©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Couples Thyrotropin-releasing Hormone Receptors Expressed in Xenopus Oocytes to Phospholipase C (*)

(Received for publication, July 19, 1994; and in revised form, November 18, 1994)

Pilar de la Peña (§) Donato del Camino (¶) Luis A. Pardo Pedro Domínguez Francisco Barros

From the Departamento de Biología Funcional, Area de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Coupling of thyrotropin-releasing hormone (TRH) receptors to individual G-proteins has been studied in Xenopus oocytes injected with receptor cRNA and antisense oligonucleotides to mRNA encoding different G-protein alpha- and beta-subunits. Injection of antisenses which target mRNA sequences shared by several G-protein alpha or beta polypeptides effectively blocked Ca-dependent Cl currents induced by TRH through activation of phospholipase C. Three different alpha(s)-specific antisense oligonucleotides complementary to sequences located in different positions along the coding region of the alpha(s) protein mRNA were highly effective in inhibiting TRH-induced responses. Anti-alpha(o), -alpha(q), -alpha(i), or -alpha(z) oligonucleotides were not able to modify the TRH-evoked response. In contrast, anti-alpha(o), but not anti-alpha(s), oligonucleotides blocked the response to serotonin in oocytes injected with serotonin 5-HT1(c) receptor cRNA. Cholera toxin catalyzed the [P]ADP-ribosylation of 40-42- and 50-52-kDa proteins in GH(3) cell plasma membranes. [P]ADP-ribosylation of oocyte membranes with the toxin labeled several proteins. These include a single 50-55-kDa substrate, which is clearly diminished in membranes from anti-alpha(s)-injected oocytes. Amplification of oocyte RNA in a polymerase chain reaction system and sequencing of the amplified products demonstrated that anti-alpha(s) oligonucleotides selectively recognize the message for the Xenopus alpha(s) polypeptide. It is concluded that G(s), but not G(o), G(q), G(i), or G(z), couples TRH receptors expressed in oocytes to activation of phospholipase C and subsequent inositol 1,4,5-trisphosphate-dependent stimulation of Ca-dependent Cl currents.


INTRODUCTION

Seven G-protein^1-linked transmembrane spanning receptors generally couple to the same G-proteins and signaling pathways in different species and cell types(1) . Furthermore, it has been argued that although receptors can be promiscuous in their choice of G-protein, they appear to be so only under extreme conditions(2) . However, recent studies with G-protein-linked receptors both endogenously expressed and transfected into heterologous systems indicate that receptors are likely to interact with more than one of the G-proteins present in the membrane (3, 4, 5, 6, 7, 8) (for reviews, see (9) and (10) ). Recent cloning and sequencing of the TRH receptor from anterior pituitary cells has revealed that it belongs to the seven hydrophobic helices G-protein-linked family of receptors(11, 12, 13) . In GH(3) rat anterior pituitary cells, the initial response triggered by TRH involves activation of a PLC through an heterotrimeric CTX- and PTX-insensitive G-protein recently identified as G(q) and/or G(14, 15) . The PLC-mediated stimulation of IP(3) production induces a transient increase in cytosolic Ca, leading to an acute enhancement of secretion, accompanied by a transient hyperpolarization of the cell membrane due to activation of Ca-dependent K channels(16, 17, 18) . This transient phase of TRH effects is followed by an enhanced generation of action potentials for an extended period, causing a sustained plateau of elevated Ca and secretion(16, 17, 19, 20) . It has been suggested that stimulation of voltage-dependent Ca channels participates in regulation of sustained Ca entry in response to TRH. The effect on the Ca channels is mediated mainly by a PTX-sensitive G-protein identified as G(21) . On the other hand, we have recently shown that hormone-induced reductions on an inwardly rectifying K current play a major role in regulation of GH(3) cell's electrical activity and hence of sustained elevations of Ca and secretion in response to TRH(22, 23) . Our results indicate that, besides the well known toxin-insensitive transduction pathway linked to PLC activation, a CTX-sensitive pathway (i.e. G(s) or a G(s)-like protein) could link the TRH receptor to regulation of inwardly rectifying K currents(22, 24) . To obtain a better knowledge of the role of individual G-proteins in coupling TRH receptors to PLC, we have used recombinant receptors functionally expressed in Xenopus oocytes. Both PTX-sensitive and -insensitive G-proteins have been shown to mediate coupling of exogenous receptors to PLC in oocytes(24, 25, 26, 27, 28) . It has been proposed that G(o) and G participate in PTX-sensitive coupling of expressed receptors to PLC (27, 28) . However, the nature of the transducer involved in PTX-insensitive responses remains obscure. It has been also shown that fidelity of coupling to G-proteins can be lost after functional expression in oocytes. Thus, several mammalian receptors that mediate activation of PLC by members of the G(q) family in their native environment display a PTX-sensitive response via G(o) in the oocyte system(24, 27) . The alpha-subunits of G-proteins bind guanine nucleotides and define the different G-protein subtypes. In this report, selective ``knocking out'' of individual G-proteins has been achieved by injection of antisense oligonucleotides that specifically hybridize with mRNAs of the distinct alpha-subunits of G-proteins. Our results indicate that G(s), but not G(q), G(o), G(i), or G(z), couples TRH receptors expressed in oocytes to activation of PLC and subsequent IP(3)-dependent stimulation of Ca-dependent Cl currents.


MATERIALS AND METHODS

The serotonin 5-HT1(c) receptor clone was obtained from Dr. H. Lübbert (Sandoz Pharmaceuticals Ltd., Basel, Switzerland). Isolation and handling of the TRH receptor cDNA has been previously described(12, 29) . Unless otherwise indicated, the long isoform (TRH-R in (29) ) of the two alternatively spliced variants of the TRH receptor was used for experiments. In vitro synthesis of RNA was performed as described elsewhere (12, 29) except that for 5-HT1(c) receptor cDNA KpnI was used for linearization and T7 RNA polymerase (Boehringer Mannheim) was used for cRNA synthesis. Procedures for microinjection and electrophysiology of oocytes have been described elsewhere(12, 29) . Sense and antisense oligonucleotides were synthesized by the phosphoamidite method in an Applied Biosystems 381A DNA synthesizer. After extraction with NH(3), they were dried under a N(2) atmosphere, resuspended in sterile water, extracted again with phenol/chloroform, and precipitated with ethanol. Final resuspension of the samples was performed in water at a concentration of 200 pmol oligonucleotide/µl. 50 nl of oligonucleotide suspension were injected per oocyte 48 h before starting recordings, followed by TRH receptor cRNA 12-24 h later. We found the 5-HT1(c) maximal response to develop more slowly than TRH receptor expression. Subsequently, 5-HT1(c) receptor cRNA was routinely injected 3-6 h after microinjection of the oligonucleotides. For total RNA extraction, oocytes were homogenized in a buffer containing 20 mM NaCl, 5 mM MgCl(2), 2% SDS, 1 mg/ml proteinase K, and 200 mM Tris-HCl at pH 8.0. After several phenol/chloroform extractions, the samples were precipitated with ethanol and resuspended in water until use. To check the sequences recognized by the anti-alpha(s) oligonucleotides, the anti-alpha(s)3 (see below for nucleotide sequences and nomenclature used) was initially used for priming an avian myeloblastoma virus reverse transcriptase (Boehringer Mannheim) with oocyte total RNA as a template. The resulting single-stranded cDNA was amplified (30 cycles; 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C) using a Gene Ataq Controller (Pharmacia Biotech Inc.) either in the presence of that primer plus the sense counterpart of anti-alpha(s)2, or in the presence of anti-alpha(s)2 plus the sense counterpart of anti-alpha(s)1. PCR products were analyzed by electrophoresis on agarose gels, in which two prominent major bands of near 0.54 and 0.56 kilobase pairs were obtained. The PCR products were directly cloned in pGEM-T vector (Promega) and recombinant plasmids containing the 0.54-kilobase pair fragment were subsequently sequenced with Sequenase (U. S. Biochemical Corp., Cleveland, OH) and the sense and antisense primers used for amplification. The sequence obtained corresponded exactly to that located between positions 697 and 1191 of the Xenopus alpha(s)-subunit gene sequence previously reported(30) .

GH(3) cells (ATCC CCL 82.1) were grown and harvested as described previously(12, 29) . For isolation of cell membranes, either 7 times 10^7 GH(3) cells or 150 defolliculated oocytes (12, 29) were homogenized in buffer A (150 mM NaCl, 10 mM MgCl(2), 0.1 mM PMSF, and 20 mM Tris-HCl at pH 7.5) containing 10% (w/v) sucrose. The PMSF is added from a 0.4 M stock in ethanol immediately before the homogenization, performed with 30 strokes in a Dounce homogenizer. The homogenate was layered on top of a discontinuous sucrose gradient made up of a cushion of 50% sucrose and a layer of 20% sucrose in buffer A. After centrifugation at 15,000 times g for 30 min in a Beckman SW 40 Ti rotor, the membranes located at the 20-50% sucrose interface were removed with a Pasteur pipette, diluted with 5 volumes of buffer A, and pelleted at 100,000 times g for 30 min. Finally, the pellet was rehomogenized in an appropriate volume of buffer containing 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2 mM PMSF, 1 µg/ml aprotinin, and 20 mM Tris-HCl at pH 7.5.

CTX (2 mg/ml, Sigma) was dissolved in 80 mM NaCl, 0.4 mM EDTA, 50% (v/v) glycerol, and 20 mM Tris-HCl at pH 7.4. The toxin (0.2 mg/ml) was activated shortly before the ADP-ribosylation reaction by dilution in an activation buffer containing 0.13 M NaCl, 0.5% SDS, 4 mM dithiothreitol, 2% bovine serum albumin, and 10 mM Hepes at pH 6.8. Membranes were incubated with 20 µg/ml activated CTX or the same amount of activation buffer in 10 mM Hepes-NaOH (pH 7.0) containing 5 mM ATP, 50 µM GTP, 2 mM EDTA, 10 mM thymidine, 10 mM isoniazid, 5 mM MgCl(2), 150 mM KCl, and 4 µM [alpha-P]NAD (about 50 Ci/mmol, Amersham Corp.). The reaction was carried out at 25 °C for 40 min in a final volume of 50 µl and stopped by dilution with 2 ml of ice-cold buffer containing 0.13 M NaCl and 10 mM Hepes-NaOH at pH 7.4. The mixture was centrifuged at 50,000 times g for 30 min, and the pellet containing the ADP-ribosylated membranes was washed with stopping buffer. After dissociation, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis in 10% gels. The gels were stained for protein and dried, after which autoradiographies were taken by exposure of films to the dried gels.

Data are expressed in the bar histograms as means plus 95% confidence intervals, with values of current in a logarithmic scale. A multifactor analysis of variance was applied to the values of current in the initial maximum. The distribution of current magnitudes was previously normalized for analysis by means of a logarithmic transformation. This allowed us to make the effects of oligonucleotides independent from those caused by the significant differences in the magnitude of responses found in populations of oocytes from different frogs. Total number of oocytes assayed is represented on top of the bars for the number of donors indicated by a capital N in the graphs. The nucleotide sequences of synthetic oligomers are as follows.

Anti-alpha

5`-AT(T/C)TTCAT(C/T)TG(C/T)TT(C/T)ACAAT(T/G)GTGCTTTT-3`, corresponding to nucleotides 237-265 of the identical strand of the Xenopus alpha(s) gene sequence(30) . It also hybridizes with messages for the rest of the alpha-subunits of G-proteins, but not with those of beta(1)- to beta(4)-subunits.

Anti-alpha(o)

5`-TGGGACCTGTGTATTCCGGAAA-3`, corresponding to nucleotides 989-1011 of the identical strand of the Xenopus alpha(o) gene sequence(31) . This sequence is also present with three mismatches in the genes for alpha-subunits from human and rat origin(32, 33) .

Anti-alpha(q)1

5`-CTCGATCTCG(T/G)CG(T/C)TGAT(G/C)C(G/T)C-3`, corresponding to nucleotides 93-114 and 113-134 of the identical strand of the alpha(q) and alpha gene sequences, respectively(34) .

Anti-alpha(q)2

5`-GAGTTC(T/A)G(A/G)AACCAGGGGTAGGTGA-3`, corresponding to nucleotides 812-836 and 832-856 of the identical strand of the alpha(q) and alpha gene sequences, respectively(34) .

Anti-alpha(q)3

5`-ACCAGGTTG(A/T)A(T/C)TCC(T/C)TCAGGTT-3`, corresponding to nucleotides 1110-1132 of the identical strand of the alpha(q) and alpha gene sequences, respectively (34) . Sequences similar to those of anti-alpha to -alpha with less than five mismatches are not present in the genes for other cloned alpha-subunits.

Anti-alpha(i)

5`-AC(G/A)AT(G/C)CC(C/T)GT(A/G)GTCTTC-3`, corresponding to nucleotides 721-738 and 509-527 of the identical strand of the Xenopus alpha and alpha gene sequences, respectively(30) . This sequence is also found with 2 mismatches in Xenopus alpha(o) mRNA(31) , with three or four mismatches in the genes for other alpha(o)-subunits from mammals and with two mismatches in sequences corresponding to alpha(z)-subunits from rat and human origin(35, 36) .

Anti-alpha(s)1

5`-TTGTTGGCCTC(A/G)CGCTG-3`, corresponding to nucleotides 135-151 of the identical strand of the Xenopus alpha(s) gene sequence(30) .

Anti-alpha(s)2

5`-ATGTGGAA(G/A)TTGACTTTGTCCACC-3`, corresponding to nucleotides 674-697 of the identical strand of the Xenopus alpha(s) gene sequence(30) .

Anti-alpha(s)3

5`-GCTC(A/G)TATTGGCG(C/G/A)AG(G/A)TGCAT-3`, corresponding to nucleotides 1191-1212 of the identical strand of the Xenopus alpha(s) gene sequence(30) . Sequences similar to those of anti-alpha(s)1 to -alpha(s)3 are not present with less than five mismatches in genes of any of the other cloned alpha-subunits.

Anti-alpha(z)

5`-GAGTTGCTGGTGCCCAGCAG-3`, corresponding to nucleotides 1312-1331 and 126-145 of the identical strand of two alpha(z) gene sequences(35, 36) . It does not hybridize with any of the genes for other alpha-subunits.

Anti-beta

5`-GT(C/G)C(T,G)CAT(C/T)TGGATTCT(C/T)CCCACTGGGTC-3`, corresponding to nucleotides 332-360, 259-287, 118-146, and 152-180 of the identical strand of beta(1) to beta(4) gene sequences, respectively(37, 38, 39) . It does not hybridize with genes for any of the alpha-subunits.

Apart from the selectivity indicated above for the different subunits of G-proteins, no sequences similar to those of the antisense oligonucleotides are found in the sequence of the expressed receptor messages.


RESULTS

To test the possible interaction of TRH receptors with individual G-proteins, the response induced by TRH in control oocytes injected with TRH receptor cRNA was compared with that of oocytes also injected with antisense oligonucleotides to the mRNA encoding different G-protein alpha- and beta-subunits. As shown in Fig. 1, the responses consisted in a large, rapid, and transient inward current, followed by a second phase of prolonged depolarizing current, often associated with current fluctuations. These responses have been largely recognized as due to activation of Ca-dependent Cl currents after stimulation of PLC and subsequent IP(3)-dependent mobilization of Ca from intracellular stores(11, 12, 13) . Responses indistinguishable from those recorded in oocytes injected exclusively with the TRH receptor cRNA were obtained in oocytes also injected with water, oligonucleotides sense, or an oligonucleotide carrying the sequence of a fragment from a voltage-dependent K channel gene present in GH(3) cells. In contrast, the response to TRH was almost abolished in oocytes injected with an antisense oligonucleotide (alpha) which targets mRNA of all known G-protein alpha-polypeptides ( Fig. 1and Fig. 2). Similar results were obtained with an antisense oligonucleotide (beta, see Fig. 2) directed against G-protein beta(1)- to beta(4)-subunit sequences. This indicates that beta-subunits are also required in the interaction between receptor and transducer, suggesting that an heterotrimeric G-protein is involved in the coupling of receptors to the oocyte response.


Figure 1: Effect of antisense oligonucleotides to mRNA encoding different G-protein alpha-subunits on TRH-induced oocyte responses. Inward currents in voltage-clamped oocytes microinjected with antisense oligonucleotides 48 h before recording and with TRH receptor cRNA 24 h later. Duration of perfusion with saline plus 1 µM TRH is signaled by horizontal lines above the traces. Holding potential -60 mV. The alpha-subunit to which each antisense oligonucleotide is directed is also indicated. Currents recorded from an oocyte injected with an oligonucleotide in sense orientation (Control) is also shown for comparison.




Figure 2: Inhibition by antisense oligonucleotides of TRH-induced oocyte responses. Averaged maximal responses of oocytes microinjected with antisense oligonucleotides 48 h before recording (solid bars) are compared with those of control oocytes from the same donors without antisenses (hatched bars). TRH was always used at 1 µM. TRH receptor cRNA was injected 24 h after oligonucleotides. Three different anti-alpha(q) and anti-alpha(s) oligonucleotides numbered 1-3 were used. For sequences of injected oligonucleotides, see ``Materials and Methods.'' Significant differences versus controls are indicated by an asterisk.



To study the preferential interaction between the expressed receptors and G-proteins containing a given alpha-subunit, oocytes were injected with antisense oligonucleotides that can specifically hybridize with the mRNA of one particular alpha-subtype. Fig. 1shows that only an oligonucleotide directed against alpha(s)-subunits, but not those directed against alpha(q)-, alpha(i)-, or Xenopus alpha(o)-subunits, is able to reduce the TRH-evoked response. Previous work with exogenous and native receptors indicated that G(o) might serve as the signal transducer to PLC in oocytes(27, 40) . Furthermore, G(q) has been recognized as the G-protein coupling the TRH receptor to PLC in GH(3) cells(14, 15) . The antisense oligonucleotide against alpha(o) is based in a sequence found in the alpha-subunit of the G(o)-type protein of Xenopus oocytes(31) . However, similar information is not available for oocyte G-proteins of the G(q)-type. To minimize the possibility that the lack of effect of anti-alpha(q) oligonucleotides is due to choice of a sequence not present in the hypothetical alpha(q) of the oocyte, three different antisense oligonucleotides containing sequences complementary to those of the cloned alpha(q) polypeptides were used. As shown in Fig. 2, none of them was able to modify the TRH-evoked response. The oocyte response was not altered either by injection of antisense oligonucleotides to regions of alpha(z) and alpha(i) mRNAs. It is interesting that the anti-alpha(i) oligonucleotide can hybridize perfectly with the two alpha(i)-subunits cloned from Xenopus (alpha(i)1 and alpha(i)3)(30) , and with human alpha(i)2(33) , but it is also complementary with nucleotides 667-684 of Xenopus alpha(o) mRNA, except for two mismatches in positions 676 and 679(31) . This adds further support to our conclusion that G(o) is not the signal transducer of the TRH receptor-regulated PLC in Xenopus oocytes.

The aforementioned results suggest that coupling of the TRH receptor to oocyte responses is performed through a G-protein of the G(s)-type, but not through G(o), G(q), G(i), or G(z). To prevent the possibility that the anti-alpha(s) oligonucleotides could act by knocking out a protein distinct from the alpha(s) polypeptide, three different alpha(s)-specific antisense oligonucleotides were used. Fig. 2shows that all three antisenses were highly effective in inhibiting TRH-induced responses. The sequences complementary to these oligonucleotides are located within the coding region of the alpha(s) protein mRNA sequence either close to the NH(2)-terminal end, in the middle, and few nucleotides from the stop codon, respectively. This demonstrates that the situation of the antisense sequences along the target mRNA is not an important determinant for effectiveness. On the other hand, since sequences recognized by other antisense oligonucleotides are located in positions equivalent to those in which anti-alpha(s) are effective, failure to detect an effect is not due to inappropriate choice of the hybridizing sequence. It is noteworthy that similar results were obtained with both isoforms of the TRH receptor previously described (29) . Thus, an oligonucleotide directed against the alpha(s)-subunit, but not that directed against alpha(q), was able to reduce the TRH-induced responses in oocytes expressing the short isoform (TRH-R) of the TRH receptor (data not shown). This indicates that, at least in the oocyte system, the coupling specificity is the same for the two variants of the receptor.

As stated under ``Materials and Methods,'' injection of antisense oligonucleotides was routinely performed 2 days before recording, followed by TRH receptor cRNA injection 12-24 h later. We found this situation as maximally effective for inhibiting the TRH-induced responses. In fact, the inhibition was very small when oligonucleotides were injected only 1 h before cRNA (not shown). This opened the possibility that the inhibitory effect was not caused by depletion of the transducer, but due to elimination of a G-protein necessary for correct operation of the expression machinery of the oocyte. The results shown in Fig. 3argue against this interpretation. As for TRH receptors, functional expression of 5-HT1(c) serotonin receptors was largely blocked by the anti-alpha oligonucleotide. On the other hand, the serotonin-induced responses were significantly reduced by the antisense against the alpha(o) polypeptide message. However, neither the anti-alpha(s) hybridizing with the portion of the alpha(s)-mRNA near the carboxyl terminus of the protein (Fig. 3), nor that to the sequences near the amino terminus of the alpha(s) polypeptides (not shown), altered the response to serotonin in oocytes expressing the serotonin receptor. This demonstrates that it is the coupling transducer, and not the expression machinery, that is affected by the antisenses. It also indicates that the inhibitory effects are specific, the anti-alpha(o) selectively blocking serotonin responses and the anti-alpha(s) oligonucleotides exclusively minimizing the TRH-evoked responses.


Figure 3: Effect of antisense oligonucleotides to mRNA for different alpha-subunits on serotonin-induced oocyte responses. Averaged maximal responses of oocytes microinjected with antisense oligonucleotides 48 h before recording (solid bars) are compared with those of control oocytes from the same donors without antisenses (hatched bars). Serotonin was used at 1 µM. Serotonin 5-HT1(c) receptor cRNA was injected 2-6 h after antisenses. Significant differences versus controls are indicated by asterisks. Representative current traces for oocytes control and injected with antisenses against the indicated alpha-subunits are shown at the top. Application of serotonin is indicated by a horizontal line above the current traces.



Further support to the conclusion that antisenses to mRNAs from alpha(s)-subunits inhibit TRH responses by depletion of these polypeptides would come from the demonstration that alpha(s) is knocked out of the cell by injection of the oligonucleotides. Preliminary experiments performed with a specific anti-alpha(s) antibody against the COOH-terminal portion of alpha(s) (kindly supplied by Dr. G. Milligan, University of Glasgow, Scotland) failed to demonstrate any clear inhibition of TRH responses in antibody-injected oocytes. On the other hand, we were unable to detect any protein band specifically recognized by the antibody in the 40-60-kDa range after Western blotting of oocyte membranes (not shown). Similar results were obtained with a commercial antibody (Upstate Biotechnology, Inc., Lake Placid, NY), even though a clear doublet of protein bands (40-41 and 50-52 kDa, respectively) was detected in the same blots using GH(3) cell membranes (data not shown). To detect the presence of the alpha(s) polypeptides by other means, membranes were treated with [alpha-P]NAD in the presence of activated CTX (see ``Materials and Methods''). As shown in Fig. 4, two prominent bands corresponding to 40-42- and 50-52-kDa polypeptides are labeled by CTX when GH(3) cell membranes are used (lanes 5 and 6). By contrast, several oocyte membrane proteins are ADP-ribosylated in a CTX-dependent way, mainly one protein in the 50-55-kDa range. This CTX substrate is clearly diminished in membranes obtained from anti-alpha(s) oligonucleotide-injected oocytes (Fig. 4, compare lanes 2 and 4). Similar patterns of CTX-catalyzed ADP-ribosylation have been previously described in membranes from manually defolliculated oocytes(41) . However, they differ from the single 42-kDa protein band labeled by CTX in membranes of oocytes obtained from collagenase-treated pieces of ovary, previously proposed to correspond to the oocyte equivalent to mammalian alpha(s)(42) . The reason(s) for these differences are not known, but they are not due to use of follicles versus nude oocytes since we have not detected the single 42-kDa protein band in membranes obtained from follicle-enclosed oocytes (not shown).


Figure 4: Cholera toxin-catalyzed ADP-ribosylation of oocyte and GH(3) cell membranes: effect of microinjection of anti-alpha(s) oligonucleotides. Membranes from oocytes (500 µg protein, lanes 1 to 4) or GH(3) cells (300 µg, lanes 5 and 6) were incubated in the presence of [alpha-P]NAD with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) preactivated CTX as described under ``Materials and Methods.'' They were then analyzed on a SDS-10% polyacrylamide gel, and the radioactivity was detected by autoradiography after 20 (lane 6) or 96 h (lanes 1-5) of exposition. Arrowheads on the left indicate the positions of the 30-, 43-, 55-, 67-, and 94-kDa molecular mass markers.



As a final demonstration that the anti-alpha(s) oligonucleotides selectively recognize the sequences of the messages for these polypeptides, the alpha(s) carboxyl terminus antisense oligonucleotide was used to prime a reverse transcriptase with oocytes RNA as a template. The resulting single-stranded cDNA was amplified using a PCR system in the presence of 1) that oligonucleotide plus the sense counterpart of the anti-alpha(s)2 located in the center of the alpha(s) coding region and 2) anti-alpha(s)2 plus the sense counterpart of the anti-alpha(s)1 located near the amino terminus of the alpha(s) coding region. In both cases, a single prominent band of the size expected from separation between the oligonucleotide sequences in the Xenopus alpha(s) message (i.e. 539 and 563 bp) was obtained. It is important to note that the detection of only a 563-bp product using the second couple of oligonucleotides correlates with the results obtained after ADP-ribosylation of oocyte membranes. Thus, since only long isoforms of the alternatively spliced alpha(s) messages are detected, only alpha(s) polypeptides in the 50-55-kDa range would be expected. Finally, subsequent cloning and sequencing of the 539 fragment identified it as corresponding exactly to Xenopus alpha(s) mRNA (not shown; see (30) ).


DISCUSSION

The data presented here indicate that G(s), but not G(o), G(q), G(z), or G(i), acts as the signal transducer of the TRH receptor expressed in Xenopus oocytes. This conclusion is based in the ability of antisense oligonucleotides, hybridizing with specific sequences of different G-protein mRNAs, to block the response to TRH and serotonin in oocytes injected with TRH or serotonin 5-HT1(c) receptor cRNA, respectively. Attempts to substantiate this conclusion using bacterial toxins, either exogenously added or injected after activation with dithiothreitol, yielded very variable results from day to day and were not pursued further. Participation of an heterotrimeric G-protein in TRH- and serotonin-induced responses is suggested by the fact that hormonal responses are effectively antagonized by antisenses that recognize sequences common to several alpha or beta G-protein subunits. The possibility that the effect of these common antisenses is not caused directly by depletion of the transducer coupling PLC to the expressed receptors, but is due to elimination of a G-protein necessary for elaboration of a correct oocyte response, cannot be excluded. However, our results demonstrate that, at least for anti-alpha(o) and anti-alpha(s) oligonucleotides, the effect is direct and specific, since only TRH and not serotonin responses are blocked by antisenses against alpha(s) polypeptides. In contrast, only serotonin-induced responses are inhibited by the anti-alpha(o) oligonucleotide, which is not able to modify significantly the TRH response. This excludes that, unless the transduction cascade to Ca-dependent currents is different for both types of receptor, the antisense oligonucleotides are affecting the signaling from PLC to native Cl channels.

Coupling of native and exogenous receptors to PLC via G(o) has been previously reported in oocytes(27, 28, 40) . This includes receptors that, like the alpha-adrenergic receptor, use a pertussis toxin-insensitive pathway to activate PLC in its native environment(27) . Since both TRH and alpha-adrenergic receptors are able to activate PLC via G(q) in other cells (14, 15, 43) , it could be expected that they would use also the same transducer in the oocyte system. However, the Xenopus G(o) alpha-subunit is involved in the alpha(1)-adrenergic response in oocytes(27) , but G(s) and not G(o) is transducing the TRH response. This happened even though 5-HT1(c) receptor-mediated responses, also shown to depend on G(o) in oocytes(28) , are effectively blocked by the anti-alpha(o) oligonucleotides. This demonstrates that failure to antagonize TRH responses in anti-alpha(o)-injected oocytes is not due to inability of the oligonucleotide to deplete alpha(o). In fact, a recently cloned new form of PLC (44) seems to be involved in oocyte responses mediated by pertussis toxin-sensitive G(o) proteins. However, we have found that the TRH-induced response is significantly reduced by two antisense oligonucleotides recognizing mammalian PLC-beta1 or PLC-beta2 mRNA sequences, which are not present in the newly reported, G(o)-regulated, oocyte PLC. (^2)It is tempting to speculate that, besides the PLC activated by G(o) involved in serotonin and alpha-adrenergic responses, additional PLCs are present in the oocyte that mediate the previously described PTX-insensitive responses (e.g.m(3)-muscarinic responses)(25) . If some of these responses are also ablated by depletion of the alpha(s)-subunits remains to be established.

The reason for the apparently paradoxical dependence of TRH responses on the presence of alpha(s) polypeptides is not known. In addition to its classical role as component of the stimulatory cascade to adenylate cyclase, G(s) alpha-subunits have been shown to transduce stimulatory effects to Ca channels and inhibitory effects to Na channels and plasma membrane Ca-ATPase (see (45) and Refs. therein). In GH(3) cells, TRH receptors seem to couple not only to G(q) to activate PLC(14, 15) , but also to G(s) to inhibit inwardly rectifying K currents(22, 23) . Thus, it is possible that if G(q) proteins are not abundant in the oocyte or the oocyte G(q) is not similar to its pituitary counterpart, effective coupling of TRH receptors to G(s) can be enhanced, much the same as coupling to G(o) of alpha-adrenergic receptors, which also interact with G(q) in other cell types.

It is important to note that our results do not allow us to discriminate between a direct effect of alpha(s)-subunits and an indirect effect of a particular combination of beta-subunits associated with alpha(s) as activators of the oocyte PLC. However, it is clear that the native G(s) in Xenopus oocytes is involved in coupling of the expressed TRH receptor to the PLC pathway. Further studies on the determinants of this coupling using the oocyte system as a model could help in understanding the reasons for the choice of transducer by the apparently promiscuous TRH receptor in pituitary cells.


FOOTNOTES

*
This work was supported by Grant PB90-0789 from Comisión Interministerial de Ciencia y Tecnología of Spain and an Incorporation Contract from Spanish Ministerio de Educacíon y Ciencia (to L. A. P.). 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.

§
To whom correspondence should be addressed. Tel.: 34-8-5103565; Fax: 34-8-5103534.

Recipient of a fellowship from Fundacíon para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnologia of Asturias (Spain).

(^1)
The abbreviations used are: G-protein, guanine nucleotide-binding protein; TRH, thyrotropin-releasing hormone; PLC, phospholipase C; CTX, cholera toxin; PTX, pertussis toxin; IP(3), inositol 1,4,5-trisphosphate; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction.

(^2)
P. de la Peña and F. Barros, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. H. Lübbert (Sandoz Pharmaceuticals Ltd., Basel, Switzerland) for the 5-HT1(c) receptor clone, Drs. G. Milligan (University of Glasgow, Scotland) and S. Gutkind (National Institutes of Health, Bethesda, MD) for the anti-alpha(s) antibodies, Dr. J. M. Martín for invaluable assistance with Western blots, and Drs. L. M. Sierra, M. A. Comendador, and J. Alvarez-Riera for help with statistical analysis.


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