(Received for publication, March 29, 1995; and in revised form, May 9, 1995)
From the
Cells of clones of rat 1 fibroblasts transfected to express the
molecularly defined
There are multiple closely related
Although it
has been well established that sustained exposure of many
G-protein-coupled receptors to agonist can result in a reduction in
cellular levels of the receptor (a process known as down-regulation),
it has only been in the recent past that agonist-mediated reduction in
cellular levels of G-proteins has also been observed (see (8) for review). Even in such cases, information on the
mechanism(s) responsible for these effects is fragmentary(8) .
To attempt to address this point directly, in the present report we
have used clonal cell lines derived from rat 1 fibroblasts following
stable transfection with the cloned rat
These data indicate that
G-proteins activated by a receptor are degraded considerably more
rapidly than those in the inactive state and provide a mechanistic
explanation for how receptor agonists can control the cellular content
of G-proteins, which interact with that receptor.
Amplifications were
performed in 50 µl of buffer containing 25 pmol of primers and 2.5
units of Taq polymerase (Promega) using a HYBAID Omnigene
temperature cycler. Amplifications of
Rat 1 fibroblast cells transfected to stably express each of
the three cloned
Membranes derived from all three clonal cell lines were
examined for their levels of expression of the
Figure 1:
The ability of phenylephrine to
displace [
Figure 2:
The
Figure 3:
Phenylephrine - mediated down-regulation
of G
Figure 4:
Time course of agonist-mediated
down-regulation of G
Figure 5:
Quantitation of
G
Figure 6:
Phenylephrine treatment of
Figure 7:
Time course of
Figure 8:
Time course of
Figure 9:
Time course of
Although the basic observation that maintained agonist
activation of a G-protein-linked receptor can result in a marked and
selective reduction in cellular levels of the G-protein(s) activated by
that receptor is now well established (see (8) for review),
far less is known about the mechanisms responsible for such phenomena.
Studies from Hadcock et al.(23, 24) havenoted complex regulation of
G-proteins following receptor stimulation, including alterations in
both protein and mRNA stability of a variety of G-proteins, which is
then integrated to result in up-regulation of some G-proteins and
down-regulation of others. By contrast, Mitchell et al.(25) noted that muscarinic m1 acetylcholine
receptor-mediated down-regulation of the
In such cells, expressing one of the
rat
An
epitope-tagged constitutively activated mutant of G
The data provided herein demonstrate that agonist
occupation of
,
, or
adrenoreceptors and prelabeled with myo-[
H]inositol were each shown to
generate high levels of inositol phosphates when exposed to the
adrenoreceptor agonist phenylephrine. Maintained
exposure of each of these cells to phenylephrine resulted in a large
down-regulation of the receptors and also a marked down-regulation of
cellular levels of both of the phosphoinositidase C-linked G-proteins
G
and G
. To examine the mechanism of
phenylephrine-induced down-regulation of G
and
G
, pulse-chase
S-amino acid labeling
experiments were performed with each of the
,
, and
adrenoreceptor-expressing
cell lines. The rate of degradation of
G
/G
, which was adequately modeled by
a monoexponential with half-life between 33 and 40 h in each of the
cell lines in the absence of agonist, was accelerated substantially
(some 4-fold) in the presence of phenylephrine. By contrast, the rate
of degradation of the G-protein G
2
, which would not be
anticipated to be activated by members of the
adrenoreceptor family, was unaltered by the presence of
phenylephrine. Levels of mRNA encoding G
and
G
were not substantially altered by exposure of the
cells to phenylephrine in any of the cell lines studied.
adrenoreceptor subtypes that have been indicated by comparisons
of the pharmacological profiles of ligands in different
tissues(1) . Three distinct
adrenoreceptor
cDNA species have currently been
isolated(2, 3, 4, 5) ,
,
, and
, but
there has been considerable debate as to how closely these reflect the
pharmacologically defined subtypes (see (1) for review), even
if all of these cDNA species show the same signal transduction
mechanisms following their heterologous expression in cell lines. This
has arisen partially because of the relative pharmacological similarity
of the subtypes and partially because the first isolated cDNA species
were derived from a number of different
species(1, 6) . Current opinion favors the view that
the cloned
adrenoreceptor corresponds to the
pharmacologically defined
adrenoreceptor(1) , that the cloned and pharmacologically
defined
adrenoreceptors are identical(1) ,
and that the cloned
adrenoreceptor may be the
equivalent of the pharmacologically defined
adrenoreceptor(1) .
(
)Despite
these ongoing concerns, it is generally accepted that the primary
signaling function of
adrenoreceptor subtypes is to
stimulate the hydrolysis of inositol-containing phospholipids via
interaction with pertussis toxin-insensitive G-proteins of the
G
/G
family (7) with subsequent
activation of phospholipase C
activity(7) .
, the
hamster
, and the bovine
adrenoreceptor cDNA species. We note that in cells expressing
each of the three receptor species, sustained exposure to phenylephrine
results in a large, selective down-regulation of G
and G
as well as in down-regulation of the receptors.
These are the G-proteins that have been demonstrated to couple
adrenoreceptors to phosphoinositidase C activity and
the hydrolysis of inositol-containing phospholipids(7) . In
each case, we demonstrate that the basal rate of turnover of these
G-proteins is described adequately by a monoexponential with a t
between 33 and 40 h, while upon exposure to
agonist, this rate of degradation is markedly increased, such that a
substantial fraction of the cellular content of these two G-proteins
now has a t
of between 7 and 10 h. In contrast,
no marked alterations in amounts of mRNA encoding the G-proteins was
observed following agonist treatment.
Materials
All materials for tissue
culture were supplied by Life Technologies, Inc. (Paisley, Strathclyde,
Scotland). [H]Prazosin (24 Ci/mmol) and myo-[2-
H]inositol (17.6 Ci/mmol) were
obtained from Amersham International. Tran
S-label (1180
Ci/mmol) was purchased from ICN Biomedicals Inc. All other chemicals
were from Sigma or Fisons plc and were of the highest purity available.
Cells
Rat 1-fibroblasts transfected to
stably express the rat , the hamster
, and the bovine
adrenoreceptor
cDNA species were obtained from Dr. D. E. Clarke (Syntex, Palo Alto,
CA) under license to Syntex from Dr. L. F. Allen (Duke University, NC).
Cell Culture
Rat 1 fibroblasts stably
expressing adrenoreceptor subtypes were maintained in
tissue culture in Dulbecco's modified Eagle's medium
(DMEM)
(
)containing 5% (v/v) newborn calf serum,
glutamine, penicillin, and streptomycin. For most experiments, cells
were grown until close to confluency and then either harvested or
subcultured in a 1:10 ratio. In preparation for labeling with
Tran
S-label for the pulse-chase experiments, cells were
trypsinized and seeded in 6-well culture plates. At about 70%
confluency, 2/3 of the growth medium was replaced with DMEM lacking
methionine and cysteine, supplemented with glutamine, antibiotics, and
50 µCi/ml Tran
S-label (final concentration in well).
After the labeling period (16 h), the radioactive medium was removed,
and the now close to confluent cell layer was washed once with 1 ml of
normal DMEM culture medium. They were subsequently incubated in 1.5
ml/well normal DMEM culture medium in the presence or absence of
phenylephrine (100 µM). At appropriate times, the medium
was removed and cells were dissolved in 1% (w/v) SDS (200 µl/well).
The cell suspension was heated in a screw-cap test tube to 100 °C
for 20 min to denature proteins and nucleic acids; then, samples were
either stored at -20 °C or processed directly for
immunoprecipitation.
Immunoprecipitation of
To the 200 µl of SDS-denatured cell
suspension was added 800 µl of solubilization buffer (1% (w/v)
Triton X-100, 10 mM EDTA, 100 mM
NaHS-Labeled
G-proteins
PO
, 10 mM NaF, 100 µM Na
VO
, 50 mM HEPES, pH 7.2) and
100 µl of Pansorbin (Calbiochem). Samples were incubated at 4
°C with continuous rotation for 1-2 h for nonspecific
preclearing. Following centrifugation of the samples (16,000
g, 2 min, 4 °C), the supernatant was collected and
subjected to immunoprecipitation by addition of 100 µl of protein
A-agarose along with 10 µl of the specific G-protein antiserum and
incubated at 4 °C for 4 h. Immune complexes were then recovered by
centrifugation (16000
g, 30 s at 4 °C) and washed
by resuspension-centrifugation three times each with 1 ml of wash
buffer (1% (w/v) Triton X-100, 100 mM NaCl, 100 mM NaF, 50 mM NaH
PO
, 50 mM HEPES, pH 7.2, 0.5% (w/v) SDS). The final protein A-agarose pellet
was resuspended in Laemmli sample buffer, heated at 100 °C for 5
min, and electrophoresed by SDS-PAGE (10% (w/v) acrylamide). Following
resolution of the proteins, the gel was stained with Coomassie Blue and
then dried. The dried gels were exposed either to photographic film or
to phosphor storage screen autoradiography according to
manufacturer's instructions using a Fujix bio-imaging analyzer
linked to an Apple Macintosh Quadra 650 personal computer.
Autoradiographs were analyzed such that the time-course data were
expressed as a percentage of the control-specific
G
/G
protein radiolabeling found at
zero time.
Immunological Studies
The generation and
specificities of the various antisera used in this study have been
fully described previously(9, 10) . Each antiserum was
produced in a New Zealand White rabbit using a conjugate of a synthetic
peptide with keyhole-limpet hemeocyanin (Calbiochem) as antigen.
Membrane samples were resolved by SDS-PAGE (10% (w/v) acrylamide gels)
overnight at 60 V. Proteins were subsequently transferred to
nitrocellulose (Schleicher and Schuell) and blocked for 3 h in 5% (w/v)
gelatin in phosphate-buffered saline (PBS), pH 7.5. Primary antisera
were added in 1% gelatin in PBS containing 0.2% (v/v) Nonidet P-40 and
incubated overnight. The primary antiserum was removed, and the blot
was washed extensively with PBS containing 0.2% Nonidet P-40. Secondary
antiserum (donkey anti-rabbit IgG coupled to horseradish peroxidase) in
1% gelatin, PBS, 0.2% Nonidet P-40 was added and left for 3 h. After
removal of the secondary antiserum, the blot was washed extensively as
above and developed with o-dianisidine hydrochloride (Sigma)
as substrate for horseradish peroxidase as described(11) .
Preparation of Membranes
Membrane
fractions were prepared from cell pastes that had been stored at
-80 °C following harvest. Cell pellets were resuspended in 5
ml of 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 (buffer A),
and rupture of the cells was achieved with 25 strokes of a hand-held
Teflon on-glass homogenizer. Unbroken cells and nuclei were removed
from the resulting homogenate by centrifugation at 500 g for 10 min in a Beckman L5-50B centrifuge with a Ti-50
rotor. The supernatant fraction was then centrifuged at 48,000
g for 10 min, and the pellet was washed and resuspended in 10
ml of buffer A. Membrane fractions were recovered following a second
centrifugation at 48,000
g for 10 min, and pellets
were resuspended in buffer A to a final protein concentration of
1-3 mg/ml and stored at -80 °C until required.
[
Binding assays were initiated by the addition
of 5-15 µg of protein to an assay buffer (50 mM
Tris-HCl, 0.5 mM EDTA, pH 7.4) containing
[H]Prazosin Binding
Experiments
H]prazosin (12, 13) (0.005-1 nM in saturation
assays and 0.1 µM for competition assays) in the absence
or presence of increasing concentrations of the test drugs (500 µl,
final volume). Nonspecific binding was determined in the presence of 10
µM phentolamine. Reactions were incubated for 30 min at 25
°C, and bound ligand was separated from free by vacuum filtration
through GF/B filters. The filters were washed with 2
5 ml assay
buffer, and bound ligand was estimated by liquid scintillation
spectrometry.
Expression of G
An expression system utilizing the promoter for
bacteriophage T7 RNA polymerase in the prokaryotic expression vector
pT7.7 (14) was employed to overexpress cDNA encoding hamster
G in Escherichia
coli
in E. coli. To ensure maximal expression of
G
, a PCR-based strategy described in (15) was
used to position the cDNA downstream from the T7 promoter at the
optimum distance from the ribosome binding site. The expression
construct was transformed into E. coli BL21 (DE3) cells, which
contain a single copy of the gene for T7 RNA polymerase under control
of the isopropyl-1-thio-
-D-galactopyranoside-inducible lac UV5 promoter.
Isopropyl-1-thio-
-D-galactopyranoside-induced cells were
recovered by centrifugation and stored as pastes at -80 °C.
Cells were seeded in 24-well
plates and labeled to isotopic equilibrium by incubation with 1
µCi/ml myo-[2- Adrenoreceptor Regulation of
Inositol Phosphate Production
H]inositol in 0.5 ml
inositol-free DMEM containing 1% (v/v) dialyzed newborn calf serum for
36 h. On the day of experiments, the labeling medium was removed, and
cells were washed twice with 0.5 ml of Hank's buffered saline, pH
7.4, containing 1% (w/v) bovine serum albumin and 10 mM glucose (HBG). Cells were then washed twice for 10 min with HBG
supplemented with 10 mM LiCl (HBG/LiCl) and subsequently
stimulated with agonist in HBG/LiCl for 20 min. All incubations were
performed at 37 °C. Reactions were terminated by the addition of
0.5 ml of ice-cold methanol. Cells were then scraped and transferred to
vials, and chloroform was added to a ratio of 1:2
(CHCl
:MeOH). Total inositol phosphates were extracted for
30 min prior to the addition of chloroform and water to a final ratio
of 1:10:9 (CHCl
:MeOH:H
O). The upper phase was
taken, and total inositol phosphates were analyzed by batch
chromatography on Dowex-1-formate as previously
described(16, 17) .
Reverse Transcriptase-Polymerase Chain
Reaction
The reverse transcriptase-PCR procedure was
essentially as previously described(18) .
RNA Extractions
Total RNA was extracted
using RNAzol B (Biogenesis). Purity and quantification of RNA were
assessed by spectrophotometric A/A
ratios.
Reverse Transcription
Samples of
5-10 µg of RNA (20 µl) were denatured by incubation at 65
°C for 10 min followed by chilling on ice and reverse transcribed
in 33 µl of reaction mixture using first strand cDNA synthesis kit
(Pharmacia Biotech Inc.) as detailed by the manufacturer. Incubation
was carried out at 37 °C for 1 h. The reactions were terminated by
heating samples at 95 °C for 5 min followed by transfer to ice.
Polymerase Chain Reaction
PCR reactions
were carried out using the following primers: sense, 5`-TTGGAAGGAGCCAGTGC3`;
/
antisense, 5`-GAAGGCGCGCTTGAACT-3`;
sense,
5`-ACAAGGAATGCGGAGTC-3`;
sense,
5`-TTCTCCGTGAGACTGCT-3`;
antisense,
5`-CCAGGTCCTTGTGCTGT-3`; G
sense,
5`CCACCTGAATTCGAGCATGCC-3`; G
antisense,
5`-GCGTGGGTCCTCTCCGGGCTCGGG-3`; G
sense,
5`-ATGACTTGGACCGTGTAGCCGACC-3`; G
sense,
5`-ACGTGGACCGCATCGCCACAGTAG-3`; G
/G
antisense, 5`-CCATGCGGTTCTCATTGTCTGACT-3`.
adrenoreceptor
subtypes were carried out as follows: 95 °C/5 min, 49 °C/30 s,
72 °C/1 min (1 cycle); 95 °C/30 s, 49 °C/30 s, 72 °C/1
min (30 cycles); 95 °C/30 s, 49 °C/30 s, 72 °C/5 min (1
cycle). Conditions used for amplification of G
,
G
, and G
were: 95 °C/5 min, 60
°C/30 s, 72 °C/1 min (1 cycle); 95 °C/30 s, 60 °C/30 s,
72 °C/1 min (30 cycles); 95 °C/30 s, 60 °C/30 s, 72
°C/5 min (1 cycle). Reaction products were then separated by
1.5-1.75% agarose gel electrophoresis. In each case, the size of
the generated product was that anticipated from the selected primers.
Northern Analysis
Total RNA (20-30
µg) was run on 1% (w/v) formaldehyde gels and transferred to Hybond
N nylon membrane (Amersham). G-, G
-,
and G
-specific probes were made by PCR-amplifying
reverse transcribed total RNA as described above. Amplified products
were electrophoresed on 1% (w/v) agarose gels, excised, and labeled by
random priming (Rediprime labeling system, Amersham International). RNA
blots were hybridized at 42 °C in 50% (v/v) formamide, 2
Denhardt's solution (0.04% (w/v) Ficoll, 0.04% (w/v)
polyvinylpyrrolidone, 0.04% (w/v) bovine serum albumin), 5
SSPE
(750 mM NaCl, 5 mM EDTA, 44 mM NaH
PO
, pH 7.4), 0.1% (w/v) SDS, and 50
µg/ml fragmented salmon sperm DNA and then washed at 55 °C in 2
SSC (300 mM NaCl, 30 mM
Na
C
H
O
, pH 7.0), 0.1%
(w/v) SDS.
Data Analysis
Analysis was performed
using the Kaleidagraph (version 2.1) curve fitting package with an
Apple Macintosh computer.
adrenoreceptor subtypes were used
for the present study. The presence of
,
, and
adrenoreceptor mRNA in
appropriately transfected cells only was confirmed by reverse
transcriptase-PCR analysis of RNA isolated from untransfected parental
rat 1 cells and each of the clonal cell lines examined in this study
using oligonucleotide primers specific for each of the three
molecularly defined
adrenoreceptor subtypes (data not
shown).
adrenoreceptor subtypes by measuring the specific binding of the
adrenoreceptor antagonist
[
H]prazosin.
,
, and
adrenoreceptor subtypes were
found to be expressed at levels of 2.1 ± 0.1, 2.8 ± 0.5,
and 7.0 ± 0.9 pmol/mg membrane protein, with K
values for the binding of
[
H]prazosin of 110 ± 20, 76 ± 13,
and 150 ± 26 pM, respectively (data not shown).
Displacement of specific [
H]prazosin binding by
varying concentrations of the
adrenoreceptor agonist
phenylephrine was achieved with pIC
values (and Hill
coefficients) of 5.1 ± 0.1 (n
= 0.76
± 0.09), 4.7 ± 0.2 (n
= 1.06
± 0.07), 5.2 ± 0.1 (n
0.90 ±
0.06), respectively, for the
,
,
and
adrenoreceptor subtypes (Fig. 1).
Addition of the poorly hydrolyzed analogue of GTP, Gpp(NH)p (100
µM), to such assays produced no significant alterations in
the positions of such displacement curves (5.1 ± 0.1 (n
= 0.99 ± 0.03), 4.6 ± 0.1 (n
= 0.89 ± 0.05), 4.9 ± 0.2 (n
1.12 ± 0.16) for the
,
, and
adrenoreceptor subtypes) (Fig. 1).
H]prazosin binding in membranes of
adrenoreceptor-expressing cells. The ability of
phenylephrine in the absence (filledsymbols) or
presence (opensymbols) of Gpp(NH)p (100
µM) to displace the specific binding of
[
H]prazosin to membranes of
(I),
(II), and
(III) adrenoreceptor-expressing rat 1 fibroblasts was
assessed as described under ``Experimental Procedures.''
Typical examples, representative of four independent experiments, are
displayed (see ``Results'' for
details).
Accumulation of
inositol phosphates in LiCl (10 mM)-treated adrenoreceptor subtype-expressing cells, which had been labeled
for 36 h with myo-[2-
H]inositol, was
found to be markedly stimulated by phenylephrine (Fig. 2),
however, in a manner that was insensitive to pretreatment of the cells
with pertussis toxin (25 ng/ml, 16 h) (data not shown), confirming a
functional coupling of these receptors to the cellular G-protein
machinery. Half-maximal stimulation of inositol phosphate generation in
response to phenylephrine was produced with between 0.3 and 1
µM agonist in a range of experiments with each of the
three
adrenoreceptor subtype-expressing cell lines (Fig. 2).
adrenoreceptor
subtypes all cause stimulation of inositol phosphate production. Rat 1
fibroblasts transfected to stably express the
(I),
(II), and
adrenoreceptor subtypes (III) were labeled with myo-[2-
H]inositol (1 µCi/ml) for 36
h prior to treatment with varying concentrations of phenylephrine for
20 min. Total inositol phosphates were measured as described under
``Experimental Procedures.'' Stimulation of inositol
phosphate generation was found to be dose dependent with an EC
for phenylephrine between 0.3 and 1.0 µM for all
three cell lines in a range of experiments. The data displayed are the
mean of triplicate assays from representative experiments; bars represent S.E.
Sustained exposure of the three cell lines to
phenylephrine (100 µM, 6 h) led to a marked reduction of
detectable adrenoreceptors in all three cases (Table 1). Sustained exposure (16 h) of cells expressing each of
the three
adrenoreceptor subtypes to varying
concentrations of phenylephrine also resulted in a reduction of
membrane-associated levels of a combination of the phosphoinositidase
C-linked G-proteins G
and G
by,
maximally, some 50-70% as determined by immunoblotting of
membranes of agonist-treated and untreated cells with antiserum
CQ(9) , which identifies the C-terminal decapeptide, which is
entirely conserved between these two closely related G-proteins (Fig. 3). No significant alterations in immunologically detected
levels of other G-protein
subunits expressed by these cells
(G
, G
) were noted to be associated
with phenylephrine treatment (data not shown). Down-regulation of
G
/G
was found to be dose dependent
with an EC
for phenylephrine close to 600 nM for
all three
adrenoreceptor subtype-expressing cells (Fig. 3). In cells expressing the
and
adrenoreceptor subtypes, half-maximal effects in
response to a maximally effective concentration of phenylephrine (1
mM) were produced after 8 h, whereas in cells expressing the
subtype, this figure was determined to be 4 h (Fig. 4), a difference that might reflect the higher levels of
expression of the receptor in these cells (see above). In all cells
treated with agonist over a sustained period (16 h), a similar
membrane-bound plateau level of G
/G
was established at some 30-50% of that present in untreated
cells.
/G
levels in membranes of
adrenoreceptor subtype-expressing rat 1 fibroblasts.
Membranes (15 µg) derived from
(a),
(b), and
(c)
adrenoreceptor-expressing rat 1 fibroblasts, which were either
untreated or had been treated with varying concentrations of
phenylephrine for 16 h, were resolved by SDS-PAGE (10% (w/v)
acrylamide) and then immunoblotted using the
anti-G
/G
antiserum CQ as primary
reagent. Quantitative analysis from these studies on the
adrenoreceptor-expressing cell line is displayed in d.
/G
. Membranes
(15 µg) derived from
(a),
(b), and
(c)
adrenoreceptor-expressing rat 1 fibroblasts either untreated or treated
with 1 mM phenylephrine for the times indicated were resolved
by SDS-PAGE (10% (w/v) acrylamide) and subsequently immunoblotted using
antiserum CQ as described under ``Experimental Procedures.''
Quantitative analysis from these studies on the
adrenoreceptor-expressing cell line is displayed in d.
The amount of expressed recombinant G in
whole cell extracts of E. coli BL21 (DE3) cells and of
G
/G
in the
adrenoreceptor-expressing cells was estimated by comparison with
known levels of purified G
/G
following immunoblotting with antiserum CQ (Fig. 5). Standard
curves for the reaction of recombinant G
with this
antiserum was obtained using various dilutions of the prokaryotically
expressed G-protein
subunit. Such standard curves allowed
measurement of the amounts of G
/G
in
membranes of all three
adrenoreceptor
subtype-expressing rat 1 fibroblast clones. The steady-state level of
G
/G
was found to be similar in all
three clonal cell lines (46 pmol/mg membrane protein). In a typical
example, as displayed in Fig. 5, sustained challenge of the
adrenoreceptor-expressing cells with a maximally
effective concentration of phenylephrine (1 mM, 16 h) resulted
in membrane-associated immunodetectable
G
/G
being reduced to 16 pmol/mg
membrane protein.
/G
levels in
adrenoreceptor subtype-expressing rat 1 fibroblasts. E. coli BL21 DE3 cells were transformed with hamster G
cDNA subcloned into the prokaryotic expression vector pT7.7 (see
``Experimental Procedures''). Cultures of transformants were
induced to express recombinant G
by the addition of 1
mM isopropyl-1-thio-
-D-galactopyranoside and
grown for a further 4 h prior to recovery by centrifugation. Known
quantities of purified G
/G
from liver (lane1 = 0, lane 2 = 10, lane 3 = 50, lane 4 = 75, lane 5 = 100, lane 6 = 150, and lane 7 = 200 ng) were resolved by SDS-PAGE together with varying
amounts of E. coli extract-expressing recombinant
G
(lane8 = 125, lane9 = 250, and lane10 =
500 ng) and membranes (15 µg) from
adrenoreceptor-expressing rat 1 fibroblasts (lanes11 and 12), untreated (lane11) or treated with phenylephrine (1 mM, 16 h) (lane12). In the example displayed, levels of
G
/G
were calculated to be 46 pmol/mg
membrane protein in untreated cells, whereas in cells treated with
agonist over a sustained period, levels of
G
/G
were reduced to 16 pmol/mg
membrane protein.
G and G
comigrate in 10% (w/v) acrylamide SDS-PAGE. Therefore, to determine
both the relative levels of expression of these two phosphoinositidase
C-linked G-proteins in the rat 1-derived clonal cell lines and whether
phenylephrine-mediated down-regulation of
G
/G
in any of these cells was
selective for either of these highly homologous G-protein
subunits, membranes from untreated and phenylephrine (1
mM)-treated cells were resolved by SDS-PAGE using 12.5% (w/v)
acrylamide gels containing 6 M urea, conditions that we have
previously shown to be effective in resolving these
G-proteins(19, 20, 21) . Fig. 6demonstrates that steady-state levels of
G
were substantially (over 2-fold) greater than
G
in these cells and that phenylephrine-driven
down-regulation of membrane-associated G
and
G
from the clonal cell lines expressing each of the
three
adrenoreceptor subtypes was non-selective
between these G-protein
subunits.
adrenoreceptor subtype-expressing cells results in
down-regulation of both G
and G
.
Membranes (60 µg) from
(lanes1 and 2),
(lanes3 and 4), and
(lanes5 and 6) adrenoreceptor-expressing rat 1 fibroblasts, untreated (lanes2, 4, and 6) or treated with
phenylephrine (1 mM, 16 h) (lanes1, 3, and 5), were resolved by SDS-PAGE in a 12.5% (w/v)
acrylamide, 0.0625% (w/v) bis-acrylamide gel system containing 6 M urea and subsequently immunoblotted as in Fig. 4.
To address the mechanism of
phenylephrine-mediated reduction in membrane-bound levels of
G/G
, cells of the
adrenoreceptor subtype-expressing clones were incubated with
Tran
S-label for 16 h, and subsequently the decay of
S with time in immunoprecipitated
G
/G
was monitored in cells that were
either untreated or treated with phenylephrine (100 µM) (Fig. 7-9). Analysis of the rate of decay of
S-labeled G
/G
indicated
that in the untreated
(Fig. 7),
(Fig. 8), and
(Fig. 9) adrenoreceptor subtype-expressing cells, this
process was described adequately by monoexponentials with estimated
half-time (t
) ranging from 33 to 40 h. However,
the decay of
S-labeled
G
/G
in each of the cell lines in the
presence of phenylephrine was more rapid (Fig. 7-9), and
the data were more effectively modeled by a two-component fit than by a
single exponential (Fig. 7-9). Addition of phenylephrine
was associated with a substantial component of the decay in which t
for G
/G
was
markedly accelerated to 10.2, 10.9, and 7.7 h in rat 1 fibroblasts
expressing
,
, and
adrenoreceptor subtypes, respectively. There was, however, a
second component of the decay rate for
G
/G
that was not enhanced compared
to the single phase decay observed in the untreated cells. Because of
the lower steady-state level of expression of G
relative to G
, we did not attempt to examine whether
the agonist-induced acceleration of
G
/G
degradation could be observed
independently for both of these G-proteins. In contrast to the effect
of phenylephrine on the rate of removal of
S-labeled
G
/G
, this agonist had no effect in
any of the
adrenoreceptor subtype-expressing cells on
the rate of decay of
S-labeled G
2
, which
had been immunoprecipitated with antiserum SG (data not shown). This
G-protein has been shown to be involved in receptor-mediated inhibition
of adenylyl cyclase(11, 22) .
adrenoreceptor-stimulated enhancement of
G
/G
protein degradation. Whole cell
S-amino acid pulse-chase analysis of
G
/G
from
-expressing rat-1 cells isotopically labeled and
experimentally processed as described under ``Experimental
Procedures'' is shown. In the chase phase, cells were incubated
with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Cell
activity was stopped at the indicated time, and the cell extract was
processed for immunoprecipitation and analysis of radiolabeled
G
/G
. Results represent the means
from four independent experiments.
adrenoreceptor-stimulated enhancement of
G
/G
protein degradation. Whole cell
S-amino acid pulse-chase analysis of
G
/G
from
-expressing rat-1 cells isotopically labeled and
experimentally processed as described under ``Experimental
Procedures'' is shown. In the chase phase, cells were incubated
with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Results
represent the means from four independent
experiments.
adrenoreceptor-stimulated enhancement of
G
/G
protein degradation. Whole cell
S-amino acid pulse-chase analysis of
G
/G
from
adrenoreceptor-expressing rat 1 cells isotopically labeled and
experimentally processed as described under ``Experimental
Procedures'' is shown. In the chase phase, cells were incubated
with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Results
represent the means from four independent
experiments.
To investigate
whether transcriptional or translational controls were likely to
contribute to the process of agonist-driven G-protein down-regulation,
Northern blot analysis of G, G
, and
G
mRNA levels in cells expressing the
adrenoreceptor subtype was performed in the absence or presence
of phenylephrine treatment. Levels of mRNA encoding each of the above
subunits were not substantially altered by exposure of the cells
to phenylephrine (data not shown).
subunits of the
phosphoinositidase C-linked G-proteins G
/G
was
accompanied by a selective accelerated rate of degradation of these
G-proteins. In the present study, we have sought to further analyze
such effects by examining the process of down-regulation of the
subunits of G
and/or G
in rat 1 cells
transfected to express individual molecularly defined
adrenoreceptor subtypes.
, the hamster
, and the
bovine
adrenoreceptors and labeled with myo-[
H]inositol, exposure to the
adrenoreceptor agonist phenylephrine resulted in
stimulation of inositol phosphate production in a fashion that was
resistant to pretreatment of the cells with pertussis toxin. Such a
feature was hardly unexpected but is the pattern anticipated for
receptors that couple to phosphoinositidase C-linked G-proteins of the
G
family. Maintained exposure of these cells to
phenylephrine resulted in down-regulation of each of the receptor
subtypes (Table 1) and selective down-regulation of some
combination of the
subunits of G
/G
.
Agonist-induced down-regulation of G-protein-linked receptors is a
common regulatory feature. However, in the subfamily of
adrenoreceptors, while both the
C10 and
C2 receptor are readily down-regulated by agonist
treatment, the
C4 receptor has been reported to be
largely resistant to down-regulation (26) . Mutation of the
site for palmitoylation in the C-terminal tail of the
C10 adrenoreceptor has been reported to render it
resistant to agonist-mediated down-regulation without altering its
ability to couple to the G
-like G-proteins (27, 28) . Although direct information is not
currently available, all three
adrenoreceptor cDNA
species used in this study have cysteine residues in their predicted
C-terminal tail in a context that makes them likely sites of
palmitoylation. It would be interesting to examine if mutation of these
residues results in an agonist-mediated down-regulation resistant form
of these receptors and whether this interferes with agonist-mediated
down-regulation of G
/G
. Agonist-mediated
down-regulation of the
subunits of G
/G
has now been reported for a variety of receptors, including the
muscarinic m1 acetylcholine receptor(19, 25) , the
long isoform of the thyrotropin-releasing hormone
receptor(29) , and the gonadotropin-releasing hormone receptor (21) . However, only for the first of these has any mechanistic
analysis been provided. In Chinese hamster ovary cells transfected to
express the rat muscarinic m1 receptor, accelerated degradation of a
combination of G
/G
was recorded without
detectable alteration in levels of mRNA of either of these polypeptides (25) . In the present study, we expand those observations to
show that in the genetic background of rat 1 fibroblasts, the basal
half-life of the
subunits of G
/G
can be
adequately modeled as a single monoexponential consistent with t
in the region of 33-40 h and that
agonist occupancy of any of the
adrenoreceptor
subtypes leads to a proportion of the cellular G
/G
population being degraded much more rapidly. Data from each of
the systems, however, are not consistent with all of the cellular
content of these G-proteins being degraded more rapidly in the presence
of agonist. A maximally effective concentration of phenylephrine was
able to cause down-regulation of between 50 and 70% of the total
G
/G
population in these cells in a
range of experiments. As the immunoprecipitation experiments that were
performed in the G-protein turnover studies made use of an antiserum
that identifies G
and G
equally(9) , as it is directed at an epitope that is identical
in these two G-proteins, and these two G-proteins are widely
co-expressed(30) , then we wished to examine the possibility
that each of the
adrenoreceptor subtypes was able to
cause down-regulation of only one of these two G-proteins. To do so, we
took advantage of our previous observations that separation of these
two polypeptides can be achieved in SDS-PAGE systems that incorporate
high concentrations of urea (19, 20, 21) .
Immunoblotting of membranes of the clones used in this study with
antiserum CQ following their resolution in such gels demonstrated that
the steady-state levels of G
expression was over
2-fold higher than that of G
. Furthermore, they
indicated that sustained phenylephrine occupancy of each of the
adrenoreceptor subtypes resulted in a down-regulation
of both G
and G
. Although we have
not analyzed the relative cellular distribution of G
and G
in these clonal cell lines, we have previously
been able to show in cellular fractionation studies of Chinese hamster
ovary cells on sucrose density gradients that the subcellular
distribution of these two G-proteins is identical(20) .
has been demonstrated to have a substantially reduced half-life
compared with the epitope-tagged wild type protein when expressed in
S49 lymphoma cyc
cells(31) , and agonist
activation of an IP prostanoid receptor can result in down-regulation
of this epitope-tagged variant of G
when this
G-protein is expressed in neuroblastoma NG108-15 cells(32) .
Thus, although it has not been formally demonstrated for any G-protein
other than G
, it is reasonable to surmise that
activation of the G-protein might be the key feature that controls its
rate of degradation. The palmitoylation status of both the activated
mutant of G
and the wild type protein following
activation of a G
-linked receptor is altered compared
with the basal state of the wild type
protein(33, 34, 35) . Kinetic evidence
indicates that this is likely to reflect accelerated
depalmitoylation(35) . How relevant this is to agonist-mediated
down-regulation of G
remains to be explored, but it is
certainly true that in a number of systems, agonist occupation of
G
-linked receptors has been noted to result in a large
selective down-regulation of this
G-protein(36, 37, 38) . It will thus now be
of considerable interest to examine whether agonist activation of the
adrenoreceptor subtypes in these cells results in an
alteration in the palmitoylation status of the
subunits of
G
/G
. As with the previous studies on
muscarinic m1 receptor regulation of
G
/G
levels(25) , Northern
blot analysis of mRNAs corresponding to these G-proteins in untreated
and phenylephrine-treated cells did not provide evidence for regulation
at the mRNA level.
adrenoreceptor subtypes can selectively
regulate the cellular levels of both G
and
G
. The mechanism of this effect is a selective
acceleration of the rate of degradation of these G-proteins.
adrenoreceptor
subtypes using the nomenclature originally assigned to the cDNA species
that were used in the study, except that the cDNA originally named the
adrenoreceptor is referred to as the
adrenoreceptor, as this is now widely accepted to be equivalent
to the pharmacologically defined
adrenoreceptor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.