence of I on [Ca](i) (n = 19; data not shown).

Galphaq Antisense Nucleotides Blocks the m3 Response

Xenopus oocyte G protein alpha subunits have a region of high sequence identity corresponding to nucleotides 228-260 of Galphas(34, 35, 36) . Antisense oligonucleotides targeted to this region (named common1 and common2), were reported previously to block synthesis of all G protein alpha subunits(37) . Control oocytes injected with nonsense or sense oligonucleotides to this region and expressing the m3 receptor, responded to 5 µM ACh with an average inward I measuring 3.2 µA (Fig. 1A). Injection of common1 antisense nucleotides (40 ng/oocyte) decreased the m3-mediated response to 19% of control by the 4th day after injection (mean peak I, 0.6 µA, Fig. 1A). Normal m3-mediated responses returned by day 7 in antisense nucleotide-injected oocytes, indicating degeneration of the oligonucleotides. Both common1 and common2 antisense oligonucleotides decreased the ACh-induced current by comparable amounts (average of 78 and 83% inhibition, respectively). Exposure to ACh failed to elicit a significant response in cells devoid of muscarinic receptors either in the presence or absence of antisense oligonucleotides (Fig. 1A, n = 8) ruling out any direct effect of the antisense or sense oligonucleotides on the native whole-cell current. During the 7 days of testing, the mean amplitude of the ACh response in sense oligonucleotide-injected oocytes did not change significantly, consistent with our previous work(20) . Furthermore, injection of antisense or sense oligonucleotides did not alter the resting membrane potential of the cells (-34 ± 4, -37 ± 5, and -38 ± 4 mV in water-, sense-, and antisense-injected cells, respectively; n = 349), demonstrating that antisense nucleotides had no deleterious, nonspecific effects on the oocytes. The time course of the antisense inhibition is consistent with previously measured slow turnover rates for Galpha subunits in mammalian cells of 21 (38) to 55 h (39) . In summary, antisense block of the m3 response was specific and greater than 80% 4 days following injection of antisense to Galpha common regions.

Specific antisense oligonucleotides for the individual Galpha subtypes were injected into oocytes using the protocol described above. Only those cells receiving antisense nucleotides to the Galphaq family (Galphaq/11-com) or to its specific members (Galphaq or Galpha11) exhibited a decrease in the ACh-induced response when compared to water- and sense-injected cells (Fig. 1B). A 70-84% decline in the ACh-induced current was measured in oocytes injected with antisense oligonucleotides designed to Galpha11 and Galphaq, respectively. We could not distinguish between Galphaq and Galpha11 subunits in the ACh response, since injection of antisense oligonucleotides to either Galphaq or Galpha11 resulted in statistically indistinguishable suppression of the m3 response. The antisense nucleotides may have interacted with both Galpha11 and Galphaq mRNAs, since they have a high identity, or both Galphaq and Galpha11 may participate in the m3 response. Such an interaction has been described for the thyrotropin-releasing hormone coupling to G proteins(40) . The dose-response relation illustrates similar efficacies of common1 antisense and Galphaq/11 common antisense (Fig. 1C). The same concentration of sense oligonucleotides did not alter peak I when compared to water-injected cells (n = 65). Thus, antisense oligonucleotides directed to members of the Galphaq family, specifically Galphaq and Galpha11, blocked the m3 muscarinic receptor mediated response.

Antisense Nucleotides Decreased the Amount of Protein Recognized by Anti-Galphaq Antibodies

Immunoblots of oocyte membranes using antibodies specific to Galphaq demonstrated that antisense treatment decreased endogenous oocyte Galphaq protein levels by roughly 40% compared to controls (n = 18). Two antibodies, each specific to the C terminus of mammalian Galphaq/11 (26, 27) and one antibody raised to the internal portion of Galphaq (28) recognized a single 35-kDa protein from Western blots of Xenopus oocyte membranes. The same antibody recognized a 42-kDa protein in human embryonic kidney cells (HEK A293; Fig. 2A) and Xenopus brain and liver (data not shown). Recognition of p35 was specific since it was blocked by antigenic peptides. Overexpression of the Xenopus oocyte Galphaq clone (provided by Dr. K. Guttridge) in oocytes resulted in a 3-fold increase in p35 levels with no detectable 42-kDa immunostaining (Fig. 2B, n = 3), although the nucleotide sequence of the clone predicted a 42-kDa protein. We conclude that p35 is the oocyte homologue of the mammalian 42-kDa Galphaq and that the difference from the predicted molecular mass may be a result of post-translational modifications (as is the case for the cGMP-gated cation channel(41) .


Figure 2: Immunoblots of Galphaq family proteins in oocyte membranes. A, human embryonic kidney (A293 or HEK) and oocyte membranes were immunoblotted with antibodies specific to members of the Galphaq family of proteins. The first two lanes show immunostaining with antibody Z811 in HEK (H) and oocytes (O) membranes. This recognition was inhibited by the C-terminal peptide (lane 3). The remaining lanes illustrate immunostaining of both HEK and oocyte membranes with the C-terminal Galphaq family antibody, CQ2 and the Galphaq-specific antibody, W082 (designed to an internal portion of Galphaq). B, the level of protein recognized by Galphaq antibodies increased 3-fold in oocytes expressing the exogenous Xenopus oocyte Galphaq clone. Protein levels were quantified by densitometry and normalized to water-injected oocytes.



Anti-Galphaq Antibody Blocked the m3 Response

Injection of the C-terminal Galphaq family antibodies (Z811 and CQ2) prior to voltage clamp diminished the peak m3-mediated response to ACh. Maximal inhibition (90%) of the ACh-induced I current was obtained with injection of 3.5 ng/oocyte of Z811 antibody (Fig. 3A). The same concentration of pre-immunoglobulins did not significantly inhibit the ACh response. Seventy seven percent inhibition was obtained using the second C-terminal Galphaq antibody, CQ2 (Fig. 3B). Neither the m3 response, nor its inhibition by Galphaq antibodies, was affected by PTX pretreatment (Fig. 3B). Concurrent experiments revealed no PTX-mediated ADP-ribosylation of p35 despite the presence of PTX-induced ribosylation at 40 kDa, presumably of Galphai/Galphao proteins (data not shown). In conclusion, experiments using both Galphaq/11-specific antisense oligonucleotides and antibodies attest to the requirement for Galphaq in the m3 response. However, anti-Galphaq antibody or antisense nucleotide block of the response alone cannot distinguish which G protein heterotrimer subunit (Galpha or Gbeta) transduces the signal to PLCbeta.


Figure 3: Galphaq-family antibodies blocked the m3-mediated response. A, the m3-mediated I response was inhibited 83% by Z811 antibody (mean peak I = 0.2 ± 0.1 µA) at a dose of 70 µg/ml. Preimmune control sera at the same concentration did not significantly decrease the ACh-induced response. Average n = 36 oocytes/group. B, the mean peak I was 3.1 ± 0.3 and 3.6 ± 0.6 µA for preimmune- and vehicle-injected oocytes, respectively. Antibody CQ2 blocked the ACh response by 77% (mean peak I = 0.7 ± 0.3 µA). Injection of activated PTX followed by incubation in PTX-containing medium did not alter the response in control or antibody-injected cells (mean peak I = 3.1 ± 0.7 and 0.3 ± 0.1 µA for PTX-treated cells injected with KCl or Z811, respectively). Average n = 34 oocytes/group.



Injection of Galpha-GDP Attenuated the m3 Response

To determine which G protein subunit transduces the m3 signal to PLCbeta, we injected proteins into oocytes that specifically bound and, presumably inactivated, free Gbeta(42, 43, 44) . Purified bovine brain Galpha-GDP subunits were injected into m3-expressing oocytes 5 min prior to voltage clamp. Galpha-GDP (15 nM) significantly attenuated the ACh-induced response in a dose-dependent manner with complete inhibition of the response in the presence of 1.75 µM Galpha-GDP (Fig. 4A). To determine whether specific Galpha subunits were more effective in blocking the muscarinic response, we injected individual Galpha subunits obtained either from recombinant DNA (Galphai-1, Galphai-2, and Galphai-3 (45) and Galphaq(46) ) or a mixture of Galphao/i (consisting primarily of Galphao) purified from bovine brain. All of the Galpha-GDP subunits tested (10-25 nM) decreased the ACh-induced activation of I to comparable levels (Fig. 4B). Injection of the vehicle alone, or use of boiled Galphao/i-GDP, had no effect on the muscarinic response. As a control, injection of purified brain Galpha-GDP (15 nM) did not change oocyte ionic currents in the absence of ACh. These results suggest that the m3 receptor transduces its signal to PLCbeta through the endogenous Gbeta subunits and that injection of Galpha-GDP inhibited the response by binding free Gbeta.


Figure 4: Galpha-GDP blocked the m3-mediated response. A, injection of purified Galpha-GDP (14 nM calculated intracellular concentration) 5 min prior to voltage-clamp attenuated the m3-mediated response at all ACh concentrations. Higher doses of Galpha-GDP (1.75 µM intracellular concentration) completely blocked the ACh-induced response. Injection of a fragment of the Gbeta-binding protein betaARK (200 µM) greatly attenuated the ACh-induced response. The amplitude of the peak current (vehicle) did not increase with higher doses of ACh (100 µM). Average n = 5 oocytes/group. B, Galpha subtypes bound to GDP equally inhibited the I response. Galphai-1 (17 nM), Galphai-2 (10 nM), Galphai-3 (25 nM) (Iniguez-Lluhi et al., 1992), and Galphaq (10 nM) (Hepler et al., 1993) were recombinant proteins. Galphao/i (10 nM) was purified from bovine brain and consisted predominantly of Galphao. Average n = 18 ooyctes/group.



To rule out the possibility that Galpha-GDP interacted directly with oocyte PLCbeta, thereby blocking access of Galpha-GTP to the PLCbeta molecule, we injected oocytes with the Gbeta-binding region of betaARK. The betaARK fragment has been used to bind Gbeta and block activation of the muscarinic-gated K channel(19) . Injection of this betaARK fragment (200 µM) abolished the 1 µM ACh-induced response (Fig. 4A, n = 5). With higher doses of ACh (10 µM, n = 7), more than 75% of the response was blocked compared to buffer-injected cells (mean peak I = 0.7 µA, n = 10). Thus, agents known to bind free Gbeta (Galpha-GDP and betaARK fragment) blocked the ACh-induced current in m3-expressing oocytes.

Gbeta Injection Released Intracellular Ca^2

If Gbeta transduces the m3 signal to PLCbeta, it should activate the I response in the absence of receptor stimulation. Injection of bovine brain Gbeta (60 nM) increased [Ca](i) and activated I (peak 2.3 µA; Fig. 5A), while injection of the vehicle alone had no effect. Preincubation of Gbeta with Galpha-GDP decreased the response by an average of 80% (Fig. 5B). Boiled Gbeta (700 nM) failed to elicit an increase in I. The Gbeta-induced current required [Ca](i) since inclusion of 10 mM EGTA in the injection buffer inhibited any Gbeta-induced increase in I. To determine whether injection of Gbeta protein increased I through the same pathway as the m3 receptor, namely release of Ca via the inositol trisphosphate (InsP(3)) receptor, Gbeta was coinjected with heparin at a concentration known to block oocyte InsP(3) receptors(46) . Heparin blocked the Gbeta-induced response, indicating that InsP(3) was the second messenger of Gbeta-mediated Ca release. To rule out a direct effect of Gbeta on the InsP(3) receptor, we applied Gbeta directly to isolated nuclei. We have shown previously that the outer nuclear membrane of isolated oocyte nuclei contains abundant InsP(3) receptors and that these receptors can be accessed readily by isolating intact nuclei and patch clamping the outer nuclear membrane or measuring InsP(3)-induced Ca release by confocal microscopy(46) . Gbeta (1 µM) did not release Ca via the oocyte nuclear InsP(3) receptors when applied to isolated nuclei (n = 4), indicating the Gbeta effect required an intermediate, presumably PLCbeta. In m3-expressing cells, application of ACh during the elevated plateau of the Gbeta response elicited a small (<0.4 µA) increase in I (n = 5), suggesting that the ACh-sensitive Ca store was already released or had desensitized following Gbeta injection. The results demonstrate that injection of Gbeta released Ca in a heparin-sensitive manner and that subsequent stimulation by ACh caused a minimal increase in I. Thus m3 receptor stimulation and Gbeta injection activates the same final pathway, the release of Ca through the InsP(3)-sensitive receptor.


Figure 5: Gbeta alone released [Ca]. A, an increase in I was elicited following injection (arrow) of 60 nM (estimated final concentration) purified Gbeta into a single oocyte. B, the mean amplitude of the Gbeta (700 nM estimated final concentration) response was 2.2 ± 0.3 µA. Injection of the holomultimeric G protein complex (700 nM estimated final concentration) did not cause a significant increase in I (0.4 ± 0.1 µA). Boiled Gbeta failed to induce a response (0.2 ± 0.1 µA) as did Gbeta co-injected with heparin (10 µg/ml, 0.3 ± 0.2 µA) or EGTA (10 mM, 0.1 ± 0.0 µA). Average n = 14 oocytes/group



Activated brain Galpha-GTPS (85 nM) resulted in a small increase in I when injected into oocytes (average peak = 0.5 µA, n = 18), less than one fourth the amplitude of the Gbeta response. Galpha and especially Galphaq subunits bind poorly to GTPS in vitro(8) , therefore excess GTPS was included in the solution (10 times > GTPS versus Galpha). Injection of equal concentrations of GTPS alone increased I to the same extent as Galpha-GTPS (n = 17). Confocal imaging of oocytes following injection of 0.5 µM GTPS revealed Ca waves near the injection site (data not shown, n = 6; see also (4) ). The Ca release by GTPS and subsequent activation of I was likely due to activation of the endogenous oocyte G protein subunits. These results indicate that the small increase in I with Galpha-GTPS may be due to the presence of free GTPS, rather than any direct activation by the exogenous Galpha protein subunit.

To further examine the possible role of Galphaq on intracellular Ca release, we overexpressed the constitutively active Galphaq subunit in oocytes(63) . ACh was applied to oocytes 3 days after coinjection of constitutively active Galphaq mRNA and the m3 receptor mRNA (n = 30) or the m3 receptor mRNA alone (n = 20). The ACh-induced change in I averaged 6-fold larger in oocytes expressing the m3 receptor alone compared to oocytes coexpressing m3 and constitutively active Galphaq. This effect was similar to the effect of Galpha-GDP injection (Fig. 4B). Thus, contrary to what might be expected if Galphaq were the direct activator of phospholipase Cbeta, I responses were inhibited by the expressed Galphaq. Although several interpretations are possible, such as the induction of crosstalk, this result can be explained by trapping of free Gbeta by overexpressed Galphaq. This is consistent with our observations that active Galphaq-GTPS can bind Gbeta, albeit with lower affinity. (^2)If constitutively active Galphaq activated phospholipase Cbeta, we would also expect a reproducible shift in membrane potential as intracellular calcium levels activate I. Coexpression of Galphaq and the m3 receptor changed the membrane potentials of the oocytes only slightly (average of -36 ± 3 mV and -45 ± 3 mV, for m3 and m3 + Galphaq, respectively; these values were not significantly different in a Student's paired t test). These results do not support the hypothesis that constitutively active Galphaq is the major direct activator of oocyte phospholipase Cbeta.

Diffusion of Gbeta and Ca^2 Waves within Oocytes

The high concentration of Gbeta used to activate I in Fig. 5B suggests that the majority of the injected Gbeta may not have had access to the plasma membrane-bound oocyte PLCbeta. To test this hypothesis, we fluorescently labeled Gbeta and monitored its diffusion through oocytes using confocal microscopy. Previously, G protein subunits fluorescently labeled in this manner retained their activity(33) . Fluorescence microscopy measurements indicated that Gbeta (60 nM estimate based on whole oocyte volume) did not diffuse freely within cells, and remained localized to the space surrounding the site of injection (Fig. 6A, n = 12). This pattern of static Gbeta localization was maintained for at least 30 min. In contrast, fluorescently-tagged bovine brain Galpha (60 nM) diffused evenly throughout the oocyte within 15 s after injection (Fig. 6A) and its distribution was unchanged after 20 min. Oocytes loaded with the Ca-sensing ultraviolet dye Indo-1 were injected with fluorescent G protein subunits, and localization of Gbeta and intracellular Ca concentrations monitored simultaneously (n = 8). In all cases there was a direct correlation between the spatial distribution of Gbeta and local increases in [Ca](i) that was not dependent on extracellular Ca (Fig. 6B). In addition to Gbeta-evoked local increases in [Ca](i), oocytes frequently displayed regenerative Ca waves that propagated from the region of tagged Gbeta fluorescence (Fig. 6B). In control cells, there were no measurable increases in [Ca](i) following injection of inactive Galpha (Galpha-GDP; n = 5).


Figure 6: Galpha subunits diffused freely in the cytoplasm, but Gbeta subunits did not. A, fluorescently labeled Gbeta recorded at 15 s and 5 min after injection demonstrates that the hydrophobic Gbeta subunit does not readily diffuse in oocyte cytoplasm. In contrast, Galpha fluoresced throughout the oocyte only 15 s after injection. B, the site of high Gbeta concentration coincided with the region of intracellular Ca release.




DISCUSSION

Our results indicate that the G protein subunit Gbeta alone may activate PLCbeta to initiate the cascade for intracellular Ca release; the Galphaq subunit couples to the m3 muscarinic receptor providing specificity for the activated pathway. This conclusion is based on experiments that isolated the function of endogenous oocyte Galphaq and Gbeta subunits using: 1) antisense nucleotides to block protein production, 2) antibodies to block protein interactions, 3) direct injection of activated G protein subunits, and 4) specific Gbeta binding compounds (Galpha-GDP and betaARK fragment) that compete with Gbeta's ability to interact with other molecules.

Antisense oligonucleotides have been used previously to identify the involvement of both Galpha and Gbeta subunits in inhibition of Ca voltage-dependent channels(37, 47, 48) . However, antisense oligonucleotide block alone cannot distinguish which G protein subunit was required for interaction with the muscarinic receptor or with the effector, PLCbeta. Antisense oligonucleotide (Galphaq) treatment of the oocytes effectively suppressed the m3 muscarinic signal by 80%, while reducing the protein levels immunostained by Galphaq/11 antibodies by only 40%. These results may be explained by a population of stained, but inactive, Galphaq present in oocytes, or by the known nonlinearity of signal transduction. Signal transduction steps between the muscarinic receptor and the measured I include the G protein, PLCbeta enzyme, InsP(3) generation, release of Ca, and Ca-dependent activation of I. The requirement of Galphaq/11 for the signal appears to lie in its ability to couple to the muscarinic receptor. In this model the specificity for the pathway lies in that Galpha/receptor interaction. This suggestion is supported by the fact that antibodies that inhibit binding of Galphaq to the receptor, but do not interfere with the interaction with PLCbeta(27) , blocked the m3 signal. The addition of exogenous Gbeta-binding proteins has been used previously to block the function of the Gbeta(42, 43, 44, 49) . Complete block of the m3 response by factors that bind free Gbeta (Galpha-GDP and betaARK fragment) suggests that most, if not all, of the m3 muscarinic signal is transduced via Gbeta.

The results are in contrast with the common perception, based on reconstitution of PLCbeta subtypes with purified Galpha or Gbeta subunits, that both may transduce the signal(17, 50) , but Galphaq/11 is responsible in PTX-insensitive pathways(10, 11) . We find that Gbeta transduces the signal even when coupled to the PTX-insensitive Galphaq subunit. Depending on the cell type and assays used, laboratories have concluded that several different Galpha activate PLCbeta including Galphao (51, 52) , Galphai-1(53) , Galphai-2 and Galphai-3(54, 55) , Galphaq(36, 40, 56, 57, 58) , Galpha11(40, 59) , and Galphas(60) . Our finding that the endogenous activator of oocyte PLCbeta is Gbeta may explain these variations since there is apparently little specificity of Gbeta interactions with effector proteins(43, 61) . We cannot rule out the possibility that Gbeta interacts with oocytes PLCbeta in a unique manner, however the oocyte expression system has been used frequently to identify receptor/G protein specificity(51, 52, 53, 60) . The results of this investigation are a cautionary reminder that the interpretation of such experiments is not simple. Overexpression of Galpha subunits in mammalian cell lines is another common approach to identifying receptor/G protein specificity(9, 40) . However, overexpressed Galpha subunits may bind endogenous Gbeta, thereby increasing the available Gbeta that is activated following the appropriate receptor stimulation. Overexpression studies determine whether a G protein subunit is a component of the receptor-activated pathway, but they cannot identify which G protein subunit (Galpha or Gbeta) interacts with PLCbeta.

Other investigators have speculated that PTX-sensitive stimulation of PLCbeta is via Gbeta, because in vitro experiments show no direct effect of Galphai-1, Galphai-2, Galphai-3, or Galphao on PLCbeta(62) , while the PTX-insensitive stimulation of PLCbeta is through the Galphaq/11 family of Galpha subunits. In contrast to earlier reports showing large, transient increases in I with injection of purified Galpha-GTPS into oocytes(52) , we found no evidence for specific activation of the PLC pathway by injection of similar concentrations of Galpha-GTPS. We cannot completely exclude Galphaq/11 as carrying a portion of the m3 signal to PLCbeta, but all activation that we measured with Galpha-GTPS could be ascribed to the activity of free GTPS, which activates intrinsic G protein pathways. Our results demonstrate that even in the presence of activated Galphaq following stimulation of the m3 muscarinic receptor, inhibition of Gbeta blocked signal transduction to oocyte PLCbeta.

The simplest interpretation of the results is that both Galpha and Gbeta subunits are necessary for m3 muscarinic signal transduction; Galphaq/11 provides the specificity of the signal through its interaction with the receptor and Gbeta freed during activation, transduces the signal to the effector. These experiments do not exclude other interpretations. If Galpha or Gbeta subunits do not dissociate in the membrane following activation, but rather form a macromolecular complex with the receptor and effector enzyme, these experimental results may be explained by a constrained, activated heterotrimer with activator sites on Gbeta accessible to inhibitory proteins. In any case, signal transduction in intact membranes does not appear to behave solely as predicted from experiments with purified subunits in solution.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL41303 (to D. E. C.), HL07094, and HL08848 (to L. S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7601.

To whom correspondence should be addressed: Dept. of Pharmacology, Mayo School of Medicine, 711 Guggenheim, Rochester, MN 55905. Tel.: 507-284-5881; Fax: 507-284-9111.

(^1)
The abbreviations used are: Ach, acetylcholine; PTX, pertussis toxin; I, Ca-sensitive chloride current; betaARK, beta-adrenergic receptor kinase; GTPS, guanosine 5`-O-(thio)triphosphate; PLC, phospholipase C; HEK, human embryonic kidney; InsP(3), inositol trisphosphate.

(^2)
G. Krapivinsky, L. Krapivinsky, K. Wickman, and D. E. Clapham, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. P. Sternweis and G. Milligan for generously donating Galphaq antibodies, Dr. R. Lefkowitz for betaARK fragment, Dr. A. Gilman and J. Hepler for recombinant Galpha subunits, Dr. K. Guttridge for oocyte Galphaq cDNA clone, Drs. E. Peralta and T. Bonner for m3 muscarinic receptor cDNA clone, and Dr. Gary Johnson for GalphaqQ209L.


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