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Laboratoire de Physiologie de la Reproduction, ESA Centre National de la Recherche Scientifique 7080, Université Pierre et Marie Curie, 75252 Paris CEDEX 05, France
Address all correspondence and requests for reprints to: Joëlle Cohen-Tannoudji, Laboratoire de Physiologie de la Reproduction, ESA Centre National de la Recherche Scientifique 7080, Université Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris CEDEX 05, France. E-mail: jtannoud{at}snv.jussieu.fr
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Introduction |
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s protein (4), thus
resulting in a higher coupling between ß2-AR
and adenylyl cyclase (5). In rat myometrium, production of
cAMP is further enhanced by the cross-regulation between
2- and ß2-AR pathways
(6).
In several species, including human, mice, and rat, desensitization of
the ß-AR/adenylyl cyclase pathway occurs at the end of pregnancy
(5, 7, 8). The molecular basis for the attenuation of
myometrial cAMP production in response to ß-agonists at parturition
is not fully understood. In the rat, we have demonstrated that
desensitization relies on alterations of ß-AR coupling rather than on
receptor expression (5). Indeed, ß-AR coupling with
Gs/adenylyl cyclase, as evaluated by competition binding experiments
between an agonist and a radiolabeled antagonist, was severely impaired
at parturition as compared with midpregnancy. In contrast, the number
of ß-AR was not significantly modified. In vitro studies
have established that the main mechanism underlying ß-AR uncoupling
is a rapid receptor phosphorylation (9, 10). Two types of
kinases are known to mediate ß-AR phosphorylation: second
messenger-dependent kinases and G protein-coupled receptor kinases
(GRK). The GRK family consists of six members that have been further
classified into three subfamilies according to their sequence homology
and functional similarity: 1) the rhodopsin kinase (GRK1) which is
predominantly localized to the retina and phosphorylates light-bleached
(agonist-activated) rhodopsin in rod outer segments; 2) the GRK2
subfamily including GRK2 and GRK3, which are more widely distributed;
and 3) the GRK4 subfamily including GRK4, GRK5, and GRK6. GRK4 is
localized primarily to the testes whereas GRK5 and GRK6 are more
ubiquitously expressed. In unstimulated cells, members of the GRK2
family are predominantly localized to the cytosol. Plasma membrane
association is triggered by receptor activation and mediated by
interactions with G protein ß
-subunits. Conversely, members of the
GRK4 subfamily exhibit substantial association with plasma membrane in
the absence of agonist stimulation. Plasma membrane association is
mediated either by palmitoylation (GRK4 and GRK6) or by interactions
with membrane phospholipids (GRK5) (10). GRK specifically
phosphorylate the agonist-activated form of the receptor and, unlike
second messenger-dependent kinases, promote the binding of cytosolic
proteins, arrestins, which further uncouple the receptors by preventing
receptor-G protein interaction. Several arrestin family members have
been identified and, among them, only ß-arrestin 1 and ß-arrestin 2
are ubiquitously expressed (9, 10).
Growing evidences support the hypothesis that GRK are important modulators of ß-adrenergic signaling in vivo. Indeed, myocardial overexpression of GRK2 or GRK5 in mice impairs ß-AR/G protein coupling (11, 12). Furthermore, ß-AR desensitization taking place in human heart failure is related to an increased GRK activity (13). In myometrium, only very few investigations have been performed on GRK expression (14, 15), and none of these studies have investigated possible alterations of GRK activity near parturition. The aim of the present study was thus 1) to assess GRK activity and expression in rat myometrium in the last stages of pregnancy; and 2) to investigate a possible temporal relationship between GRK activity and ß-AR/G protein uncoupling.
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Materials and Methods |
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-32P]ATP (6,000 Ci/mmol) were obtained
from NEN Life Science Products (Les Ulis, France).
Cesium-trifluoroacetate and prepacked oligo(dT)-cellulose columns were
from Pharmacia LKB Biotechnology Inc. (Pharmacia Biotech, Orsay, France). Urea-treated bovine rod outer segments
were obtained from Dr. N. Bennett (CEA, Grenoble, France) and
prepared as described (16). The pCMV-bovine GRK2 and the
pRK5-human GRK5 were kindly provided by Dr. S. Cotecchia (Lausanne,
Switzerland). The pBSKS-rat GRK3 was provided by Dr. R. Lefkowitz
(Durham, NC). The pBSKS-rat GRK4 and the rat GRK6 probe were kind gifts
from Dr. J. M. Elalouf (CEA, Saclay, France). Cell culture
reagents were purchased from Life Technologies, Inc.
(Cergy Pontoise, France). Polyclonal antibody anti-GRK2 was kindly
donated by Dr. F. Mayor, Jr. (Universitad Autõnoma, Madrid,
Spain) and was raised against the N-terminal amino acids 50–145 of the
bovine GRK2. Polyclonal antibody anti-GRK5 (N-terminal peptide) was a
kind gift of Dr. F. Boulay (CEA, Grenoble, France). Antibodies against
GRK3 and GRK6 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibodies (mAbs) directed
against glutathione-S-transferase-fusion proteins
containing C-terminal domains of GRK2/3 or GRK4–6 were obtained from
Upstate Biotechnology, Inc. (Euromedex, Souffelweyersheim,
France). The mAb F4C1, raised against the highly conserved epitope
DGVVLVD, identical in ß-arrestins, was provided by Dr. L. A.
Donoso. Antirabbit and antimouse IgG horseradish peroxidase conjugates
were from Amersham Pharmacia Biotech (Les Ulis, France).
Immunoprecipitation of GRK3 was performed with the IMMUNOcatcher kit
from CytoSignal (Interchim, Montluçon, France).
N-ethylmaleimide (NEM), (-)-isoproterenol-HCl,
(±)-propranolol-HCl, 5'-guanylylimidodiphosphate (Gpp(NH)p), and all
other reagents of the highest grade commercially available were from
Sigma (L’Isle d’Abeau, France).
Animals
Sprague Dawley rats (250–300 g) were obtained from Iffa-Credo
(L’Arbresle, France). They were maintained in accordance with the
guidelines for care and use of laboratory animals (NIH Guide). The
females were caged with males overnight, and successful mating was
determined by the presence of spermatozoa in the vaginal smear (day 1
of pregnancy). In our breeding colony, parturition occurs between
1200 h and 1900 h of day 22 of pregnancy for 80% of rats
(17). Animals were killed by cervical dislocation either
in the morning of day 22 of pregnancy (preparturient, P) or in the
afternoon when the first pup is expelled (term, T), unless otherwise
indicated. Uterine horns were immediately excised and the fetoplacental
units were removed. Myometrial tissues were then rapidly trimmed of fat
and connective tissues and scraped from adherent endometrium. They were
then stored at -80 C until used.
GRK activity
Assessment of GRK activity was made both on plasma membrane and
cytosolic fractions. Myometrial plasma membranes were prepared as
previously described (18). Briefly, myometrium was
homogenized in 4 vol of ice-cold lysis buffer A (20 mM
Tris-HCI, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1
mM phenylmethylsulfonylfluoride, 20 µg/ml leupeptin, 20
µg/ml benzamidine) containing 250 mM sucrose. The
supernatants of two successive 20,000 x g
centrifugations were pooled and centrifuged at 50,000x g
for 60 min at 4 C to obtain the membrane pellet. The cytosolic fraction
was obtained after a final centrifugation of 250,000 x
g during 60 min at 4C. GRK activity in both fractions was
assessed by light-dependent phosphorylation of purified urea-treated
rod outer segments as substrate. For membrane fractions, extraction of
peripheral proteins with 200 mM of NaCl
was performed before GRK assay to dissociate membrane-associated GRK
(19). Protein concentration of the fractions was
determined by the method of Bradford (20) using BSA as a
standard. Myometrial cytosolic or plasma membrane proteins (100 µg)
were incubated with purified urea-treated rod outer segments (300–400
pmol of rhodopsin) in a 50 µl reaction buffer containing 20
mM Tris-HCl, pH 7.5, 2 mM
EDTA, 6 mM MgCl2, and 100
µM [-32P] ATP
500–3(2,500–3,500 cpm/pmol). The phosphorylation reactions were incubated
for 20 min at room temperature, in the dark or in the light as
indicated, and then quenched by diluting 20-fold with ice-cold buffer A
and centrifuging at 12,000 x g for 15 min. The
resulting pellets were electrophoresed on a 10% SDS polyacrylamide gel
and autoradiographed 15–18 h at -80 C. Quantification of GRK activity
was done by densitometric scanning and computer analysis using the NIH
image 1.62 program. GRK activity was also quantified by measuring the
radioactivity associated with the excised rhodopsin band in the dried
gel by liquid scintillation spectrometry. In some experiments,
rhodopsin phosphorylation was assessed in the presence of heparin (1
mg/ml) in the reaction buffer. Rhodopsin phosphorylation assay was also
performed after preincubation of extracts for 30 min at 37 C with 37.5
µg of specific mAbs directed against GRK2/3 or GRK4–6
(21).
Characterization of myometrial GRK in term pregnant rat
Transcripts. Northern blot analysis was performed as
previously described (18). Poly(A)+
RNA (8 µg) isolated from myometrium or several other tissues by the
guanidium isothiocyanate/cesium trifluoroacetate gradient method were
subjected to electrophoresis in 1% agarose-formaldehyde gel and
transferred to GeneScreenPlus membranes (NEN Life Science Products). Hybridizations were performed using random-primed
cDNA fragments including most of the catalytic domain. Probes
corresponded to coding regions: bp 545–1,262 for bovine GRK2, bp
584–1,057 for rat GRK3, bp 423–1,034 for rat GRK4, bp 159–1,316 for
human GRK5. The probe used for GRK6 corresponded to bp 591–976 of rat
GRK6 coding sequence (22). The probes were obtained either
by digestion of cDNA by restriction enzymes or by selective
amplification of the sequence by PCR. RNA blots were hybridized as
described previously (23) and subjected to autoradiography
at -80 C for 1–3 days. Transcript size was determined by comparison
with an RNA kilobase ladder (Life Technologies, Inc.).
GRK3 expression was also studied by RT-PCR analysis using kits from Life Technologies, Inc. cDNA was generated from 5 µg of myometrial total RNA according to the manufacturer’s instructions and the RT products were stocked at -80 C. No PCR product was detected in the absence of RT indicating that the RNA preparations were free of genomic DNA. Amplification of a 474-bp sequence from nucleic acids 584 to 1,057 was generated using specific 21-bp primers. Reactions were cycled 40 times for 1 min at 94 C, 1 min at 56 C, and 1 min at 72 C followed by a final 10-min extension at 72 C. The amplified fragment was visualized by electrophoresis of the reaction mixture on ethidium bromide containing 2% agarose gel. RT-PCR was also conducted on brain total RNA as control.
Proteins. For immunoblot analysis, proteins (100 µg) from plasma membranes or cytosol were subjected to 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane filters (NEN Life Science Products). Blots were probed with the antibodies indicated above, and the resulting bands were visualized by enhanced chemiluminescence (NEN Life Science Products). Recombinant GRKs were used as positive controls for immunoblotting. For GRK2 and GRK3, COS-7 cells were grown in DMEM with 10% FBS, streptomycin (100 ng/ml), and penicillin (100 U/ml) and transfected by the diethylaminoethyl-dextran method as previously described (24). COS-7 cells were incubated for 3 h with 10 µg pCMV plasmids expressing either bovine GRK2 or GRK3. After this period, cells were incubated in DMEM with 10% FBS, streptomycin (100 ng/ml), and penicillin (100 U/ml) for 48 h. Cytosolic fractions containing the overexpressed proteins were then obtained by a 10,000 x g centrifugation for 20 min. Cytosol of 293 cells transfected by GRK 5 and 6 was kindly provided by Dr. E. Reiter (Nouzilly, France).
Immunodetection of myometrial GRK3 was performed on detergent-solubilized extract after immunoprecipitation using the IMMUNOcatcher kit (CytoSignal, Interchim, France). Briefly, myometrium was homogenized in ice-cold mild lysis solution. Extract obtained after centrifugation was precleared by adding preimmune serum. GRK3 was immunoprecipitated with the anti-GRK2/3 mAb (1:20) for 12 h at room temperature. The antibody-antigen complex was then precipitated using protein A/G Sepharose for 30 min. The immune complex was electrophoresed on 7.5% SDS-PAGE. GRK3 was immunoblotted with the anti-GRK2/3 mAb (1:500) used for immunoprecipitation.
ß-AR radioligand binding studies
Radioligand binding studies were performed as described
previously (3). Briefly, membranes (150 µg proteins)
were incubated with [125I]-cyanopindolol
(25–600 pM), and nonspecific binding was determined in the
presence of 10 µM (±)-propranolol. Assays were performed
in duplicate, incubated for 1 h at 25C, and terminated by rapid
filtration on APF/C filters (Millipore Corp., St-Quentin
en Yvelynes, France). Low-affinity (RL) and
high-affinity (RH) states of ß-AR were
quantified as previously described (23). Low-affinity
ß-AR were quantified after preincubation (20 min at 30 C) of
membranes (150 µg proteins) in the binding buffer containing 10
µM isoproterenol and 0.5 mM NEM, which
stabilizes the ternary agonist-receptor-Gs protein complex
(25). The membranes were then washed twice and the
remaining low-affinity receptors were quantified by
[125I]-cyanopindolol binding. The number of
total ß-AR was determined by incubating myometrial membranes with
[125I]-cyanopindolol in the presence of 0.1
mM Gpp(NH)p. High-affinity ß-AR density was estimated as
total minus low-affinity ß-AR. Data from saturation binding
experiments were analyzed according to the method of Scatchard.
Data analysis
Results are expressed as mean ± SEM.
Statistical analyses were performed using unpaired Student’s
t test. Values were considered statistically different when
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
consistent with the phosphorylation of rhodopsin. In both fractions,
rhodopsin phosphorylation was dependent on light exposure since only
faint phosphorylation could be detected in the dark. Furthermore, the
light-dependent phosphorylation of rhodopsin was completely inhibited
by the addition of 1 mg/ml of heparin (Fig. 1), which is known to
inhibit GRK phosphorylation activity (26). Altogether,
these results argue for the existence of a GRK activity in both
fractions from late pregnant rat myometrium. Quantification of GRK
specific activity in the two subcellular fractions indicated that
cytosolic activity was approximately 2- to 3-fold higher than plasma
membrane-associated GRK activity whatever the stage of pregnancy
(midpregnant, preparturient, or parturient stage). In myometrium from
late pregnant rats, we measured an average specific activity of
0.049 ± 0.009 pmol phosphate/min/mg of proteins in the cytosol
and of 0.018 ± 0.003 pmol phosphate/min/mg of proteins in plasma
membranes.
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). In plasma membrane extracts,
only anti-GRK4–6 mAbs significantly inhibited GRK activity (Fig. 2).
These results, which characterized GRK subfamilies in the myometrium of
late pregnant rats, also demonstrated that members of the GRK2
subfamily are predominantly expressed in the cytosol whereas members of
the GRK4 subfamily exhibit substantial membrane association.
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). As expected,
the predominant signals were visualized in the brain. In comparison,
GRK2 transcripts were also strongly expressed in the myometrium. In all
the tissues studied, GRK5- and GRK6-specific probes both detected
single transcripts of approximately 3 and 2.8 kb, respectively (Fig. 3), with a higher density in the myometrium as compared with heart or
with brain and testis. The GRK4-specific probe identified the expected
3-kb transcript in testis only (Fig. 3), confirming the restricted
expression of this kinase. Using a GRK3-specific cDNA probe, a
transcript of approximately 7.2 kb was detected in brain but not in the
myometrium. However, using a more sensitive technique, i.e.
RT-PCR, GRK3 transcript was detected in myometrium. The PCR products
amplified from brain and myometrial RNA were obtained as single bands
of the expected length. The amount of PCR products, evaluated by
ethidium bromide staining, was considerably lower in myometrium than in
brain (data not shown).
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).
Using the same experimental approach, GRK3 was not detected in the
myometrium (Fig. 4), while the antibody efficiently labeled the
recombinant protein of 79 kDa. Further examination of GRK3 expression
by immunoprecipitation of solubilized myometrial and brain homogenates
using monoclonal anti-GRK2/3 revealed two immunoprecipitated proteins.
One predominant band comigrated with recombinant GRK2 and another faint
band comigrated with recombinant GRK3 (Fig. 5). These results revealed that
GRK3 is weakly expressed in rat myometrium. In plasma membranes,
antibodies against GRK5 and GRK6 specifically detected proteins of 68
and 66 kDa, respectively, which both comigrated with the respective
recombinant proteins (Fig. 4).
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Alterations of GRK activity and expression at parturition
We investigated possible alterations of GRK activity in the
myometrium at the end of pregnancy. For this purpose, we evaluated GRK
activity both in cytosolic and plasma membrane fractions obtained from
rats in the preparturient state or at parturition. Our results
demonstrated that plasma membrane-associated GRK activity was
approximately 2-fold higher at parturition (+190 ± 32%
vs. preparturient state, P < 0.05) whereas
cytosolic GRK activity was simultaneously decreased (-46 ± 8%
vs. preparturient state, P < 0.05) (Fig. 6A).
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).
Enhanced levels of membrane-associated GRK2 may contribute to the
observed increase of membrane-associated GRK activity.
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Coupling of ß-AR was thus assessed at the same stages of pregnancy by
evaluating the number of receptors in the high-affinity state, using
[125I]-cyanopindolol in the presence of NEM. In
myometrial plasma membranes from preparturient rats, the percentage of
high-affinity ß-AR was 64 ± 1% (Fig. 6B
). At an earlier stage
of pregnancy (day 15), the number of high-affinity ß-ARs was in the
same proportion, i.e. 72 ± 3%. At parturition, this
percentage was markedly reduced (-30%) since we measured only 42
± 5% of high-affinity state ß-AR (Fig. 6B). This uncoupling
occurred without any alterations in the number of total myometrial
ß-AR. Indeed, the number of myometrial ß-AR, as assessed by
[125I]-cyanopindolol binding, was 156 ±
11 fmol/mg protein in the preparturient stage and 167 ± 11
fmol/mg protein at parturition. Furthermore, the dissociation constant
(KD) value for the
[125I]-cyanopindolol was unchanged over this
period of time (0.13 ± 0.02 nM and
0.17 ± 0.03 nM in preparturient and
parturient myometrium, respectively).
These results revealed that, at parturition, myometrial ß-AR uncoupling is concomitant with the increase of membrane-associated GRK activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GRK characterization was performed using three different and complementary technical approaches: functional assay using specific mAbs, Western and Northern blotting. The fact that anti-GRK2/3 and anti-GRK4–6 mAbs efficiently blocked rhodopsin phosphorylation demonstrates that members of both subfamilies are present in rat myometrium. Moreover, this approach gives information on myometrial GRK subcellular distribution. Indeed, marked inhibition of rhodopsin phosphorylation by GRK2/3-specific mAbs in soluble fractions is in agreement with the well-established concept of a cytosolic localization of these kinases. Significant inhibition of GRK activity in plasma membranes by anti-GRK4–6 mAbs revealed that myometrial native GRK5 and/or 6 are mainly associated with plasma membranes. Such localization was demonstrated by Premont et al. (27) using purified GRK5. Small amounts of myometrial native GRK6 appear to be also present in the cytosol as evidenced by immunodetection (data not shown) and may explain why anti-GRK4–6 mAbs also blocked some cytosolic GRK activity.
We have characterized both GRK transcripts and proteins in the pregnant rat myometrium. All three transcripts identified by Northern blotting were of the reported size (22, 28, 29) and were highly expressed in pregnant rat myometrium as compared with testis and heart used as controls. As mentioned in Results, the GRK4 transcript was not present in rat myometrium, and the expression of this transcript was restricted to the testis, in agreement with previous works (30). Characterization of GRK by Western blot analysis confirmed the presence of GRK2, GRK5, and GRK6 in the pregnant myometrium. All protein identifications were validated by coimmunoblotting of the respective recombinant proteins. We have also identified in the pregnant myometrium ß-arrestin 1 and ß-arrestin 2 (data not shown), which co-act with GRK to uncouple G protein-coupled receptors.
Amplification of GRK3 transcript by RT-PCR or immunoprecipitation before protein detection have revealed that GRK3 is only slightly expressed in pregnant rat myometrium. Previously, Benovic et al. (31) indicated that the GRK3 transcript is present at much lower levels than GRK2 (<10%) in peripheral tissues. Low expression of GRK3 has also been reported in the heart, where GRK2 and GRK5 appear to be predominantly expressed (27, 32). In pregnant term and nonpregnant human myometrium, GRK3 was not found in myometrial tissue by using immunoblotting or RT-PCR analysis (15). Our findings concerning GRK3 are different from those reported by Ruzycky and DeLoia (14), who identified as GRK3 a transcript of 1.4 kb and a protein of 69 kDa in pregnant rat myometrium. Such identification remains questionable since reported size and apparent molecular mass values are not in agreement with the established ones (31) and thus probably do not indicate GRK3 myometrial expression. Using all GRK antibodies, we have detected, in most cases, a protein of an apparent molecular mass close to 69 kDa (data not shown). The result obtained by Ruzycky and DeLoia could thus correspond to a nonspecific signal or to a GRK-related protein.
In rat uterus, we have clearly demonstrated a GRK activity by the
ability of myometrial extracts to phosphorylate rhodopsin. Myometrial
GRK activity was detected both in cytosolic and plasma membrane
fractions, and we reported significant changes of GRK activities in
those fractions in the last 6–10 h before parturition. Interestingly,
at term, plasma membrane-associated GRK activity was markedly
increased. This event is linked to a concomitant decrease of GRK
activity in soluble extracts, demonstrating an apparent translocation
of GRK activity. GRK2 contributes, at least in part, to the observed
increase of GRK activity in myometrial membranes at parturition.
Indeed, increase of GRK activity is associated with a parallel increase
of the amounts of plasma membrane-associated GRK2 but not of GRK5 or 6.
Since translocation of GRK2 is a prerequisite of its activation
(10), this argues for its involvement in a total increase
of GRK activity. In addition, the increased responsiveness of the
uterus, near parturition, to contractile factors acting via the
phospholipase C/protein kinase C (PKC)/calcium pathway further argues
for a contribution of GRK2. Indeed, GRK2 is activated by PKC and
relatively insensitive to calcium/calmodulin inhibition
(10). Conversely, GRK5 is inhibited by PKC and much
sensitive to calcium/calmodulin inhibition (IC50
50 nM for GRK5 vs. 2
µM for GRK2). Nevertheless, this does not
exclude a contribution of GRK5/6 in increased myometrial GRK activity
at parturition.
GRK-mediated G protein-coupled receptor phosphorylation plays a key role in receptor/G protein uncoupling and desensitization (10). An important finding of our work is that the increase in plasma membrane-associated GRK activity takes place concomitantly with ß-AR uncoupling. Indeed, at this ultimate phase of pregnancy, the proportion of ß-AR in the high-affinity state sharply decreases in the myometrium. In vivo, coincidence of GRK activation and ß-AR uncoupling, has been exclusively described in rat neonatal liver immediately after birth (19) and in myocardium during experimentally induced ischemia (33). Interestingly, the extent of changes of GRK specific activities is similar to that obtained in our present study on myometrium. Based upon recent findings of Dodge et al. (34), implication of PKA, which also contributes to phosphorylation and uncoupling of ß-AR, may be of limited importance. Indeed, these authors reported that PKA concentration and activity are dramatically reduced in myometrial plasma membranes of late pregnant rat. Altogether, these results suggest that GRK activation may be an important mediator of myometrial ß-AR desensitization at parturition. This hypothesis is further supported by the fact that GRK2 translocation is induced by incubation of myometrial strips with the ß-agonist isoproterenol and blocked by coincubation with a ß-antagonist (our preliminary unpublished data). Our results, however, do not exclude a potential involvement of GRK2 in the desensitization of other G protein-coupled receptors, such as oxytocin or muscarinic receptors, which are activated at term.
In summary, this study shows that uncoupling between the ß-AR and Gs proteins takes place concomitantly with an increase in plasma membrane-associated GRK activity at the end of pregnancy. This suggests that alterations of ß-adrenergic signaling at parturition may be triggered by GRK and that such a mechanism may be relevant for the initiation of uterine contractions and normal delivery in the rat.
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Acknowledgments |
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Received July 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2/ß2-adrenergic receptor-coupled adenyl cyclase system in rat
myometrium during pregnancy. J Biol Chem 270:11012–110161b-adrenergic
receptor mutants display different phosphorylation and internalization
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38 kDa) is shown.
