From the Institute of Physiology, Czech Academy of
Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic, the
§ Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and
the ¶ Medical Research Council Reproductive Biology Unit,
Edinburgh, EH3 9EW Scotland, United Kingdom
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ABSTRACT |
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Using a combination of confocal
immunofluorescence microscopy and subcellular fractionation, we
demonstrate for the first time active internalization, trafficking, and
down-regulation of a G protein subunit subsequent to agonist
occupation of a receptor. This proceeds on a much slower time scale
than internalization of the corresponding receptor. In intact E2M11
HEK293 cells that express high levels of murine
G11
and the rat thyrotropin-releasing hormone
(TRH) receptor, the immunofluorescence signal of G11
was
restricted almost exclusively to the plasma membrane. Exposure to TRH
(10 µM) resulted first in partial relocation of
G11
to discrete, segregated patches within the plasma
membrane (10-60 min). Further exposure to TRH caused internalization
of G11
to discrete, punctate, intracellular bodies (2-4
h) and subsequently to a virtually complete loss of G11
from plasma membranes and the cells (8-16 h). Short-term treatment
with TRH followed by wash-out of the ligand allowed G11
immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G11
was centered
on plasma membranes, and this was not altered by up to 1-2 h of
incubation with TRH. Further exposure to TRH (2-4 h) resulted in
transfer of a significant portion of G11
to
light-vesicular and cytosol fractions. At longer time intervals (4-16
h), an overall decrease in G11
content was observed.
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INTRODUCTION |
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The capacity of agonist ligands to cause internalization of G protein-coupled receptors (GPCRs)1 has been actively studied for a number of years. Studies using fluorescent ligands, antireceptor antibodies, antibodies to epitope tags introduced into the cDNA sequences of GPCRs, and a GPCR-Green fluorescent protein fusion protein have demonstrated many of the mechanisms that contribute to such processes (1-14). Despite the central role of G proteins in transducing GPCR-mediated signal transduction, potential agonist-induced subcellular redistribution of heterotrimeric G proteins has not been widely examined. This is despite a large range of studies indicating that sustained treatment of cells with agonists at GPCRs can cause a reduction in total cellular levels of the G protein(s) activated by the receptor (15-23)
We have recently produced a clonal cell line derived from human
embryonic kidney, HEK, 293 cells, in which both the long isoform of the
rat thyrotropin-releasing hormone (TRH) receptor and murine G11 are expressed to high levels (24). The principal
mechanism of action of TRH receptors is via activation of
phosphoinositidase C in a pertussis toxin-insensitive manner, a process
which proceeds via interaction of the receptor with G proteins of the
Gq/G11 family (23-26). Using this cell line
and fractionation of cell homogenate on sucrose density gradient, we
were able to demonstrate that long-term agonist treatment results in
subcellular redistribution and down-regulation of this G protein
(24).
Herein we demonstrate the active internalization and trafficking of a phosphoinositidase C-linked G protein in response to agonist activation and that this proceeds on a much slower time scale than internalization of the TRH receptor.
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EXPERIMENTAL PROCEDURES |
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Materials
[3H]TRH (74 Ci/mmol) was from NEN Life Science Products (NET-577). Goat anti-rabbit IgG FITC conjugate (T-6005), goat anti-mouse IgG TRITC conjugate (T-5393) and phenylarsine oxide were from Sigma, sucrose (Aristar grade) was obtained from BDH. HEK-293 cells were obtained from the American Tissue Type Collection.
Methods
Generation and Isolation of Clone E2M11
Clone E2M11 of HEK 293 cells that stably express high levels of
the rat TRH receptor and murine G11 was prepared as
described before (24). Briefly, a full-length rat (long isoform) TRH
receptor cDNA (2.2 kilobases) (27) was subcloned into the
eukaryotic expression vector pcDNA1 (Invitrogen) which is driven by
the cytomegalovirus (CMV) promoter. HEK-293 cells were co-transfected
with linearized pcDNA1/TRH receptor and pSP neo (Invitrogen) using
Lipofectin reagent (Life Technologies, Inc., Paisley, Strathclyde, UK).
Resultant Geneticin-resistant clones were picked, and TRH
receptor-containing clones were identified as those in which TRH
produced a rise in total inositol phosphate production. Expression of
the TRH receptor in membranes from these clones was assessed by the
specific binding of [3H]TRH. Clone E2, which expresses
some 14 pmol of the receptor/mg of membrane protein (23), was selected
for further transfection with plasmid pCMV, into which a cDNA
encoding murine G11
was inserted, and with the plasmid
pBABE hygro, which allows expression of resistance to the antibiotic
hygromycin B. Clones were selected on the basis of resistance to
hygromycin B, and the continued expression of the TRH receptor and
novel expression of murine G11
were examined (24).
Cell Growth Clone E2M11 cells were grown in tissue culture in Dulbecco's modified Eagle's medium (DMEM) containing 5% (v/v) newborn calf serum and were maintained in the presence of Geneticin sulfate (800 µg/ml) and hygromycin B (200 µg/ml). Prior to confluency, they were split 1:5 into fresh tissue culture flasks or were harvested.
Production of Antisera
Antisera 452 and CQ are rabbit polyclonal antipeptide antisera
that were raised in rabbits obtained from VELAZ, Prague (452) and New
Zealand White rabbits (CQ). Rabbits were immunized with a synthetic
peptide QLNLKEYNLV (C-terminal decapeptide conserved between
Gq and G11
) (28) conjugated by the
glutaraldehyde method to keyhole limpet hemocyanin (Calbiochem) as
described before (29).
Immunofluorescence and Confocal Microscopy
E2M11 cells were grown on ethanol-sterilized glass coverslips.
Before confluency, they were pre-treated in the absence or presence of
10 µM TRH for various times. The cells were washed three
times with PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM
Na2HPO4, pH 7.2) (pH 7.5) at 37 °C and fixed
with methanol for 5 min at 20 °C and with acetone for 1 min at
20 °C. Cell membranes were permeabilized with 0.15% (w/v) Triton
X-100 in PBS at room temperature (Rt) and washed
three times with PBS at Rt for 5 min.
G11
and Gq
are expressed to very similar
levels by HEK 293 cells (23), and these are both activated by the TRH receptor in E2M11 cells (23, 24). The level of overexpression of
G11
in E2M11 cells (24) means that although the
antiserum used should identify Gq
and G11
equally, at least 90% of the immunological signal is derived from the
stably introduced murine G11
. The G11
was
labeled with antiserum 452 (details above) for 1 h at 37 °C
with a 1:500 dilution of the antiserum in PBS with 0.2% (w/v) BSA. In
the cases in which double labeling procedures were employed, vimentin
and
-tubulin were detected by mixing primary mouse monoclonal
antibodies (1:500 dilution) directed against these proteins with the
anti-G11
/Gq
antiserum. After primary
labeling, coverslips were washed three times with PBS at
Rt for 5 min and then incubated with secondary
antibodies (goat anti-rabbit IgG whole molecule conjugated to FITC and
goat anti-mouse IgG whole molecule conjugated to TRITC) diluted 1:100
in PBS for 1 h at 37 °C. Finally, the coverslips were washed
three times with PBS at Rt for 5 min, covered
with 40% (v/v) glycerol in PBS, and laid on slides. Detection was
performed using a Bio-Rad MRC 600 confocal laser scanning microscope.
FITC (green color fluorescence) and TRITC (red color fluorescence) were
exited at 468 and 567 nm, respectively.
Subcellular Fractionation on Sucrose Density Gradients
E2M11 HEK-293 cells (3 × 75 cm2 flasks per
sample) were harvested by low speed centrifugation, washed twice in 140 mM NaCl, 20 mM Tris-HCl (pH 7.4), 3 mM MgCl2, and 1 mM EDTA, and
homogenized in 2.5 ml of 20 mM Tris-HCl (pH 7.4), 3 mM MgCl2, and 1 mM EDTA using a
Potter-Elvehjem (Teflon-glass homogenizer). Two ml of homogenate (after
freezing at 80 °C for at least 1 h) was layered on the top of
a discontinuous sucrose density gradient consisting of (from top to
bottom) 19, 23, 27, 31, 35 (all 5 ml), and 43% (10 ml) (all w/w)
sucrose, 20 mM Tris-HCl (pH 8.0), 3 mM
MgCl2, and 1 mM EDTA. The gradient was
centrifuged for 30 min at 27,000 rpm in a Beckman SW 28 rotor and
fractionated manually from the meniscus (fractions 1-7, 5 ml each).
The first 5 ml (fraction 1) represented an interphase between the
overlaid homogenate and 19% (w/w) sucrose. To separate the low density
membranes (light-vesicles) from cytosol, fraction 1 was diluted 1:1
with redistilled water, centrifuged for 120 min at 50,000 rpm in
Beckman Ti-50 rotor, and the resulting pellet (fraction 1P) was
suspended by rehomogenization in 0.3 ml of 20 mM Tris-HCl
(pH 7.4), 3 mM MgCl2, and 1 mM EDTA (TME buffer). The supernatant (fraction 1S) represented the cytosol fraction. The gradient fractions were frozen at
80 °C until use (see Ref. 24 for further details).
Immunoblotting of Sucrose Density Gradient Fractions Sucrose density gradient fractions were precipitated with TCA (6% w/v, 1 h on ice), and the precipitate was solubilized in Laemmli buffer. Standard (10% w/v acrylamide, 0.26% w/v bisacrylamide) or urea (12.5% w/v acrylamide, 0.0625% w/v bisacrylamide containing 6 M urea) SDS-PAGE was carried out overnight at 60 V (standard gel) or 100 V (urea gel) as described before (24). Molecular mass determinations were based on prestained molecular mass markers (Sigma, SDS 7B).
After SDS-PAGE, proteins were transferred to nitrocellulose and blocked for 1 h in 4% (w/v) BSA in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl). The 452 antiserum was added in 1% (w/v) BSA in TBS containing (0.2% w/v) Tween 20 and incubated for at least 2 h. The primary antibody was then removed and the blot washed extensively in TBS with 0.2% (v/v) Tween 20. Secondary antiserum (goat anti-rabbit IgG conjugated with alkaline phosphatase) was applied for 1 h, and after three 10-min washes, the blots were developed in TNM buffer (100 mM Tris-HCl, pH 9.0, 100 mM NaCl, and 5 mM MgCl2) containing 5-bromo-4-chloro-3-indolyl phosphate (100 µg/ml) and nitro blue tetrazolium (200 µg/ml). The developed blots were scanned with a Bio-Rad GS 670 imaging densitometer to enable quantification of the immunoblots.Radioligand Binding Assays of TRH Receptor Internalization
Protocol I, Direct Binding Assay-- [3H]TRH binding and internalization was measured according to Hinkle and Kinsella (30). E2M11 cells were incubated with 10 nM [3H]TRH in serum-free DMEM medium for 0-120 min at 37 °C, the binding medium was then aspirated, and the cells washed three times with ice-cold 0.15 M NaCl to remove unbound [3H]TRH. Total binding was measured after solubilizing the cells with 1% SDS and 1% Triton X-100. When the internalized (acid-resistant) pool of TRH receptors was to be determined, the cells were incubated for 1 min with ice-cold 0.5 M NaCl, 0.2 M acetic acid, pH 2.5 (acid/salt solution), and washed once with 0.15 M NaCl. The radioactivity was determined by liquid scintillation. Nonspecific binding was assessed in parallel dishes which contained a 1000-fold molar excess of unlabeled TRH and represented less than 5% of total binding.
Protocol II, Binding Assay after Preincubation with Unlabeled TRH-- This was done according to Petrou et al. (31). After preincubation with 10 µM TRH for 1-120 min at 37 °C, E2M11 cells were rinsed once with 0.15 M NaCl, incubated for 1 min with acid/salt solution, washed twice with 0.15 M NaCl (everything on ice), and incubated further with 10 nM [3H]TRH for 90 min at 0 °C. The cells were subsequently washed three times with ice-cold 0.15 M NaCl, solubilized with 1% SDS and 1% Triton X-100, and counted for bound radioactivity.
Inositol Phosphate Assay E2M11 cells were split into 24-well plates. The next day they were labeled with [3H]inositol (1 µCi/ml) in inositol-free DMEM supplemented with 2% (v/v) dialyzed new born calf serum and 1% (w/v) glutamate for 24 h. On the day of experiment, cells were washed two times with Krebs-Ringer-Hepes buffer (KRH/LiCl: 120 mM NaCl, 5 mM KCl, 10 mM LiCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 20 mM Hepes, 1.2 mM Na2HPO4, 10 mM glucose, 0.1% BSA, pH 7.4), incubated for 10 min with KRH/LiCl, and stimulation was performed with 10 µM TRH in the same buffer for 10 min. All manipulations were done at 37 °C. Reactions were stopped by aspiration of KRH/LiCl buffer with TRH, and cells were lysed by 0.75 ml of 20 mM formic acid on ice (30 min). Supernatant fractions were loaded on Dowex (Sigma, Dowex 1 × 8-200) columns, followed by immediate addition of 3 ml of 50 mM NH4OH ([3H]inositol fraction). The columns were then washed with 4 ml of 40 mM ammonium formate, followed by 5 ml of 2 M ammonium formate ([3H]inositol phosphate (IP) fraction). Data were presented as the quotient of [3H]IP divided by [3H]inositol plus [3H]IP.
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RESULTS |
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Internalization of G11 after Long-term Agonist
Treatment Detected by Confocal Fluorescence Microscopy--
E2M11
cells grown on glass coverslips were prepared for immunofluorescence
and confocal microscopy as described under "Experimental Procedures," and G11
was detected using antiserum 452 which identifies the extreme C-terminal decapeptide of this G protein.
The immunofluorescence signal was essentially entirely restricted to
the plasma membranes of E2M11 cells, forming a sharp homogeneous
barrier (Fig. 1a). Dual
labeling with an antibody directed against
-tubulin indicated this
cytoskeletal protein to be distributed within the cytoplasm and that
the cells had large distinct nuclei (Fig. 1a). Addition and
maintenance of TRH (10 µM, 16 h) prior to
preparation of the cells for the immunofluorescence studies resulted in
a vastly different cellular distribution of the G protein (Fig.
1b). Very little of the G11
protein could be
detected then at the plasma membrane, whereas the vast majority of the
immunodetectable G11
had a punctate, intracellular
location. By contrast, the cytoplasmic distribution pattern of
-tubulin was unchanged (Fig. 1b).
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Time Course of Internalization of G11 Determined by
Confocal Fluorescence Microscopy--
E2M11 cells grown on glass
coverslips were incubated with TRH (10 µM) for increasing
time intervals (10, 30, and 60 min, 2, 4, and 16 h), and samples
were prepared for immunofluorescence and confocal microscopy (Fig.
2). Within 10-60 min of addition of TRH
(10 µM), the G11
immunofluorescence signal
(green) was restricted exclusively to plasma membranes;
however, a fraction of the G11
immunofluorescence
appeared to cluster to discrete segregated patches of the plasma
membrane (Fig. 2, b-d) compared with the largely homogeneous
plasma membrane distribution prior to addition of TRH (Fig.
2a). Further maintenance of TRH (2 h) resulted in a distinct
loss of plasma membrane-associated G11
with the marked
appearance of distinct, punctate patterns of staining with
intracellular location (Fig. 2e). Subsequently (4 h), plasma membrane-localized G11
immunofluorescence all but
disappeared (Fig. 2f) and was largely confined to the areas
of punctate, intracellular staining, and eventually (16 h) much of the
total cellular G11
immunofluorescence was depleted (Fig.
2g), consistent with the major immunodepletion previously
recorded in immunoblotting studies (24).
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Time Course of Redistribution of G11 Analyzed by
Sucrose Density Gradient Centrifugation--
E2M11 cells were
untreated or treated with TRH (10 µM) for the same time
intervals (10, 30, and 60 min and 2, 4, and 16 h) as described
above for immunofluorescence analysis. The control and TRH-treated
cells were collected by low speed centrifugation and then homogenized,
and subcellular fractionation on sucrose density gradients was
performed as described under "Experimental Procedures" and in Ref.
24. The cytosol (1S), light vesicular (1P and 2), plasma membrane (3 and 4), mitochondrial (5 and 6), and nuclear (8) fractions were
precipitated by 6% TCA and resolved by 12.5% (w/v) acrylamide
SDS-PAGE containing 6 M urea, and G11
was
detected by immunoblotting with 452 antiserum. The immunoblots were
quantified by densitometric scanning.
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Time Course of TRH Receptor Internalization Measured by Radioligand
Binding--
To compare the time course of G11 and TRH
receptor internalization, the kinetics of [3H]TRH binding
were measured in acid-washed E2M11 cells according to both Hinkle and
Kinsella (30) and Petrou et al. (31). According to protocol
I, [3H]TRH binding was measured by direct radioligand
binding assays performed with untreated E2M11 cells (Fig.
5a); in protocol II, E2M11
cells were preincubated with unlabeled TRH to saturate the internal
pool of TRH receptors before initiation of the internalization process.
The remaining surface (membrane)-associated receptors were then
determined by the [3H]TRH binding reaction (Fig.
5b). Both approaches produced virtually the same results. In
untreated cells (protocol I), about 73% of receptors were transferred
to the acid-resistant (internalized) pool within 60 min of agonist
exposure (half-time ~ 29 min). The cells pre-treated with
unlabeled TRH exhibited very similar results
after 1 h of TRH
exposure, about 70% of binding sites were lost from the surface
(half-time ~ 25 min). After prolonged exposure (up to 16 h)
of E2M11 cells to the hormone, surface membrane-associated receptors
were almost completely depleted. Incubation of E2M11 cells with 10 µM TRH for 0.5 h followed by wash-out of TRH and further incubation for 3 h without TRH resulted in recovery of 84% of receptors back to the surface (data not shown).
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The Concentration Dependence of TRH-induced Redistribution of
G11--
The agonist concentrations used in these
(Figs. 1-4) as well as previous experiments to demonstrate
down-regulation and/or redistribution of G proteins (for review, see
Ref. 32), undoubtedly represents a supramaximal dose when compared with
physiological hormone concentrations. Therefore, E2M11 cells were
treated with increasing concentrations of TRH (0.1 nM-10
µM, 16 h), and the membrane-cytosol balance of
G11
was measured after centrifugation of the cell
homogenate for 1 h at 250,000 × g (Fig.
6). The first decrease in membrane-bound
G11
was observed with 1 nM TRH, with half-maximal response at 5 and 3 nM TRH for membrane
(decrease in content) and cytosolic (increase in content)
G11
, respectively. Distribution of G11
was not substantially altered by further increase of TRH concentration
from 0.1 to 10 µM (Fig. 6).
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Recovery of Plasma Membrane-associated G11 following
Removal of TRH and Effect of Cytoskeletal Inhibitors--
In
preliminary experiments to determine the minimum time of TRH exposure
sufficient to induce marked internalization of G11
, E2M11 cells were treated with TRH (10 µM) for 5, 10, 20, and 30 min, washed three times with DMEM to remove the agonist and
further incubated for 3 h without TRH. The combination of 0.5 h TRH(+) and 3 h TRH(-) incubation periods was found to be
sufficient to induce marked internalization of G11
(Fig.
7b) when compared with control
signal (Fig. 7a). Prolongation of the incubation period
without TRH (after constant 0.5 h TRH exposure) to 5 h (Fig.
7c), 8 h (Fig. 7d), and 12 h (Fig.
7e) was associated with a step-wise increase of
G11
immunofluorescence such that after 12 h, the
plasma membrane was again richly endowed with a uniform population of
G11
. Further prolongation of recovery period up to
16 h did not further increase the plasma membrane-associated fluorescence signal of G11
(Fig. 7f).
Recovery of G11
immunofluorescence signal obtained after
0.5 h TRH(+) and 8 h TRH(-) incubation periods was
significantly blocked by 100 µM cycloheximide (Fig.
7g). Therefore, recovery of plasma membrane
G11
seems to require de novo protein synthesis. The cytoskeletal inhibitors nocodazole (microtubules), cytochalasin B (microfilaments), and colcemide (microtubules and intermediate filaments) exhibited clear inhibitory effects on internalization of G11
in response to TRH (data not
shown).
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Inositol Phosphate Generation-- E2M11 cells prelabeled with [3H]inositol (1 µCi/ml) were incubated with TRH (10 µM) for 0, 0.5, 1, 2, 8, and 16 h, and production of [3H]inositol phosphates was subsequently measured over a 10-min period following wash-out of TRH and reexposure to TRH (10 µM). In the absence of preexposure to the ligand, addition of TRH resulted in a 3.9 ± 0.1 (n = 2)-fold stimulation of [3H]IP generation (Table I). However, with as little as 0.5 h preexposure to TRH, fresh ligand was unable to cause any stimulation above basal levels, demonstrating a complete desensitization of the response. Interestingly, similar nonresponsiveness to TRH was observed in E2M11 cells preincubated with 10 µM TRH for 0.5 h, which were subsequently allowed to recover for 3 h in medium without the hormone (Table II).
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DISCUSSION |
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Continuous exposure of cells to agonists often causes a rapid waning of the stimulated response, an effect which is termed desensitization, refractoriness, tolerance, or tachyphylaxis. Agonist-promoted desensitization appears to be a general homeostatic mechanism by which target cells modulate responsiveness to agents acting on cell surface receptors, and molecular mechanism(s) of this process may be located at any step of the signal transmission pathway which is initiated by a receptor and proceeds into the cells. Among these, receptor-based regulations of desensitization have been studied most intensively (see Refs. 33-35 for reviews).
Agonist-induced phosphorylation of GPCRs and subsequent subcellular
redistribution, sequestration, internalization, recycling, and finally
down-regulation have frequently been found to be directly related to
desensitization of hormone action. By contrast to GPCRs, relatively
little attention has been devoted to the role of the subcellular
redistribution of heterotrimeric G proteins in such phenomena. However,
isoprenaline-stimulation of 2-adrenergic receptors in
S49 lymphoma cells redistributed the Gs
subunits from
membranes to cytosol (18). Such agonist-induced membrane-cytosol shifts
of the
subunits of heterotrimeric G proteins have also been
demonstrated for other systems including the prostacyclin receptor and
Gs
(36) and somatostatin receptor and Gi2
(37). It has also been shown that agonist exposure can induce a
transfer of G
subunits from plasma membranes to light vesicular
membrane fractions which were recovered either by sucrose density
gradient or differential centrifugation (see Ref. 24 for details).
Furthermore, Crouch (38) has indicated a capacity of mitogen-activated
Gi
to be redistributed from the plasma membrane to the
nucleus and for adrenaline to cause a redistribution of
Gi2
in platelets (39). Direct examination of the
dynamics of internalization of heterotrimeric G protein induced by an
agonist, however, has been missing.
Although not examined as widely as regulation of GPCR levels,
agonist-mediated down-regulation of G protein subunits has been
observed in a number of systems (see Ref. 32 for review). In general,
down-regulation of G proteins requires extended periods of cellular
exposure to the agonist, is restricted to the G protein(s) expected to
be activated by the agonist-GPCR complex, and requires the cell or
tissue to express relatively high levels of the GPCR. This last point
can be understood on the basis that the cellular levels of G proteins
are usually far higher than the levels of any particular GPCR. Thus,
even when considering the capacity of an agonist-occupied GPCR to
catalytically activate the G protein population, the fraction of the
cellular pool of a specific G protein which becomes activated by a GPCR
is likely to be small unless GPCR levels are high. In the studies which
have examined the mechanisms responsible for agonist-mediated reduction
in cellular G protein levels, an enhanced rate of degradation of the
cognate G protein, but not of other cellular G proteins which are not activated by that agonist, has routinely been observed (18-22). By
contrast, relatively few studies have provided evidence for decreased
rates of G protein synthesis or reduction in mRNA levels (see Ref.
32 for details).
Even in the case of widely studied GPCRs, it has not been clear whether
sequestration is or is not a prerequisite for agonist-induced down-regulation. The process of sequestration, in which receptors remain detectable by lipophilic receptor ligands but become
increasingly inaccessible to hydrophilic, membrane-impermeable ligands
(40-41), was proposed to represent internalization of receptors into
an intracellular compartment and to serve as a prerequisite for
down-regulation. Direct demonstration of dynamic internalization and
recycling pathways by confocal fluorescence microscopy was first
achieved for 2-adrenergic receptors (1-2, 33) and has
subsequently been replicated for a variety of other receptors (3-14).
As such, in the current studies, we have utilized a combination of cell biology using confocal fluorescence microscopy and biochemical analyses
of the subcellular localization of the G protein G11
following addition of TRH to cells expressing both the TRH receptor and
G11
. Using these approaches, we have demonstrated
sequestration, internalization, subcellular redistribution, and finally
down-regulation of G11
protein as subsequent steps of
TRH action and have observed good agreement of results between these
two widely different methodological approaches. These observations
provide new and original insights into the role of G protein regulation
which are likely to contribute to long-term mechanisms of
desensitization of hormone effect. Desensitization of the TRH receptor
was clearly demonstrated in our studies by measurement of inositol
phosphate generation in E2M11 cells that were pre-treated with TRH for
various time intervals (0.5-16 h). 0.5 h was sufficient to induce
complete desensitization of the inositol phosphate response to
subsequent addition of TRH (Table I).
The mechanisms of G protein internalization and the extent to which
internalization of G11 proceeds via
clathrin-dependent or other endocytotic pathways remains to
be examined in the future. Treatment with hypertonic sucrose that has
been used to interfere with clathrin-mediated endocytosis and
internalization of TRH receptors (31, 42) appeared to produce a
nonspecific alteration in the pattern of G11
immunofluorescence (data not shown). Clearly, G11
internalization does not accompany the TRH receptor along its way into
the cell interior as more than 70% of receptors were transferred into
an acid wash-resistant, intracellular pool within the first 60 min of
incubation with agonist (Fig. 5). At this time, no significant level of
G11
was internalized in response to addition of TRH.
These findings clearly indicate an uncoupling of G11
from the TRH receptor in the course of the internalization process. The
possibility that clathrin-dependent endocytosis is not
involved in G11
internalization, therefore, has to be
considered. Multiple G protein
subunits have been identified in
caveolae (43-45), and it has been shown that
-adrenergic receptors
redistribute to caveolin-rich membrane domains in response to agonist
stimulation (46-47). A similar scenario has been reported following
addition of a muscarinic acetylcholine receptor agonist to cardiac
myocytes, which resulted in movement of a proportion of the
m2 muscarinic acetylcholine receptor population to a
caveolar location and the subsequent interaction of the receptor with
caveolin-3, a muscle-specific form of caveolin (48).
Although there has been an ongoing discussion as to whether
-adrenergic receptor is internalized via clathrin-coated pits or
caveolae (49-50), which serves as a good example of how difficult it
has been to decide between these two alternative pathways of endocytosis, recent studies using GTP binding mutants of dynamin, a
GTPase which plays a key role in pinching off of clathrin-coated endocytic vesicles from the plasma membrane, have shown this to interfere with agonist-induced sequestration of the
2-adrenoreceptor when expressed in HEK 293 cells but not
with internalization of the angiotensin II type 1A receptor in the same
cells (5), arguing both for a specific role of clathrin-coated vesicles
in the regulation of the
2-adrenoreceptor and that this
is not a pathway used universally by GPCRs. Analysis at the
ultrastructural level using immunogold electron microscopy, such as
that recently achieved for the cholecystokinin receptor (51), may, in
time, provide an alternative approach to examine different membrane components in a single type of endocytic vesicle.
The results generated in this work display a host of other interesting
features. There has been great interest in potential nonuniformity of
plasma membrane G protein distribution and in the potential roles for
cytoskeletal elements in regulating G protein distribution and function
(52-56). However, in the absence of agonist, the cellular
G11 in E2M11 cells was both highly concentrated at the
plasma membrane and remarkably uniform in distribution around the
plasma membrane (Fig. 1a). The double labeling procedures to
detect
-tubulin and vimentin, were not able to detect any marked
degree of association of G11
with elements of the
cytoskeleton as has recently been reported in WRK cells (56). Indeed,
the cellular distribution of
-tubulin was essentially unaffected during the process of TRH-induced G11
redistribution
(Fig. 1) and although there were marked alterations in the pattern of
vimentin immunostaining (Fig. 3), this seemed to be separate from that observed for G11
. On the other hand, the lack of
structural evidence for G11
protein-cytoskeleton
interaction does not mean that the two processes are functionally
independent. Treatment of E2M11 cells with the cytoskeleton inhibitors
nocodazole, cytochalasin B, and colcemide resulted in significant
inhibition of G11
internalization.
In conclusion, these studies provide a dramatic new insight into the
internalization, cellular distribution, and down-regulation of the
phosphoinositidase C-linked G protein G11 in response to
the presence of an agonist at a GPCR coupled to this G protein, and
they demonstrate clearly that the series of recent studies on agonist
regulation of GPCR distribution should be extended to analysis of their
G protein partners.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. Draber, Institute of Molecular Genetics, AV CR for the anti-vimentin and anti-tubulin antisera; Dr. L. Kubinova, Division of Biomathematics, Institute of Physiology, AV CR for excellent assistance with the confocal microscopy; and Dr. H. Kovaru, 1st Medical Faculty, Charles University, for preparation of 452 antiserum.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust and the Grant Agency of Czech Republic (305/96/0678).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.
To whom correspondence should be addressed. Fax:
420-2-4719517; E-mail: svobodap{at}biomed.cas.cz.
The abbreviations used are: GPCR, G protein-coupled receptor; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; G protein, guanine nucleotide binding protein; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TCA, trichloroacetic acid; TRH, thyrotropin-releasing hormone; TRITC, tetrarhodamin isothiocyanate; IP, inositol phosphate.
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REFERENCES |
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