Sir Quinton Hazell Molecular Medicine Research Centre (D.K.G., Y.D., H.S.R., E.K., A.J.E., E.W.H.) Department of Biological Sciences University of Warwick Coventry, CV4 7AL, United Kingdom The Johns Hopkins University School of Medicine (M.A.L.) Division of Pediatric Endocrinology Department of Pediatrics Baltimore, Maryland 21287
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ABSTRACT |
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Second messenger studies of the variant receptor showed that CRH and
CRH-like peptides can stimulate the adenylate cyclase system, with
reduced sensitivity and potency by 10-fold compared with the CRH-R1.
Furthermore, CRH failed to stimulate inositol trisphosphate production.
Coexpression studies between the CRH-R1d or CRH-R1
showed that
this receptor does not play a role as a dominant negative receptor for
CRH.
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INTRODUCTION |
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The two receptors share 70% homology at the amino acid level, and each
of them exists as a family of related proteins produced from multiple
alternatively spliced forms of mRNA. The cDNA of CRH-R1 (7) encodes
a 415-amino acid protein, and two alternative forms have been
identified so far; the first variant has 29 amino acids inserted into
the first intracellular loop (CRH-R1ß), while the second (CRH-R1C)
(12) has 40 amino acids deleted from the N-terminal domain. These
structural differences reduce the receptor binding affinity for
[125I]CRH, and the CRH-R1ß appears to have reduced
coupling to adenylate cyclase, while the CRH-R1C appears to have no
signal transduction properties (12, 13). The type 2 CRH receptors have
distinct tissue distribution from the type 1 receptors and also exist
in three splice variant forms, namely CRH-R2
, -R2ß, and -R2
(9, 14, 15). Structural comparison of these CRH-R2 subtypes showed that 377
amino acids at their C terminus are identical and they differ only in
their N terminus; the 34 amino acids N terminal to CRH-R2
are
replaced by a 61-amino acid sequence to form the CRH-R2ß or a
20-amino acid sequence to form the CRH-R2
. Moreover, in adenylate
cyclase activation assays, CRH-related peptides appeared 10-fold more
potent at the CRH-R2ß than CRHR-2
or CRH-R2
, which suggests
that the N terminus of the receptor is involved in the ligand-receptor
interaction (15). In these experiments, urocortin was found to be the
most potent peptide for activation of the type 2 CRH receptors.
We have identified and characterized specific CRH-Rs in the human
pregnant and nonpregnant myometrium (16, 17, 18, 19, 20) that mediate the actions
of CRH on the myometrium during pregnancy and labor. Multiple subtypes
are present as determined by isoelectric focussing (18) and RT-PCR
(21), and our studies on the CRH-R subtype expression in the human
myometrium demonstrated that during pregnancy there is differential
CRH-R expression. These studies have led to the cloning and
characterization of a cDNA from human pregnant, but not nonpregnant
myometrial RNA that encodes a novel spliced variant of the human CRH-R1
receptor (termed CRHR-1d). This receptor isoform, which is also present
in the human fetal membranes (amnion and chorion derived from
spontaneous rupture) but not in placental biopsies at term, is
generated by deletion of a sequence that corresponds to exon 12 of the
human CRH-R1 gene (22) and encodes 14 amino acids in the putative
seventh TMD. Characterization of the CRHR-1d isoform demonstrated that
the protein has identical binding but different signal transduction
characteristics to those of R1.
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RESULTS |
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Initially, the class of G proteins coupled to the CRH-R1 and -R1d
receptors was investigated by using the nonhydrolyzable GTP analog
32P-GTP-AA to label the activated G proteins, and this was
followed by immunoprecipitation with specific G protein antibodies
(25). In preliminary experiments we found that for all classes of G
proteins maximal GTP-AA incorporation was found at a concentration
range of 100 nM CRH (data not shown). As shown in Fig. 5
our results demonstrated that the
stimulation of CRH-R1
by CRH resulted in activation of a 48-kDa
G
s-protein, as well as three more types of G proteins:
the G
q, G
i1/2, and G
o.
G
s activation was found to be the most potent (4-fold
increase over basal), followed by G
q and
G
o activation (2.5- to 3-fold increase over basal) while
G
i-protein activation was found to be the least potent
(1.2- to 1.5-fold increase over basal). When urocortin was used to
stimulate 293-R1
cells, a similar G protein activation pattern was
seen with potency comparable to CRH (data not shown). Furthermore, in
293-R1d cells CRH and urocortin could only weakly activate
G
s and G
o-proteins (0.5- to 0.9-fold
increase over basal). No G
q or G
i
activation was observed with either CRH or urocortin.
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DISCUSSION |
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Receptor-spliced variants arising from similar exon deletions resulting
in 14-amino acid deletions in the putative seventh TMD have been
described for two other members of this receptor family,
i.e. calcitonin (CTRe13) (28) and PTH/PTHrP (29)
receptors. The CRH-R1 exon/intron junctions are aligned to that of the
PTH receptor after exons 3, 5, 710, and 12, and both receptors are
similar in that amino acids 457509 (of the CRH-R1) are divided into
exons 11 and 12. The calcitonin receptor family is of particular
interest since it appears to have a very similar isoform profile to the
CRH-R1 receptor family; both receptors have splice variants that
contain inserts in the first intracellular loop (16 and 29 amino acids
in the calcitonin and CRH-R1 receptor, respectively) (7, 30) and exon
deletions in the seventh TMD. Furthermore, analysis of the nucleotide
sequence reveals that there are conserved splicing sites in the first
intracellular loop (site of insertion) and the seventh TMD (site of
exon deletion) (Table 2
).
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The deletion of the amino acids encoded by exon 12 resulted in major
differences in the signal transduction characteristics of R1d receptor.
The CRH-R1 receptors can efficiently stimulate adenylate cyclase
upon activation with subnanomolar concentrations of CRH and, like
several receptors of the R7G2 family, can generate phospholipase C and
IP3 production at high concentrations of CRH (13). In
contrast, in 293-R1d cells CRH and CRH-like peptides were less potent
in generating intracellular cAMP and were unable to stimulate
IP3 production. Again the CTR
e13 splice variant had
similar altered signal transduction characteristics (28). Previous
studies on the CRH-R1 isoforms revealed that the insertion of 29 amino
acids in the first intracellular loop of the CRH-R1ß had similar
effects on the signal transduction characteristics (12). However, the
CRH-R1ß variant has also reduced binding affinity for CRH.
Using techniques that demonstrate receptor-mediated G protein
activation, we provide direct evidence for the first time that in
HEK-293 cells upon stimulation by CRH or urocortin the CRH-R1 can
activate multiple G proteins with order of potency
G
s > G
q =
G
o > G
i. This suggests that the
CRH-R1
receptor can activate diverse intracellular signaling
pathways. Alternative candidate signaling pathways include calcium
channel modulation and the tyrosine kinase pathway, both of which have
been shown to be modulated by CRH in various types of cells (35, 36).
Interestingly, although the anti-Gs-antibody used could
detect three types of G
s protein present in the HEK293
cells, our results showed that the CRH-R1
could only activate the
long form of G
s (48 kDa). Also, consistent with the
observations on cAMP and inositol phosphate production, the CRH-R1d
variant had 75% reduced potency in stimulating Gs and
Go proteins while its ability to activate G
q
and G
i proteins was abolished. The observed reduction in
cAMP release was much greater (10 fold) than the difference in
G
s activation, which might be due to the differences in
experimental conditions and/or the different sensitivities of each
method. At present, the exact nature of signal transduction pathways
that can be activated by these G proteins is unknown, and since it is
possible that there is tissue-specific selectivity of G-protein
activation, detailed analysis in various tissues is
required.
Collectively, these results indicate that an intact seventh TMD is required for efficient coupling to G proteins, but not for high-affinity binding, and therefore it dissociates high-affinity binding of the ligand from its biological responsiveness and might provide a clue to the conformational switch that activates this receptor. It is possible that the deletion of the 14 amino acids in the seventh TMD could draw the proximal residues of the C-tail into the lipid bilayer and thus block their interaction with G proteins or that the CRH-R1d variant has a reduced hydrophobic character in its seventh TMD, which results in instability and failure to anchor the remaining sequence in the membrane. Also, our results suggest that sequences in the C terminus proximal to the membrane may be important for specific coupling to phospholipase C, while more distal residues in the C-tail are required for coupling to adenylate cyclase.
Of interest is the observation that during pregnancy there is an
alteration in the pattern of myometrial CRH-R1d expression that is
expressed only at term. This would suggest an important, yet
unidentified, role of this receptor variant in the mechanism of labor.
The cotransfection experiments in HEK 293 cells failed to demonstrate a
dominant negative action for the CRH-R1d when overexpressed in
conjunction with CRH-R1. In the native tissue, however, the
possibility exists that it could contribute to the decreased activation
of adenylate cyclase by CRH at term (19). It is also important to note
that in the fetal membranes, the CRH-R1d signal was localized mainly in
the chorion, while that of the CRH-R1
was localized mainly in the
amniotic epithelium. The functional significance of this differential
receptor expression is unknown, but again it suggests a distinct
functional role for the CRH-R1d. During pregnancy, CRH is produced by
the placenta and feto-maternal and myometrial tissues but its
biological role is unknown. Multiple CRH receptor mRNAs have been
identified in the fetal membranes, placenta, and human myometrium with
differential expression pattern during pregnancy (21), which argues for
multiple roles for CRH and/or related peptides in myometrial function
and suggests distinct functional roles for each receptor during
pregnancy. Different mechanisms may be involved to increase myometrial
sensitivity to different ligands dependent upon the presence of CRH
receptor subtypes on the membrane of myometrial smooth muscle cells.
The specific role of CRH-R1d during pregnancy is currently unknown, and
cotransfection experiments between CRH-R1
and R1d receptors suggest
that the R1d does not act as a dominant negative receptor by preventing
activation of R1
and stimulation of adenylate cyclase or
phospholipase C. However, the possibility remains that it might block
other signaling cascades.
In conclusion, we have identified a novel CRH-R1 variant generated by alternative splicing of mRNA resulting in the absence of 14 amino acids from the seventh TMD. This variant receptor has impaired signal transduction properties and is present in the human pregnant, but not in the nonpregnant, myometrium as well as in fetal membranes, which argues for a physiological role during pregnancy and provides evidence that the generation of different CRH-R isoforms by alternative splicing of the CRH-R mRNA may define the balance of diverse biological responses induced by CRH.
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MATERIALS AND METHODS |
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Subjects and Sample Preparations
Pregnant myometrial biopsies (n = 13) were obtained from
women undergoing elective cesarean section at term (n = 7) or
preterm (n = 6) before the onset of labor for nonmaternal
problems. The biopsy site was standardized to the upper margin of the
lower segment of the uterus in the midline. This provides the closest
approximation to the upper segment of the uterus. Nonpregnant
myometrial tissues (n = 8) were obtained from premenopausal
controls undergoing hysterectomy for nonmalignant conditions. The
nonpregnant myometrial biopsies were obtained from the same location as
the cesarean section myometrial biopsies to avoid possible differences
in receptor expression patterns. The relative content of myometrial and
fibrous tissue in these biopsies was identified by immunostaining using
specific smooth muscle cell and fibroblast markers (actin and vimentin,
respectively). The biopsies were immediately snap frozen in liquid
nitrogen and subsequently stored at -70 C until use. Ethical approval
was obtained from the local ethical committee and informed consent to
the study was obtained from all patients.
Culture of Myocytes
The tissue was immediately placed in 20 ml of ice-cold DMEM
culture medium containing 200 IU penicillin/ml, 200 mg streptomycin/ml.
Myocytes were prepared by enzymatic dispersion as previously described
(20). Briefly, pieces of myometrium were transferred into DMEM
containing collagenase (300 U/ml), deoxyribonuclease (DNAase) (30
U/ml), penicillin (200 U/ml), and streptomycin (200 mg/ml) and
incubated at 37 C for 30 min. After filtration and centrifugation,
single myocytes were suspended in DMEM containing 10% FCS, penicillin
(100 U/ml), streptomycin (100 mg/ml), and Fungizone (2.5 µg/ml). The
purity of myometrial muscle cells was assessed by immunocytochemical
staining. Mouse antihuman smooth muscle actin-specific monoclonal
antibody (MAb) and peroxidase-conjugated rabbit antimouse antibody (Ab)
were used. Human fibroblast cells and omission of the primary antibody
were used as negative controls, while frozen myometrial tissue was used
as a positive control. The cells were kept at 37 C in a humidified
atmosphere of 95% air and 5% CO2 until confluent.
RT-PCR, Cloning, and Sequence Analysis
Polyadenylated RNA was isolated from pregnant and nonpregnant
myometrium tissues by RNeasy Total RNA kit (Qiagen,
Crawley, UK) and reverse transcribed to synthesise cDNA, by using RNase
H Reverse Transcriptase (Life Technologies, Inc., Paisley,
UK). This was used as a template for a first-round PCR, and the
products of the first PCR reaction served as a template for the second
round of amplification reaction. All PCR reactions were carried out
using Elongase enzyme mix (Life Technologies, Inc.) with
200 ng cDNA for each amplification. Four primers (1S:
5'-AGCCGAGCGAGCCCGAGGATG-3'; 1A: 5'-GTCGACAAGCTT
(T)18-3'; 2S: 5'-CGAGGAT GGGAGGGCACCCGC-3'; and
2A: 5'-TCAGACTGCTGTGGACTGCTT-3') were used for the nested
PCR. Distilled water was used in place of the cDNA as a negative
control for each reaction.
The products of the second PCR reaction were analyzed by using 1% agarose gel electrophoresis, purified by using QIAquick Gel Extraction Kit (Qiagen) and ligated using T4 DNA Ligase Kit (Life Technologies, Inc.) into plasmid pBluescript II SK (+/-)-derived T-vector for sequence analysis. Positive isolated clones were sequenced using internal primers for the whole gene, in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information (NCBI, Bethesda, MD).
FISH
Paraffin-embedded blocks of human myometrium previously fixed in
4% (wt/vol) paraformaldehyde in 10 mmol/liter PBS, were sectioned (7
µm) and mounted onto gelatin-coated slides. At the time of
hybridization, sections were dewaxed using xylene, dehydrated by
successive washes through ethanol, and air-dried. A synthetic
oligonucleotide probe (Life Technologies, Inc.) with
fluorescein conjugated at the 5'-end was used
(F-CCGGATGGGAGAACGGACCTGGAAGGATTCCA GGAA-3').
After prehybridization, sections were covered with parafilm and incubated at 37 C for 5 h in a humidified chamber. Then hybridization solution (100 µl) containing 25% formamide, 4x SSC, 5% dextran sulfate, 0.2% dried milk powder, and 1 ng/µl of the probe was added, and the section was sealed with Sureseal (Hybaid Ltd, Teddington, Middlesex, UK) and allowed to hybridize at 37 C for 20 h. After hybridization, sections were washed twice with 2x SSC at 45 C and once with 0.1x SSC at 45 C. The sections were then rinsed in PBS, air dried, and visualized under a fluorescent microscope. Samples of pregnant and nonpregnant human myometrium were probed with both sense and antisense oligonucleotide probes of CRH receptors to ensure specificity.
Stable Transfection of HEK-293 Cells
Human CRH-R1 or the variant (R1d) CRH receptor subtypes were
subcloned into the expression vector pCI-neo (Promega Corp.) under the control of the human cytomegalovirus immediate
early promoter. Human embryonic kidney cells (HEK)-293 were transfected
using Lipofectamine reagent (Life Technologies, Inc.). The
cells were grown in DMEM in the presence of G418 (500 µg/ml) to
select for transfected cells, and those surviving were subcultured. A
number of these cell lines (293-R1
or 293-R1d) were selected for
characterization of their binding and signaling properties.
For cotransfection studies CRH-R1 or the variant (R1d) CRH receptor
subtypes were subcloned into the expression vectors pCI-neo
(Promega Corp.) and pREP4 (Invitrogen),
respectively, and transfected into HEK-293 cells. The cells were grown
in DMEM in the presence of G418 (500 µg/ml) + Hugromycin (200
µg/ml) to select for transfected cells, and those surviving were
subcultured and characterized for their signaling properties.
Membrane Preparation and CRH RRA
When confluent, 293 cells were washed with PBS and lysed with
0.2% NaCl. The cells were homogenized in extraction buffer A
containing 10 mM Tris-HCl, 1 mM EDTA, 1
mM phenylmethylsulfonyl fluoride, 10 mM
MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2. The
homogenate was centrifuged at 600 x g for 30 min at 4
C to remove nuclei and unbroken cells. The supernatant was collected
and centrifuged at 40,000 x g for 60 min at 4 C. The
pellet was rinsed twice, resuspended in binding buffer B containing 10
mM Tris-HCl, 1 mM EDTA, 10 mM
MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2, and
aliquoted (50 µg in 50 µl aliquots) in microfuge tubes.
For competitive displacement studies, 293-R1 or 293-R1d cell
membranes (50 µg) were incubated with 1 nM
125I-labeled oCRH in the presence or absence of unlabeled
peptide (0.011000 nM). Nonspecific binding was measured
in the presence of 1 µM unlabeled r/hCRH. For Scatchard
analysis, membranes (50100 µg of protein) were incubated with
125I-labeled oCRH (0.22 nM) and unlabeled r/h
CRH (1,000 molar excess) in 50 µl of binding buffer B. The tubes were
incubated at 22 C for 120 min. The reaction was terminated by adding 1
ml/tube of ice-cold 20% polyethylene glycol. After centrifugation at
10,000 x g for 15 min at 4 C, the pellets were washed
once with 20% polyethylene glycol and counted in a
-counter
(Packard Instrument Co., Meriden, CT) at 70% efficiency.
Nonspecific binding was 18 ± 5% of the total added
radioactivity.
The binding data were analyzed using the computer program EBDA (37), which provides initial estimates of equilibrium binding parameters by Scatchard and Hill analyses and then produces a file for the nonlinear curve-fitting program Ligand (38).
Second Messenger Studies
293-R1 or 293-R1d cells were plated in 96-well plates and
cultured until 95% confluency. Before treatments, cells were washed
once with 200 µl DMEM containing 0.1% BSA, followed by preincubation
with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine for
30 min. Cells were then stimulated with hCRH (0.11000 nM)
for 15 min at 37 C; reactions were terminated by addition of 0.1
M HCl. After an overnight freeze/thaw cycle, the cAMP
levels were measured in the supernatants using RIA. The sensitivity of
the assay was 0.025 pmol/liter with intraassay precision of CV 2.9%
and interassay precision of CV 9.7%.
For the inositol phosphate stimulation assay, cells were plated in six-well plates and subcultured in DMEM until 95% confluency. After incubation with inositol-free DMEM with [3H]myo-inositol (10 µCi/well) for 24 h, cells were washed with inositol-free DMEM once and preincubated with inositol-free DMEM containing 0.1% BSA and 30 mM LiCl for 30 min at 37 C. Phosphoinositide turnover was stimulated with r/hCRH (10500 nM) in the presence of 30 mM LiCl, and the reactions were stopped by addition of chloroform-methanol-hydrochloric acid (50:100:1) at specified time intervals. After transferring to borosilicate glass tubes and centrifugation, the upper phase was applied to Prefilled Poly-Prep columns (AG 1-X8 resin 100200 mesh chloride form, Bio-Rad Laboratories, Inc., York, UK) and [3H]IPs were resolved and quantified as previously described (39, 40). The radioactivity was measured by a ß-counter.
Synthesis of GTP-AA and Photolabeling of G Subunits
GTP-AA was synthesized after a method described previously (41).
Briefly, [-32P]GTP (1 mCi) was evaporated and
resuspended in 60 µl of 0.1 M
2-[N-morpholino]ethane sulfonic acid containing 30 mg/ml
1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride, pH
5.6, plus 40 µl of a suspension of azidoanilido-HCl (40 mg/ml) in
1,4-dioxane. The mixture was incubated for 6 h at room temperature
and the GTP-AA was purified using a C-18 Sep Pak cartridge. The
cartidge was prewetted with methanol and equilibrated with 97.2%
buffer A and 2.8% buffer B (buffer A was prepared by bubbling
CO2 through 100 mM triethylamine until the pH
reached 7; buffer B was prepared similarly with a 100 mM
solution of triethylamine in ethanol). The sample was dissolved in 1 ml
equilibrating buffer and applied to the cartridge. After a wash step
(with 10 ml of equilibrating buffer), the GTP-AA was eluted with 5 ml
of 10% buffer A and 90% buffer B and collected in 0.5-ml fractions.
Aliquots of each fraction were added to scintillation fluid and
32P was measured by scintillation spectrophotometry.
Fractions containing GTP-AA were combined, evaporated to dryness, and
stored at -70 C for up to 1 month. The overall yield of GTP-AA varied
from 30 to 50%. All procedures were carried out in a darkened
room.
293-R1 or 293-R1d cell membranes (100 µg) were incubated with or
without h/rCRH (1 pM to 100 nM) for 5 min at 30
C before the addition of 5 µCi of GTP-AA in 120 µl of 50
mM HEPES buffer, pH 7.4, containing 30 mM KCl,
10 mM MgCl2, 1 mM benzamidine, 5
µM GDP, 0.1 mM EDTA, in a darkened room.
After incubation for 3 min at 30 C, membranes were collected by
centrifugation and resuspended in 100 µl of the above buffer
containing 2 mM glutathione, placed on ice, and exposed to
UV light (254 nm) at a distance of 5 cm for 5 min.
G Protein Immunoprecipitation
GTP-AA-labeled 293-R1 or 293-R1d cell membranes were
precipitated by centrifugation and solubilized in 120 µl of 2% SDS.
Then 360 µl of 10 mM Tris-HCl buffer, pH 7.4, containing
1% (vol/vol) Triton X-100, 1% (vol/vol) deoxycholate, 0.5% (wt/vol)
SDS, 150 mM NaCl, 1 mM dithiothreitol, 1
mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin were added and insoluble material was removed by
centrifugation. Solubilized membranes were divided into 100-µl
aliquots, and each aliquot was incubated with 10 µl of undiluted G
protein antiserum at 4 C for 2 h under constant rotation. Then 50
µl of protein A Sepharose beads [10% (wt/vol) in the above buffer]
were added, and the incubation was continued at 4 C overnight under
constant rotation. The beads were collected by centrifugation, washed
twice with 1 ml of a 50 mM Tris-HCl buffer, pH 7.4,
containing 10% NP-40, 0.5% SDS, 600 mM NaCl, and then
were further washed twice with 1 ml of a 100 mM Tris-HCl
buffer, pH 7.4, containing 300 mM NaCl, 10 mM
EDTA and dried under vacuum in a Speed-Vac microconcentrator. The
immune complexes were dissociated from protein A by reconstitution in
Laemmlis buffer (42) (100 µl) and boiling for 5 min. Samples were
then subjected to gel electrophoresis using discontinuous SDS-PAGE slab
gels (10% running; 5% stacking). Molecular weight markers dissolved
in solubilization buffer were also electrophoresed. The gels were then
stained with Coomassie blue, dried using a slab gel dryer, and exposed
to x-ray film (Fuji Photo Film Co., Ltd., Dusseldorf,
Germany) at -70 C for 25 days.
Activated Receptor-G Protein Complex Immunoprecipitation
After the CRH binding reaction, 293-R1 or 293-R1d membranes
were centrifuged and solubilized as described above. Insoluble material
was removed by centrifugation, and solubilized ligand-receptor complex
was incubated with anti-CRH-R1 antiserum to a final concentration of
1:100 and incubated at 4 C for 3 h. Protein A-Sepharose 4B was
then added (40 µl), and the incubation was continued at 4 C
overnight. After centrifugation (15,000 rpm x 5 min), the pellet
was washed twice with 1 ml of a 100 mM Tris-HCl buffer, pH
7.4, containing 300 mM NaCl, 10 mM EDTA and
dried under vacuum in a Speed-Vac microconcentrator.
Immunoblotting
The pellet was resuspended in Laemmlis buffer (100 µl) (42)
and boiled for 5 min. Samples were then subjected to gel
electrophoresis using discontinuous SDS-PAGE slab gels (10% running;
5% stacking). Membranes from HEK293 cells were used as positive
controls for all G protein blotting experiments. Molecular weight
markers dissolved in solubilization buffer were also electrophoresed.
The resolved proteins were transferred to polyvinylidene difluoride
membrane at 100 mA for 90 min. The membrane was then blocked with 5%
nonfat dry milk at room temperature for 30 min and subsequently
incubated at 4 C for 2 h with G protein subtype-specific antisera.
Polyvinylidene difluoride membranes were washed twice with PBS-Tween 20
(0.05%) and incubated with goat antirabbit antibody conjugated with
horseradish peroxidase for 1 h. After washing twice with PBS-Tween
20 (0.05%), immunoreactivity was detected by enhanced
luminescence.
Statistical Analysis
Data are shown as the means ± SEM of each
measurement. Comparison between group means was performed by ANOVA;
P < 0.05 was considered significant. The relative
density of the bands was measured by optical density scanning using the
software Scion Image-Beta 3b for Windows (Scion Corp., Frederick,
MD).
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FOOTNOTES |
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This work was supported by Wellcome Trust, Action Research, and Sports Action Research for Kids (SPARKS). D.G. is a Wellcome Trust Career Development Fellow. E.H. is the Warwickshire Private Hospitals (WPH) Charitable Trust Chair of Medicine.
1 D.G. and Y.D. should be considered equal first authors by virtue of
their unique contributions to this work.
Received for publication March 8, 1999. Revision received July 7, 1999. Accepted for publication September 3, 1999.
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REFERENCES |
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