(Received for publication, July 19, 1994; and in revised form, November 18, 1994)
From the
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 - and
-subunits. Injection of
antisenses which target mRNA sequences shared by several G-protein
or
polypeptides effectively blocked
Ca
-dependent Cl
currents induced by
TRH through activation of phospholipase C. Three different
-specific antisense oligonucleotides complementary to
sequences located in different positions along the coding region of the
protein mRNA were highly effective in inhibiting
TRH-induced responses. Anti-
, -
,
-
, or -
oligonucleotides were not
able to modify the TRH-evoked response. In contrast,
anti-
, but not anti-
,
oligonucleotides blocked the response to serotonin in oocytes injected
with serotonin 5-HT1
receptor cRNA. Cholera toxin catalyzed
the [
P]ADP-ribosylation of 40-42- and
50-52-kDa proteins in GH
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-
-injected oocytes. Amplification of oocyte RNA in
a polymerase chain reaction system and sequencing of the amplified
products demonstrated that anti-
oligonucleotides
selectively recognize the message for the Xenopus
polypeptide. It is concluded that G
, but not
G
, G
, G
, or G
, 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.
Seven G-protein-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
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
and/or
G
(14, 15) . The PLC-mediated stimulation
of IP
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
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
or a
G
-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
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
family
in their native environment display a PTX-sensitive response via
G
in the oocyte system(24, 27) . The
-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
-subunits of G-proteins. Our results indicate that
G
, but not G
, G
, G
, or
G
, couples TRH receptors expressed in oocytes to activation
of PLC and subsequent IP
-dependent stimulation of
Ca
-dependent Cl
currents.
The serotonin 5-HT1 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
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
, they were dried under a N
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
maximal response to develop more slowly than TRH receptor
expression. Subsequently, 5-HT1
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% 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-
oligonucleotides, the
anti-
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-
2, or in the presence of anti-
2
plus the sense counterpart of anti-
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
-subunit gene sequence previously
reported(30) .
GH cells (ATCC CCL 82.1) were
grown and harvested as described previously(12, 29) .
For isolation of cell membranes, either 7
10
GH
cells or 150 defolliculated oocytes (12, 29) were homogenized in buffer A (150 mM NaCl, 10 mM MgCl
, 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
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
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, 150 mM KCl, and 4 µM [
-
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
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.
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.
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 - and
-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
-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
cells. In contrast, the
response to TRH was almost abolished in oocytes injected with an
antisense oligonucleotide (
) which targets mRNA of
all known G-protein
-polypeptides ( Fig. 1and Fig. 2). Similar results were obtained with an antisense
oligonucleotide (
, see Fig. 2) directed
against G-protein
- to
-subunit
sequences. This indicates that
-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
-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
-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- and
anti-
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
-subunit, oocytes were injected with antisense oligonucleotides
that can specifically hybridize with the mRNA of one particular
-subtype. Fig. 1shows that only an oligonucleotide
directed against
-subunits, but not those directed
against
-,
-, or Xenopus
-subunits, is able to reduce the TRH-evoked
response. Previous work with exogenous and native receptors indicated
that G
might serve as the signal transducer to PLC in
oocytes(27, 40) . Furthermore, G
has been
recognized as the G-protein coupling the TRH receptor to PLC in
GH
cells(14, 15) . The antisense
oligonucleotide against
is based in a sequence found
in the
-subunit of the G
-type protein of Xenopus oocytes(31) . However, similar information is not
available for oocyte G-proteins of the G
-type. To minimize
the possibility that the lack of effect of anti-
oligonucleotides is due to choice of a sequence not present in
the hypothetical
of the oocyte, three different
antisense oligonucleotides containing sequences complementary to those
of the cloned
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
and
mRNAs. It is interesting that the anti-
oligonucleotide can hybridize perfectly with the two
-subunits cloned from Xenopus (
1 and
3)(30) , and with
human
2(33) , but it is also complementary
with nucleotides 667-684 of Xenopus
mRNA, except for two mismatches in positions 676 and
679(31) . This adds further support to our conclusion that
G
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-type, but not through G
, G
,
G
, or G
. To prevent the possibility that the
anti-
oligonucleotides could act by knocking out a
protein distinct from the
polypeptide, three
different
-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
protein mRNA sequence either close to
the NH
-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-
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
-subunit, but not that directed against
, 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 serotonin receptors was largely blocked by the
anti-
oligonucleotide. On the other hand, the
serotonin-induced responses were significantly reduced by the antisense
against the
polypeptide message. However, neither the
anti-
hybridizing with the portion of the
-mRNA near the carboxyl terminus of the protein (Fig. 3), nor that to the sequences near the amino terminus of
the
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-
selectively blocking serotonin responses and the anti-
oligonucleotides exclusively minimizing the TRH-evoked responses.
Figure 3:
Effect of antisense oligonucleotides to
mRNA for different -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
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
-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
-subunits inhibit TRH responses by depletion of these
polypeptides would come from the demonstration that
is knocked out of the cell by injection of the oligonucleotides.
Preliminary experiments performed with a specific anti-
antibody against the COOH-terminal portion of
(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
cell
membranes (data not shown). To detect the presence of the
polypeptides by other means, membranes were treated with
[
-
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
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-
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
(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 cell membranes: effect of microinjection
of anti-
oligonucleotides. Membranes from oocytes (500
µg protein, lanes 1 to 4) or GH
cells
(300 µg, lanes 5 and 6) were incubated in the
presence of [
-
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- oligonucleotides selectively recognize the sequences of the
messages for these polypeptides, the
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-
2 located in the center of the
coding region and 2) anti-
2 plus the sense
counterpart of the anti-
1 located near the amino
terminus of the
coding region. In both cases, a
single prominent band of the size expected from separation between the
oligonucleotide sequences in the Xenopus
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
messages are
detected, only
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
mRNA (not shown; see (30) ).
The data presented here indicate that G, but not
G
, G
, G
, or G
, 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
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
or
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-
and anti-
oligonucleotides, the effect is direct
and specific, since only TRH and not serotonin responses are blocked by
antisenses against
polypeptides. In contrast, only
serotonin-induced responses are inhibited by the anti-
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 has been previously
reported in oocytes(27, 28, 40) . This
includes receptors that, like the
-adrenergic
receptor, use a pertussis toxin-insensitive pathway to activate PLC in
its native environment(27) . Since both TRH and
-adrenergic receptors are able to activate PLC via
G
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
-subunit is involved in the
-adrenergic
response in oocytes(27) , but G
and not G
is transducing the TRH response. This happened even though
5-HT1
receptor-mediated responses, also shown to depend on
G
in oocytes(28) , are effectively blocked by the
anti-
oligonucleotides. This demonstrates that failure
to antagonize TRH responses in anti-
-injected oocytes
is not due to inability of the oligonucleotide to deplete
. In fact, a recently cloned new form of PLC (44) seems to be involved in oocyte responses mediated by
pertussis toxin-sensitive G
proteins. However, we have
found that the TRH-induced response is significantly reduced by two
antisense oligonucleotides recognizing mammalian PLC-
1 or
PLC-
2 mRNA sequences, which are not present in the newly reported,
G
-regulated, oocyte PLC. (
)It is tempting to
speculate that, besides the PLC activated by G
involved in
serotonin and
-adrenergic responses, additional PLCs are present
in the oocyte that mediate the previously described PTX-insensitive
responses (e.g.m
-muscarinic
responses)(25) . If some of these responses are also ablated by
depletion of the
-subunits remains to be established.
The reason for the apparently paradoxical dependence of TRH
responses on the presence of polypeptides is not
known. In addition to its classical role as component of the
stimulatory cascade to adenylate cyclase, G
-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
cells, TRH receptors seem to couple
not only to G
to activate PLC(14, 15) ,
but also to G
to inhibit inwardly rectifying K
currents(22, 23) . Thus, it is possible that if
G
proteins are not abundant in the oocyte or the oocyte
G
is not similar to its pituitary counterpart, effective
coupling of TRH receptors to G
can be enhanced, much the
same as coupling to G
of
-adrenergic
receptors, which also interact with G
in other cell types.
It is important to note that our results do not allow us to
discriminate between a direct effect of -subunits and
an indirect effect of a particular combination of
-subunits
associated with
as activators of the oocyte PLC.
However, it is clear that the native G
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.