Galpha 14 and Galpha q Mediate the Response to Trypsin in Xenopus Oocytes*

Hagit ShapiraDagger , Ilan Amit, Merav Revach, Yoram Oron, and James F. Battey§

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel and § National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galpha q family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galpha q family, Xenopus Galpha 14, in addition to the previously cloned Xenopus Galpha q and Galpha 11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha 14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha 14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha 14) that, together with antiGalpha q and antiGalpha 11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalpha q or antiGalpha 14 reduced the response to 1 µg/ml trypsin by 70%, whereas antiGalpha 11 had no effect. A mixture of antiGalpha q and antiGalpha 14 virtually abolished the response. These data strongly suggest that Galpha q and Galpha 14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galpha q and Galpha 11, but not Galpha 14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galpha q family discriminate among different native receptors in vivo.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heterotrimeric GTP-binding proteins (G proteins) of the Galpha q family activate the phosphatidylinositol-bisphosphate-inositol-trisphosphate-calcium pathway in a pertussis toxin-insensitive manner. Among Galpha q family members, Galpha q and Galpha 11 share 88% homology and are expressed ubiquitously (2-4). Galpha 14 is 81% identical to Galpha q and is restricted in its tissue distribution mainly to spleen, lung, kidney, and testes (5). Galpha 15 and its human counterpart, Galpha 16, show only 58 and 57% amino acid identity, respectively, to mouse Galpha q and are restricted to a subset of hematopoietic cells (5, 6). Upon activation, all G proteins of the Galpha q family activate beta  isomers of phospholipase C.

The coexistence of closely related G proteins in the same cell suggests different functions or different interactions between individual G proteins and either receptors or phospholipases. However, in reconstitution systems and transfected cells, Galpha q and Galpha 11 have been shown to couple indiscriminately to a wide range of receptors, e.g. muscarinic m1 receptor (m1-R)1 (7, 8) and m3-R (9), thyrotropin-releasing hormone (10, 11), parathyroid hormone (12), gastrin-releasing peptide (GRP) and vasopressin (13), gonadotrophin-releasing hormone (14), angiotensin II and bradykinin (15), histamine (16), and alpha 1-adrenergic receptors (17). Galpha 15/16, despite restricted distribution, are capable of coupling many serpentine receptors tested, including those natively coupled to Galpha s and Galpha i (for review, see Ref. 18). Similarly, an attempt to assign different roles to Galpha q and Galpha 11 using agonist-induced down-regulation failed to distinguish between the two G proteins (19-21).

This lack of discrimination may reflect real interchangeability in vivo among the G proteins of the Galpha q family or merely be a result of the assays used. To address this question, we have used the Xenopus oocytes system to study receptor-G protein specificity in vivo. Coexpression of thyrotropin-releasing hormone receptors with either mouse Galpha q or Galpha 11 demonstrated different modulation of the response to thyrotropin-releasing hormone by each one of these G proteins (22). Applying the antisense approach, we demonstrated for the first time different coupling preferences of neuromedin B receptor for Galpha q and Galpha 11 (1).

Although some information is available for other Galpha q family members, little is known of Galpha 14-receptor coupling specificity. Wu et al. (17, 23) demonstrated in transfected COS-7 cells that Galpha 14 mediates responses evoked by the alpha 1-adrenergic and the interleukin-8 receptors. Kuhn et al. (16) showed that histamine receptors couple to all Galpha q family members.

In this study, we have used the Xenopus oocyte system to investigate the coupling of native or expressed receptors to Galpha 14. Xenopus oocytes express an endogenous protease receptor that is activated by trypsin (24). The response, typical for the activation of the phosphatidylinositol-specific phospholipase C signal transduction pathway, is manifested as Ca2+-dependent Cl- current. Trypsin, along with several other proteases, stimulates fertilization-like responses in starfish eggs (25). In Xenopus oocytes, there is an elevation of G proteins of the Galpha q family during maturation and early development (26). These data suggest that protease receptors coupling via G proteins of the Galpha q family may be of importance in fertilization and early development. Using the antisense approach, we show here that Galpha q and Galpha 14 are the main mediators of the response elicited by trypsin, in contrast to the response evoked by the activation of GRP-Rs, which utilize Galpha q and Galpha 11, and the response to activation of m1-Rs, which utilize Galpha q and Galpha o.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of Xenopus Galpha 14-- A cDNA library of Xenopus oocytes (constructed in lambda gt10) was screened at low stringency with a mouse Galpha q probe, as described previously (1). Positive plaques were isolated and sequenced with universal and gene-specific primers using fMOLTM sequencing kit (Promega), according to the manufacturer's instructions. A partial cDNA clone that displayed high homology to mouse Galpha 14 was used as a probe in a second round of screening at high stringency: overnight hybridization at 37 °C in NT hybridization buffer (27) followed by a 4 × 10-min wash at room temperature in 1 × SSC (0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS and 20 min wash at 42 °C in 0.1 × SSC, 0.1% SDS. Positive clones were isolated and either subcloned into the EcoRI site of pGEM-4 plasmid (Promega) or PCR-amplified with pfu DNA polymerase (Stratagene) and sequenced on both strands with universal and gene-specific primers, as described above.

Antisense Oligonucleotides Sequence-- antiGalpha q: 5'-ATTCTCAAAAGAGGCGACC-3'; antiGalpha 11: 5'-CTGTTCAAAGGTACATACT-3'; antiGalpha 14: 5'-GTTTCCTTTCAAGACTGGAT-3'; antiGalpha o: 5'-GCGCTCAGTCTGCAGCCCAT-3'.

Handling of Oocytes-- Oocytes were excised from Xenopus females (South African Xenopus Facility, South Africa), defolliculated with collagenase, and kept in NDE solution at 20 °C, essentially as described previously (28). Stage V oocytes were injected with the desired RNAs (0.5-2 ng/oocyte) and/or phosphorothioate DNA antisense oligonucleotides (S-oligos, 50 ng/oocyte). The injections resulted in deterioration of some oocytes, particularly those injected with antiGalpha 11. Viable oocytes were used in three assays: functional assay and Northern and Western analyses.

Functional Assay in Oocytes-- Measurements of electrophysiological responses to agonists were performed essentially as described previously (28). Briefly, individual oocytes were voltage-clamped at -70 mV. Agonists (trypsin, GRP, or acetylcholine (ACh)) were added directly to the bath, and membrane currents were continuously recorded. The mean amplitude of the control group in each experiment was set as 100%, and the effect of each treatment was calculated as the percent of that control response. N denotes the number of different donors, and n denotes the number of oocytes tested. Results are represented as means ±S.E. Paired Student's t test was used to estimate the statistical significance of the data.

Northern Analysis-- Groups of 80 oocytes were homogenized in 8 ml of cold guanidynium isothiocynate buffer, centrifuged for 5 min at 3000 × g, 4 °C, and the supernatant was loaded onto 4 ml of CsCl (5.7 M) cushion for standard total RNA isolation (27). The precipitated RNA was dissolved in 40 µl of H20. 6 µg of total RNA were loaded and resolved on RNA gel (agarose/formaldehyde) and blotted onto a nitrocellulose filter. Filters were air-dried and then baked at 80 °C in a vacuum for 1 h. Full-length clones of Xenopus Galpha q, Galpha 11, and Galpha 14 and partial clone of rat glycerol aldehyde phosphate dehydrogenase (GAPDH) were randomly labeled with 32P (Random Primer labeling kit, Life Technologies, Inc.) and hybridized to the filters at high stringency (see cloning section above). Filters were autoradiographed for 3-7 days on XAR 5 Kodak films. Quantitation of the relevant bands was done by densitometry and normalization with the 28S and the 18S bands on the RNA gels and the 1.2-kilobase GAPDH bands on the autoradiograms.

Western Analysis-- Groups of 60 oocytes were homogenized in 1 ml of cold hypotonic membrane buffer (5 mM Hepes, pH 7.4, 5 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µg/ml soybean trypsin inhibitor) and centrifuged twice in a microcentrifuge for 5 s at 5,000 rpm to pellet the yolk. The supernatant was centrifuged for 20 min at 14,000 rpm. The pelleted membranes were resuspended in 60 µl of hypotonic membrane buffer and kept at -70 °C. Before use, loading buffer was added (1:1 v/v), and samples were heated to 90 °C for 5 min. 20 µl (equivalent to membranes of 10 oocytes) were resolved on 10% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose filters. Filters were blocked for 3 h in blocking solution (TBS-T, Tris-buffered saline + 0.05% Tween 20, with 5% nonfat milk powder) and then exposed to the first antibody (1:500 dilution in blocking solution) overnight, with gentle shaking at 4 °C. Filters were then washed 5 times in washing solution (TBS-T) and exposed to the second antibody (1:1500 dilution in blocking solution of goat anti-rabbit IgG conjugated to horseradish peroxidase). Enhanced chemiluminescence system (ECL kit, Amersham Pharmacia Biotech) was used for developing the blots.

Materials-- Collagenase (Type 1A), trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated), Hepes, aprotinin, leupeptin, soybean trypsin inhibitor, goat anti-rabbit IgG, and ACh were purchased from Sigma; GRP14-27 from Bachem, S-oligos from Oligos Etc.; primers from Genemed Biotechnologies; and radiodeoxynucleotides from NEN Life Science Products. All molecular biology reagents were of molecular biology grade. All other chemicals were of analytical grade. WO82, B825, and Z808 antibodies were a gift of Dr. P. Sternweis, and QL antibody was a gift of Dr. T. Jones.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of Xenopus Oocyte Galpha 14-- Initial screening of a Xenopus oocyte cDNA library with mouse Galpha q probe at low stringency resulted in the cloning of the amphibian counterparts of Galpha q, Galpha 11, and an additional partial clone that most resembled mouse Galpha 14 (see "Experimental Procedures"). This partial clone was used as a probe in a second round of screening at high stringency to isolate a full-length clone of Xenopus Galpha 14. The cloned Xenopus Galpha 14 cDNA (accession number AF059182) had an open reading frame of 1065 bases, predicting a 354-amino acid protein. The cDNA sequence was 78% identical to mouse Galpha 14 (GenBankTM accession number M80631). The predicted protein was 90% identical to its mammalian counterpart, with most differing residues representing conservative substitutions, except for a cysteine missing at position 4 (Fig. 1). The consensus sequences for the GTP binding site and the carboxyl terminus were identical to the mammalian protein. Northern analysis of the native RNA with the cloned Galpha 14 cDNA as a probe detected a major 3.8-kilobase band and an additional 3.1-kilobase band (Fig. 2).


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Fig. 1.   Alignment of the predicted amino acid sequences of Xenopus oocyte (upper row) and mouse (lower row) Galpha 14.


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Fig. 2.   Specificity of antisense oligonucleotides in degrading cognate G protein mRNAs. Groups of oocytes were injected with each of the S-oligos (50 ng/oocyte). After 48 h, RNA was isolated, resolved on an RNA gel, and blotted onto nitrocellulose filters, as described under "Experimental Procedures." Filters were hybridized with 32P-labeled probes of Xenopus Galpha q (A), Galpha 11 (B), Galpha 14 (C), and mouse GAPDH (D) at high stringency. a-c, oocytes injected with antiGalpha q, antiGalpha 11, and antiGalpha 14, respectively. d, control (uninjected) oocytes. In most of the experiments, lower molecular mass degradation products were observed for the targeted RNAs. E, densitometric analysis of relevant G protein mRNAs bands in three independent experiments after normalization with GAPDH bands. Percent remaining mRNAs: Galpha q (open bars); Galpha 11 (gray bars); Galpha 14 (black bars).

Despite the high degree of homology among the three cloned Xenopus G proteins, alignment of their nucleotide sequences revealed several 20-base segments with less than 50% homology that were used for antisense design. The stretch of bases 559-578 (5'-atccagtcttgaaaggaaac-3') showed 42% identity to Xenopus Galpha q and Galpha 11. The antisense S-oligos used previously to deplete endogenous Xenopus Galpha q or Galpha 11 mRNAs (1) were also less than 50% homologous with the cloned Xenopus Galpha 14.

Antisense-induced RNA Degradation-- To test the antisense S-oligos selectivity for the corresponding mRNAs, we injected S-oligos into oocytes (50 ng/oocyte) and isolated RNA 48 h after injection. Northern analysis of three independent experiments after normalization with internal GAPDH transcript levels (see "Experimental Procedures") showed that all the three S-oligos were selective in promoting the degradation of their target mRNAs. Although antiGalpha 11 and antiGalpha 14 exhibited absolute selectivity, antiGalpha q was less effective and caused some degradation of Galpha 14 (Fig. 2). 48 h post-S-oligos injection, antiGalpha 14 degraded Galpha 14 RNA by 86% and had no effect on Galpha q and Galpha 11 RNA levels. antiGalpha 11 completely abolished its cognate RNA and had no effect on the other two RNAs. antiGalpha q caused degradation of both Galpha q and Galpha 14 transcripts by 62 and 42%, respectively, and had no effect on Galpha 11 mRNA. The kinetics of RNA degradation show that most of the RNA is already degraded 3 h after antisense injection. Although a 3-h treatment with S-oligos did not always result in a complete disappearance of the Galpha 11 or Galpha 14 mRNA bands, low molecular mass products indicated substantial partial degradation (Fig. 3A). In a representative experiment designed to test early effects of antisense S-oligos (3 h), densitometry of the autoradiograms after normalization with endogenous GAPDH resulted in the reduction of Galpha q by 67%, Galpha 11 by 89%, and Galpha 14 by 77%. 24 h after the injection of S-oligos, the Galpha 11 and Galpha 14 mRNAs were fully degraded. Since some reports demonstrated recovery of the response 4-7 days post-antisense oligonucleotides injections (29, 30), we checked for possible reappearance of RNAs encoding these G proteins within the time frame of our experiments. Oocytes were injected with antisense oligonucleotide and subjected to RNA isolation 1, 2, 3, and 4 days post-injection and then to Northern analysis with the respective probes. No Galpha 14-encoding RNA recovery was detected during this period (Fig. 3B). Similar results were obtained for the other two mRNAs (not shown).


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Fig. 3.   Kinetics of mRNA degradation after antisense injection. 3-96 h post-S-oligos injection (50 ng/oocyte), RNA was isolated, resolved, blotted, and hybridized with 32P-labeled probes of Galpha q (q), Galpha 11 (11), and Galpha 14 (14), as described under "Experimental Procedures." +, antisense-injected oocytes; -, control (uninjected) oocytes. A, 3 h post-injection of antiGalpha q, antiGalpha 11, or antiGalpha 14; B, 1-4 days post-injection of antiGalpha 14. GAPDH Northern analysis for normalization was performed on the same filters after the decay of G protein probes.

In all kinetic experiments, we observed a decrease in the apparent amounts of RNAs coding for the different G proteins. This phenomenon may have been related to the decreased synthetic ability of in vitro maintained oocytes. In view of the generally observed increase in response amplitudes to the stimulation of expressed G protein-coupled receptors (GPCRs), even 96 h after the injection of RNA, there is an excess of residual G proteins, and the limiting factor appears to be receptor density.

Antisense-induced Depletion of Oocyte G Proteins-- To assess the effect of S-oligos-induced mRNA depletion at the protein level, we used Western analysis with antisera developed against the mammalian G proteins of the Galpha q family. Antibodies that specifically discriminate between mouse Galpha q and Galpha 11 (WO82, B825; Refs. 15 and 31) interacted with a large number of proteins in the 30-50-kDa range and could not, therefore, detect amphibian G proteins unambiguously. Antibodies raised against the common carboxyl terminus of the Galpha q family (Z808 or QL, see Ref. 32) were more specific and, despite their interaction with a number of other proteins, we detected a faint band migrating at a position corresponding to approximately 42 kDa (Fig. 4). These data confirm the findings of Gallo et al., (26), showing only very small amounts of proteins of the Galpha q family in oocytes. The small amount of native G proteins made the quantitation of turnover rates difficult. To circumvent this problem, we overexpressed Xenopus Galpha q, Galpha 11, or Galpha 14 in oocytes by injecting (1 ng/oocyte) in vitro transcripts encoding their open reading frames. 48 h later, an increase in the intensity of the 42-kDa band was observed. The results indicated that Galpha 11 and Galpha q were overexpressed, albeit to a different extent. Galpha 14 did not seem to overexpress; however, injection of antiGalpha 14 markedly decreased the signal. This could be interpreted either as the oocyte natively expressing mainly Galpha 14 or that the expression of Galpha 14 repressed the expression of other G proteins of the family. We have used oocytes injected with the transcripts of each of the three Galpha q family members to estimate their turnover rates. 24 h post-injection of the cognate S-oligos, membranes were subjected to Western analysis with QL and Z808 antisera. S-oligos treatments caused substantial degradation of each protein (Fig. 4A). Densitometric analysis of the overexpressed 42-kDa bands showed that Galpha q, Galpha 11, and Galpha 14 were degraded by 75, 50, and 50%, respectively (Fig. 4B). Since RNA degradation was very rapid, these numbers approximate the degradation rates of the overexpressed proteins. Mitchell et al. (33) found that the turnover of Galpha q/11 in CHO cells was best described by monoexponential curve with t1/2 = 18 h. Our data indicate similar G protein turnover rates in oocytes. In control oocytes, 24 h post-injection of antiGalpha q, the endogenous 42-kDa proteins were degraded by 30%, suggesting that native G proteins of the Galpha q family include approximately 40% Galpha q. These results taken together demonstrate that antisense S-oligos treatment caused rapid and significant depletion of oocyte G proteins.


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Fig. 4.   The effect of antisense oligonucleotides on immunoreactive proteins in oocytes overexpressing Galpha q, Galpha 11, and Galpha 14. Groups of oocytes were injected with each specific in vitro transcript (1 ng/oocyte) and 48 h later with the cognate S-oligo. 1 day later, oocyte membranes were prepared (see "Experimental Procedures"), resolved on SDS-polyacrylamide gels, and immunoblotted with QL antibody. A, immunoblot; B, densitometric analysis of the data in A. Br, membranes prepared from mouse brain; C, control (no RNA injected) oocytes, + or - antiGalpha q; G14, oocytes injected with Galpha 14 in vitro transcript, + or - antiGalpha 14; G11, oocytes injected with Galpha 11 in vitro transcript, + or - antiGalpha 11; Gq, oocytes injected with Galpha q in vitro transcript, + or - antiGalpha q. Similar results were obtained using the Z808 antibody.

The Effects of Antisenses on the Endogenous Responses to Trypsin-- To study the involvement of Galpha q, Galpha 11, and Galpha 14 in the response evoked by trypsin, oocytes were injected with each of the S-oligos (50 ng/oocyte) and challenged with 0.1-5 µg/ml (1-50 benzoyl-L-arginine ethyl ester units/ml) trypsin at different time intervals after the injections. 24 h after antiGalpha q injection, the response was reduced by 69 ± 4% (n = 57, N = 7, Fig. 5). antiGalpha 14 reduced the response by 68 ± 7% (n = 58, N = 7). antiGalpha 11 decreased oocytes viability, but the response in the surviving cells was not affected. Injection of only half of the amount of antiGalpha 11 (25 ng/oocyte) did not compromise oocyte viability and had no effect on the response (81 ± 15% of control, n = 30, N = 3, not significant). In oocytes injected with a mixture of antiGalpha q and antiGalpha 14, the response to trypsin was reduced to 7 ± 3% that of control (n = 57, N = 7). Despite the relatively high amounts of S-oligos injected in the mixture (50 ng/oocyte, each), oocyte deterioration was very limited and did not exceed that of oocytes injected with each one of the S-oligos alone. Similarly, membrane potentials and holding currents did not indicate any nonspecific functional damage due to the co-injection of the two S-oligos. These results suggest that Galpha q and Galpha 14, but not Galpha 11, are the main mediators of the endogenous response to trypsin in Xenopus oocytes.


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Fig. 5.   The effect of antisense oligonucleotides on responses to trypsin. Oocytes were injected with antiGalpha q (q), antiGalpha 14 (14), antiGalpha 11 (11) (50 ng/oocyte), or a mixture of antiGalpha q/antiGalpha 14 (q/14) (50 ng/oocyte each antisense). 24 h later, oocytes were tested for responses to 1 µg/ml trypsin. Upper panel, tracings of representative responses; lower panel, average responses in seven independent experiments (n = 58). Data were calculated as percent of control (C) responses in each experiment. AS, antisense-injected.

As shown above for the S-oligos-induced degradation of mRNAs and depletion of proteins, the S-oligos-induced inhibition of the response to trypsin was time-dependent. In a representative experiment, antiGalpha q, antiGalpha 14, or a mixture of both reduced the response to 1 µg/ml trypsin after 24 h to 44, 43, and 11% and after 48 h, to 21, 20, and 6% that of control responses, respectively (Fig. 6). Neither the magnitude of the control response to trypsin nor the concentration of the agonist (0.1-50 µg/ml) had any influence on the effect of antisense treatment. For example, at 48 h, the amplitude of the response to 5 µg/ml trypsin was double that to 1 µg/ml, and the antisenses reduced the response to 36 (antiGalpha q), 51 (antiGalpha 14), and 7% (mixture of both) that of control (Fig. 6).


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Fig. 6.   Time and dose dependence of the effect of antisense oligonucleotides. Oocytes were injected with antiGalpha q (q), antiGalpha 14 (14), or a mixture of the two (q/14), as described in legend to Fig. 5, and tested 24 and 48 h later for their response to either 1 or 5 µg/ml trypsin. AS, antisense-injected; Time, time after antisense injection; [Tryp], trypsin concentration (µg/ml). The mean control response to 1 µg/ml trypsin after 24 h was 1388 ± 161 nA (n = 14) and after 48 h, 1274 ± 225 nA (n = 10). Mean control response to 5 µg/ml after 48 h was 2857 ± 410 nA (n = 15). A representative experiment is shown.

In our previous report (1) we found no effect of a 3-h treatment with antiGalpha q or antiGalpha 11 on the responses to the activation of GRP-Rs expressed in Xenopus oocytes. In view of our present results relating to the kinetics of the depletion of G proteins, we studied the effects of all three antisense S-oligos on the responses to the activation of expressed GRP and m1 receptors 24 h after S-oligos injections. In oocytes expressing the GRP-Rs, antiGalpha q or antiGalpha 11 reduced the responses to 78 ± 7% (n = 102, N = 9) or 82 ± 5% (n = 75, N = 6) of control, respectively, and these effects, though modest, were statistically significant. antiGalpha 14 had no effect (99 ± 27% of control, n = 24, N = 3, see Fig. 7). These results suggest that Galpha q and Galpha 11, but most probably not Galpha 14, couple to the GRP-R, albeit to a limited extent.


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Fig. 7.   The effect of antisense oligonucleotides on GRP-R- and m1-R-induced responses. Oocytes were injected with in vitro transcripts encoding the GRP-R and m1-R and, 18 h later, with antiGalpha q (q), antiGalpha 11 (11), antiGalpha 14 (14), or antiGalpha o (o) (50 ng/oocyte). 24 h after the injection of S-oligos, oocytes were tested for responses to 1 µM GRP or 10 µM ACh. Responses are presented as % of control responses in each experiment. *, p < 0.05.

In oocytes expressing m1-Rs, antiGalpha q or antiGalpha 11 reduced responses to ACh to 58 ± 5% (n = 80; N = 15, p < 0.05) and 61 ± 6% (n = 65; N = 6, p < 0.05) of control, respectively, whereas antiGalpha 14 had no effect (133 ± 28% that of control, n = 65, N = 8, not significant). In some experiments, co-injection of antiGalpha q and Galpha 11 markedly reduced the response to ACh (not shown). The extensive deterioration of oocytes injected with both S-oligos, however, did not allow positive conclusions from these experiments. Since injections of Galpha o has been reported to increase responses to ACh in Xenopus oocytes (34, 35), we tested the effect of antiGalpha o (complementary to the first 20 nucleotides of the open reading frame of Xenopus Galpha o, Ref. 36) 24 h after its injection. antiGalpha o caused a reduction of the responses to 79 ± 8% of control (p < 0.05). Our data confirmed the involvement of Galpha q and Galpha 11 in m1 muscarinic response in Xenopus oocytes, similarly to the previously published results in other model systems (e.g. Ref. 7). The involvement of Galpha o in the muscarinic response in oocytes, previously reported by Moriarty et al. (34) and Padrell et al. (35), was also detected, albeit to a much more limited extent. Hence, the involvement of Galpha 14 was demonstrated only for the endogenous receptor to protease in oocytes.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

All G proteins of the Galpha q family have been shown to couple to GPCRs in the pertussis-toxin-insensitive phosphatidylinositol-specific phospholipase C signal transduction pathway. With the exception of our previous report in oocytes (1), transfection and reconstitution experiments failed to functionally discriminate between Galpha q and Galpha 11. However, sequence and distribution differences (e.g. Ref. 4) between the two G proteins as well as among all the family members in general suggest individual physiological roles in regulating signaling in target tissues and organs. The antisense approach has the advantage of relying on the natural milieu of each component of the signal transduction pathway. By applying this approach in Xenopus oocytes, we have previously demonstrated functional in vivo preferences of neuromedin B receptor coupling via Galpha q (1).

To broaden the scope of the study, we have cloned Xenopus Galpha 14 in order to develop an antisense oligonucleotide that could be used to deplete cells of Galpha 14 protein. Similarly to the previously cloned Xenopus Galpha q and Galpha 11, Xenopus Galpha 14 showed a high degree of homology to its mammalian counterpart, demonstrating the conservation of these protein sequences during vertebrate evolution. Northern analysis in oocytes demonstrated the intrafamily selectivity of the designed antisenses (antiGalpha q being less selective, degrading mainly Galpha q but also Galpha 14) and the rapid elimination of the cognate mRNAs after antisense injection. Thus, proteins turnover rates were the limiting factor in the depletion process. The turnover rate of Galpha q/11 has been shown in Chinese hamster ovary cells to fit a first-order reaction with a t1/2 of 18 h (33). Our data in oocytes, obtained from both Western analysis and functional assays, exhibit similar kinetics of degradation, but distinguish between Galpha q and Galpha 11. The turnover rate of Galpha 14 was similar to that of Galpha 11. Our functional assays were executed 24-80 h after antisense injection, a time window in which most of the Galpha q and more than 50% of Galpha 11 and Galpha 14 were degraded. Therefore, a 40% reduction in the m1 muscarinic response by either antiGalpha q or antiGalpha 11 and the fact that this response was not further reduced after 48 and 80 h support the existence of an additional mediator for this receptor. Indeed, antiGalpha o caused a 20% reduction in the response to ACh 24 h after its injection. Mixtures of antiGalpha q/antiGalpha 11 caused pronounced oocytes deterioration, preventing a more conclusive interpretation. We cannot exclude the possibility that even residual amounts of any G protein (undetectable by Western analysis) can mediate a full response, provided there is a large excess of G protein over the receptor and high affinity between the two proteins.

The antisense oligonucleotides designed against the different G proteins could have achieved their effects by interfering with the synthesis of different phospholipase Cbeta isoforms. This possibility, however, appears unlikely since there is no evidence for multiple phospholipase Cbeta species in oocytes.

Since there is little information about the Galpha 14 ability to couple GPCRs, we tested a number of receptors (mouse m1, GRP and neuromedin B receptors) and the native trypsin response to investigate its specificity. Only the endogenous protease receptor exhibited sensitivity to Galpha 14 depletion. Two antisense oligonucleotides, antiGalpha q and antiGalpha 14, each diminished the responses to about 30%, and a mixture of the two reduced it to 7% that of control, whereas antiGalpha 11 had no effect. These results strongly suggest that the response to trypsin is mediated exclusively by Galpha q and Galpha 14. Although it appears that the contribution of these two G proteins to the trypsin response is similar, the limited degree of degradation of Galpha 14 mRNA caused by anti Galpha q precludes a firm conclusion regarding this issue. Thus, Galpha 14 mediates the response to protease in addition to its ability to couple alpha 1-adrenergic, interleukin-8 receptors (17, 23), and histamine receptors (16). Wu et al. (37) have shown by altering selected domains within the third intracellular loop of the alpha 1B-adrenergic receptor, sequence-related specificity for either Galpha q/11 or Galpha 14. It would have been interesting to compare the sequence of the yet uncloned Xenopus protease receptor, which appears to couple to Galpha q and Galpha 14, but not to Galpha 11, with that of the alpha 1B-adrenergic receptor.

The finding that antiGalpha 11 did not affect the response confirms our previous report that in vivo Galpha q and Galpha 11 have distinct receptor preferences. It is interesting to note that so far in reconstitution, transfection, and in vivo models, every GPCR tested (with the possible exception of neuromedin B receptor expressed in oocytes, Ref. 1) was able to couple to more than one G protein of the Galpha q family. Since different GPCRs appear to be able to discriminate among the G proteins, the interaction of a single receptor with more than a single G protein implies biological significance. In this respect, the recent report by Macrez-Lepretre et al. (38) points to discrimination between Galpha q and Galpha 11 for specific effector molecule in the signaling pathway. The G proteins specificity for the receptors on the one hand and for the distal parts of the signal transduction pathway on the other may serve to fine tune receptor-mediated biological responses.

    ACKNOWLEDGEMENTS

We are indebted to Dr. P. Sternweis for his generous donation of anti-G protein antibodies and to Dr. T. Jones for her gift of the QL antiserum.

    FOOTNOTES

* This work was supported in part by a grant of BiNational Science Foundation (BSF) (to H. S. and J. F. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF059182.

Dagger To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. Tel.: 972-3-640-9862; Fax: 972-3-640-9113; E-mail: hshapira{at}post.tau.ac.il.

1 The abbreviations used are: m1-R, m1 muscarinic receptor; GPCRs, G protein-coupled receptors; GRP, gastrin-releasing peptide; GRP-R, GRP receptor; ACh, acetylcholine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; S-oligos, phosphorothioate oligodeoxynucleotides; PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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