©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characteristics of the /-Adrenergic Receptor-coupled Adenylyl Cyclase System in Rat Myometrium during Pregnancy (*)

Sakina Mhaouty , Joelle Cohen-Tannoudji , Rachel Bouet-Alard , Isabelle Limon-Boulez , Jean-Paul Maltier , Chantal Legrand(§)

From the (1) Laboratoire de Physiologie de la Reproduction, CNRS URA 1449, Université P. M. Curie, 4 Place Jussieu, 75230 Paris Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

- and -adrenoreceptors (AR), identified by Northern blotting in rat myometrium, showed a differential expression during the course of pregnancy. Indeed, the -AR transcript was present at mid-pregnancy, whereas high levels of -AR mRNA could be detected at term. The role of these subtypes in modulating -AR-stimulated adenylyl cyclase activity was investigated on myometrial membranes from mid-pregnancy and term. At nanomolar concentrations of clonidine (full -AR agonist) or oxymetazoline (partial -AR agonist), adenylyl cyclase activity was inhibited by up to 50 ± 7% at mid-pregnancy or 75 ± 7% at term, whereas at micromolar concentrations, -AR agonists potentiate adenylyl cyclase activity by 140-170% at mid-pregnancy. Both inhibitory and stimulatory components of this biphasic response were blocked by yohimbine, a selective -AR antagonist. Preincubation of myometrial membranes with G and/or G antisera eliminated -AR mediated attenuation or potentiation of isoproterenol-stimulated adenylyl cyclase, thus indicating that both the inhibitory and stimulatory components are mediated via G and G. In addition, type II and IV adenylyl cyclases were identified by Northern blotting in the pregnant rat myometrium. Altogether these data strongly suggest that the -AR at mid-pregnancy potentiates adenylyl cyclase types II and IV through released from G and G proteins, whereas the -AR expression at term may be related to persistent inhibition.


INTRODUCTION

The rat myometrium has a rich adrenergic innervation (1) . Activation of the adrenergic nerves modulates uterine contractility and also subtle changes in sensitivity of the smooth muscle to hormonal agents (2) . Direct interaction of norepinephrine with the -AR() induces myorelaxation via the G proteins coupled -AR/cAMP cascade, whereas stimulation of the -AR signaling pathway elicited contraction. We have previously shown that progesterone, which culminates during mid-pregnancy (3) , enhances myometrial -AR responsiveness to catecholamines by controlling the rate of transcription of the gene encoding the -AR (4) and by increasing the high affinity state, R, of this receptor (5) . The existence of multiple -AR subtypes is supported by several lines of evidence, the strongest of which are the cloning of three subtypes in human and rat (6, 7, 8, 9, 10, 11, 12, 13) , the development of specific drugs, and the characterization of several cell lines that express a single subtype (14, 15) . These receptors, present in a variety of tissues or cell lines, were both defined as receptors of high affinity for the agonists clonidine and UK14304 and for the antagonists yohimbine and rauwolscine. Recently, our pharmacological data strongly suggest that rat myometrium could express the - and the -AR subtypes (16) . To date, in many systems examined, -AR have been described as being coupled to G with agonist occupancy resulting in a decrease of adenylyl cyclase activity. However, the -AR has also been reported to stimulate adenylyl cyclase or cAMP accumulation in various cell lines (17, 18) or in stably transfected cells (19, 20, 21, 22) . In addition, the -AR may couple to other intracellular pathways involving phospholipase A2, phospholipase C, and Na/H exchange ion channels (for review, see Refs. 23 and 24).

In the present study, we first provided a molecular analysis of the -AR expressed in the rat myometrium during the course of pregnancy. Then, we examined the question of whether activation of -AR regulates -AR-stimulated adenylyl cyclase activity. Finally, we identified the G proteins involved in -AR coupling and the type(s) of adenylyl cyclase expressed in myometrium in order to investigate by which mechanism -AR may cross-regulate the -AR pathway.


EXPERIMENTAL PROCEDURES

Chemicals and Reagents

I-cAMP assay system and [H]rauwolscine (88 Ci/mmol) were purchased from Amersham Corp.; [-P]dATP (3000 Ci/mmol) and G protein antisera were obtained from DuPont NEN. Pertussis toxin; isoproterenol; clonidine-, oxymetazoline-, and yohimbine-hydrochloride; and all other reagents of the highest grade commercially available were from Sigma. UK14304 tartrate was a gift from Pfizer.

Animals

Sprague-Dawley rats were obtained from Iffa-Credo (L'arbresle, France). 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 12 and 19 h on day 22 for 80% of rats.

Radioligand Binding Assays

The preparation of membranes for the radioligand binding assays from pregnant myometrium was as described previously (16) . For competition experiments, 10 nM [H]rauwolscine (a concentration near the Kvalue for that tissue) and 14-18 concentrations (1 pM to 1 mM) of unlabeled competing drugs were added to duplicate tubes. Nonspecific binding was determined in the presence of 10 µM phentolamine. Binding data were analyzed using nonlinear least squares curve-fitting program (Graph PAD Software) to obtain IC values. IC values were converted to Kvalues by the Cheng and Prussoff equation (25) .

Adenylyl Cyclase Assays

Adenylyl cyclase activity in myometrial cell membranes was measured as described previously (5) . Briefly, 20-µl membrane fractions (2.5 mg of protein/ml) were incubated in 100 µl of a medium containing 50 mM Tris (pH 7.4), 5 mM MgSO, 5 mM creatine phosphate, 12 units of creatine phosphokinase, 0.5 mM ATP, 1 mM isobutylmethylxanthine and 0.1% bovine serum albumin, for 10 min at 30 °C. Reactions were stopped by transferring the tubes to a boiling water bath for 3 min. After centrifugation at 2500 g for 30 min at 4 °C, cAMP accumulation in the supernatant was determined using the Amersham cAMP radioimmunoassay system. When present in the assay, isoproterenol was used at a concentration of 0.1 mM, which we have found to be optimal for promoting -AR stimulation of adenylyl cyclase activity in the presence of 0.1 mM GTP (5) . Clonidine or oxymetazoline was used at a range of concentrations between 0.1 nM and 1 mM. In some studies, membrane suspensions were preincubated either with polyclonal antisera directed against the carboxyl-terminal portion of G proteins (RM1, anti-; AS/7, anti-/; EC/2, anti-/; QL, anti-/) for 2 h at 4 °C at a dilution of 1:50, which is maximally effective to prevent functional interaction between G protein and activated receptor or with 10 µg/ml of preactivated pertussis toxin for 30 min at 30 °C. We have previously tested (data not shown) that, at this saturating concentration, pertussis toxin causes full inactivation of myometrial G proteins.

RNA Preparation and Blotting

Total RNA was extracted from myometrium and other tissues (brain, spleen, neonatal and adult lung, kidney) by the cesium-trifluoroacetate gradient method (26) as described previously (4) . Poly(A) RNA were purified from total RNA using prepacked oligo(dT)-cellulose columns (Pharmacia Biotech Inc.). Poly(A) RNA (10 µg) were then electrophoresed in a 1% agarose gel containing formaldehyde (27) and transferred to GeneScreen Plus membranes (DuPont NEN) by capillatory blotting.

cDNA Probes

Probes specific of each -AR subtype were chosen from regions coding for the putative third intracellular loop of the receptors since these sequences are divergent between the different subtypes. The 333-base pair KpnI- XmnI and 466-base pair NaeI- StuI fragments were derived, respectively, from the rat brain and cDNA (kindly provided by Drs D. E. Handy and C. S. Flordellis) and were used as subtype-specific probes (28) . Transformation in Escherichia coli, plasmid preparation, and cDNA purification were performed by standard molecular biology methods (29) . A fragment specific of the -subtype was amplified from rat genomic DNA using polymerase chain reaction. The primers 5`-CGCAGCCACTGCAGAGGTCT-3` (sense) and 5`-AGTCGCCCCACTAGTCCCCT-3` (antisense) were chosen and provided amplification of a 279-base pair sequence from nucleic acids 981 to 1259 (10) . The polymerase chain reaction product was subcloned into pCR II vector (Invitrogen), and its sequence was verified by the Sanger dideoxynucleotide termination method using T7 DNA polymerase (Pharmacia).

pBluescripts KS and SK with the full-length type II cDNA (3.5 kb) and type IV cDNA (3.2 kb) adenylyl cyclase respectively were kind gifts from Dr. R. R. Reed (30) and Dr. W. J. Tang (31) . For both types, hybridizations were performed with the full-length cDNA. All of the cDNA probes were labeled by random priming with [-P]dATP to a specific activity averaging 10 dpm/µg. Unbound radioactivity was separated by gel filtration using Sephadex G-50 DNA grade (Pharmacia).

Hybridization Procedures

The membranes were prehybridized at 42 °C for 4 h in a buffer containing 45% formamide, 4 SSC, 5 Denhart's solution, 100 mM NaHPO, pH 6.6, 0.5% SDS, and 75 µg/ml denatured salmon sperm DNA and then hybridized overnight at 42 °C in the same medium containing 10% dextran sulfate and 10 cpm/ml denatured cDNA probes. After hybridization, membranes were washed with a final stringency of 0.1 SSC, 0.1% SDS at 55 °C (adrenoreceptor probes) or 0.2 SSC, 0.2% SDS at 55 °C (adenylyl cyclase probes) and exposed to Kodak X-Omat AR films at -80 °C for 1-5 days. The intensities of the bands were determined by scanning densitometry. Size estimates of the RNA species were established by comparison with an RNA ladder (Life Technologies, Inc.). Hybridization with an oligo(dT) probe (Pharmacia) was used as an internal control to estimate the amount of poly(A) RNA loaded in each well and transfer efficiency.


RESULTS AND DISCUSSION

Hybridization of poly(A) RNA with DNA fragments specific for the three subtypes of the rat -AR clearly demonstrates the presence and the differential expression of the - and -AR genes in the rat myometrium during the course of pregnancy. Fig. 1 A shows a high level of the 3.8-kb mRNA of the -subtype in rat myometrium. The -AR-specific probe also hybridized with a 3.8-kb transcript in rat tissues known to express this receptor subtype (brain, spleen) and did not detect any signal in liver known to express only the -AR subtype (Fig. 1 A) (28) . A weak hybridization to a band of 4.7 kb could also be detected; the identity of this mRNA is still unknown (32, 33) . A 4.2-kb message encoding the -AR subtype was also found in myometrium (Fig. 1 A) as in other tissues, which express this subtype (for review, see Refs. 24 and 28). As expected from previous data (11) , the -AR specific probe hybridized to a single band of 3 kb in rat brain. No hybridization could be detected in rat myometrium at any stage of pregnancy (Fig. 1 A). As shown in Fig. 1 B, the -AR transcript could be detected at all of the stages of pregnancy. Quantification of the signal indicated that the -AR mRNA is significantly higher (4-fold) at mid-pregnancy (day 10 and day 12) than in the later stages, where it remains constant. Conversely, the -AR transcript could not be detected until day 17 of pregnancy. The -AR mRNA level then increases by 110% from day 17 to term (Fig. 1 B).


Figure 1: RNA blot analysis of -AR subtype expression in rat myometrium. A, identification of -AR transcripts. Poly(A) RNA (10 µg) isolated from myometrium ( M), brain ( B), spleen ( S), liver ( Li), kidney ( K), and neonatal lung ( nL) were hybridized to P-labeled -AR ( a), -AR ( b), and -AR ( c) cDNA probes, as described under ``Experimental Procedures.'' Position of co-electrophoresed RNA size markers (in kb) are shown. B, evolution of the steady-state level of -AR ( a) and -AR ( b) transcripts in rat myometrium during pregnancy. Autoradiographic signals were quantified by densitometric scanning from day 10 of pregnancy ( D10) to term ( T). The results were expressed as the changes observed in the amount of -AR subtype mRNA compared with values of day 21, as arbitrary units. Values are mean ± S.E. of three to six independent determinations. ★ indicates a statistically significant difference from day 21 ( p < 0.05; nonpaired Student's t test).



Since we have found that -AR and -AR subtypes were colocalized in the same layer of the myometrium (16, 34) , we then explored the ability of selective -AR agonists to alter isoproterenol-stimulated adenylyl cyclase activity in myometrial plasma membranes from mid-pregnancy or term. We first examined the potency of clonidine, UK14304, or oxymetazoline to inhibit [H]rauwolscine binding to myometrial cell membranes. As shown in , clonidine had a higher affinity than UK14304 for the rat myometrial -AR. On the other hand, preincubation of myometrial membranes with the selective -AR antagonist propranolol or with the antiserum RM/1 raised against the C-terminal decapeptide of s-subunits abolished the isoproterenol-mediated stimulation of adenylyl cyclase (). Fig. 2 shows the dose-response curves for the effects of clonidine on isoproterenol-stimulated adenylyl cyclase in myometrial membranes; at mid-pregnancy, clonidine induced a biphasic response exhibiting both inhibitory and stimulatory components with EC values of 1.27 ± 0.01 and 1344 ± 72 nM, respectively. Maximal inhibition (50 ± 7% versus 75 ± 7% at term, p < 0.05) obtained at low concentration (10 nM) of clonidine occurred when 20-25% -AR are occupied as indicated by [H]rauwolscine competition data. At mid-pregnancy, higher concentrations (1-100 µM) of clonidine increased adenylyl cyclase activity to 140-170% over that elicited by isoproterenol alone. Maximal stimulation of adenylyl cyclase at 10 µM clonidine ( i.e. 1000-fold greater agonist concentration) represents 85-90% -AR occupancy. Inhibition and potentiation of isoproterenol-stimulated adenylyl cyclase could be blocked by coincubation with the -AR-selective antagonist yohimbine at a concentration that occupied 99% of [H]rauwolscine binding sites (Figs. 3 and 4). These results demonstrated the implication of -AR in both inhibition and potentiation. Furthermore, at mid-pregnancy, oxymetazoline also produced a biphasic response with 55 ± 1% inhibition at 1 µM and 140 ± 7% potentiation at 1 mM. At the same range of concentrations, UK14304 elicited only very minimal inhibition (31 ± 2%) and no potentiation of isoproterenol-stimulated adenylyl cyclase activity. The lack of efficiency of this imidazoline compound could be due to its lower affinity for the rat myometrial -AR (our present data and Ref. 35). Thus, all other experiments have been conducted in the presence of clonidine. By themselves, all three -AR agonists do not significantly activate basal adenylyl cyclase (data not shown). Our results, obtained from in vivo studies, strongly suggest that at mid-pregnancy activation of -AR subtype results in a biphasic response, whereas at term the highly expressed -AR transcript could be related to persistent inhibition of adenylyl cyclase. These data resemble those reported in cell lines stably transfected with - (19, 22) or -AR (18, 21) . In addition, agonist potencies in our experiments are comparable with those from these other studies.


Figure 2: Effects of clonidine on isoproterenol-stimulated adenylyl cyclase activities in myometrial plasma membranes. Adenylyl cyclase activity was measured as described under ``Experimental Procedures.'' Data are expressed as percent of isoproterenol-stimulated adenylyl cyclase activity (control 100%). Results are mean ± S.E. of four to seven independent determinations. The dose response significance for inhibition and potentiation was analyzed by one-way analysis of variance followed by Ducan's multiple range test.



It is well known that pertussis toxin ADP-ribosylates G proteins, which then remain in an inactive conformational state, resulting in an ablation of their functional interaction with receptor or effector enzyme. After treatment of myometrial membranes with this toxin, the clonidine-induced inhibition of adenylyl cyclase activity was completely abolished, indicating that coupling to a G protein is involved. Furthermore, adenylyl cyclase activity was 2.5-3-fold higher than that measured in the presence of isoproterenol alone (Fig. 3). In the absence of clonidine, when membranes have been pretreated with pertussis toxin, isoproterenol-stimulated adenylyl cyclase was also consistently increased (110 ± 6 and 99 ± 13 pmol cAMP/mg of protein/10 min, respectively, at mid-pregnancy and term; the values are for three experiments). To explain these data, it is conceivable to assume that high concentrations (0.1 mM) of GTP, when present in the assay system, were generally effective to activate the G protein signaling pathway, even in the absence of any inhibitory input. This lead us to conclude that a tonic inhibition exists in the rat myometrium during pregnancy that could be due to the high levels of G and G proteins (36, 37) . At term, the level of ADP-ribosylated G proteins was decreased (38) , and this may account for the decline of adenylyl cyclase stimulability (). These observations suggest a physiological importance of these two competing pathways (G and G) in the coupling mechanisms that dictate adenylyl cyclase responsiveness in the pregnant myometrium.


Figure 3: Effects of yohimbine, pertussis toxin, and G antisera on clonidine inhibition of isoproterenol-stimulated adenylyl cyclase activity of myometrial membranes at mid-pregnancy ( A) or term ( B). Adenylyl cyclase activity was measured as described under ``Experimental Procedures'' in the presence of isoproterenol (0.1 mM) and GTP (0.1 mM) plus clonidine (100 nM). Yohimbine was used at the concentration of 10 µM. Membranes were preincubated with 10 µg/ml pertussis toxin ( PTX) or with EC/2 (anti-/) or/and AS/7 (anti-/) antiserum at a 1:50 dilution. Data are presented as net responses (total activity in the presence of GTP 0.1 mM minus activity in the presence of GTP alone). Results are expressed as the mean ± S.E. of the number of independent determinations indicated in parenthesis. ★ indicates a statistically significant difference from isoproterenol or from isoproterenol plus clonidine. ( p < 0.05; nonpaired Student's t test). NS, no significant difference.



Preincubation of myometrial membranes with G antiserum (AS/7) blocked the -AR inhibitory pathway on both mid-pregnancy and term, whereas G antiserum (EC/2) was only effective at term (Fig. 3). Thus, at term, the activated -AR do interact functionally with both G and G proteins to inhibit the isoproterenol-mediated effect on adenylyl cyclase activity. Indeed, Fig. 3 B demonstrates that uncoupling of G alone from -AR using AS/7 antiserum did not substantially reduced adenylyl cyclase activity in response to clonidine. Similar responses were observed when testing anti-G alone, or anti-G and anti-G together (Fig. 3 B). These data, compared with findings reported above after pertussis toxin treatment of myometrial membranes, revealed that only a small fraction of pertussis toxin-sensitive G and G proteins do couple to -AR to mediate clonidine inhibition of isoproterenol-stimulated adenylyl cyclase at term.

At mid-pregnancy, clonidine potentiation of activated adenylyl cyclase was completely abolished with G or G antiserum in isolation or with both antisera together (Fig. 4). Our conclusion from this findings is that the stimulatory component in the modulation of adenylyl cyclase activity by -AR, the only subtype detected at mid-pregnancy, is due to dual coupling of G and G proteins to the -AR. Since potentiation by clonidine of adenylyl cyclase activity was unaffected by QL antiserum, it is reasonable to consider that myometrial -AR does not exert its effect via the phospholipase C/Ca/proteine kinase C signal transduction pathway (39, 40, 41, 42) .


Figure 4: Effects of yohimbine and G antisera on clonidine potentiation of isoproterenol-stimulated adenylyl cyclase activity of myometrial membranes at mid-pregnancy. Adenylyl cyclase activity was measured as described under ``Experimental Procedures'' in the presence of isoproterenol (0.1 mM) and GTP (0.1 mM) plus clonidine (100 µM). Yohimbine was used at the concentration of 10 µM. Membranes were preincubated with AS/7 (anti-/) or/and EC/2 (anti-/) or QL (anti-/) antiserum or normal rabbit antiserum at 1:50 dilution. Adenylyl cyclase activities are presented as net responses (total activity in the presence of GTP (0.1 mM) plus adrenergic agents minus activity in the presence of GTP alone). Results are expressed as the mean ± S.E. of the number of determinations indicated in parenthesis. ★ indicates a statistically significant difference from isoproterenol or from isoproterenol plus clonidine ( p < 0.05; nonpaired Student's t test). NS, no significant difference.



Recent studies (31, 43, 44) have shown that type II and IV adenylyl cyclases may be activated by released from G proteins. Since, in our system, G and G mediate -AR dependent potentiation of adenylyl cyclase activity, we determined if one or both of these adenylyl cyclases were expressed in the pregnant rat myometrium. Hybridization of poly(A) RNA from pregnant rat myometrium with the type II adenylyl cyclase probe revealed a single band of 4.2 kb. As expected, this message was also present (Fig. 5 A) in brain and lung and very weakly expressed in kidney (30) . High levels of a 3.5-kb poly(A) RNA were also detected with the type IV probe (Fig. 5 B) in comparison with tissues known to express adenylyl cyclase IV (43) . Thus, a possible molecular mechanism for the myometrial -AR to potentiate adenylyl cyclase activity could reside in the ability of subunits released from -AR-stimulated G proteins, in association with activated , to stimulate adenylyl cyclase types II and IV. We demonstrate above that dual coupling of -AR to both G and G is required to potentiate isoproterenol-stimulated adenylyl cyclase. This lead us to suggest that a high concentration of is required for this stimulatory effect, as predicted previously in membranes from Sf9 cells expressing adenylyl cyclase II (31) . Works are under progress in our laboratory to confirm this interpretation by countering clonidine potentiation of adenylyl cyclase activity in the presence of subunit of retinal transducin (kindly provided by Dr T. Wieland) that can bind and neutralize free . At term, the absence of potentiation of isoproterenol-stimulated adenylyl cyclase activity by -AR, the highly expressed subtype at this stage of pregnancy, could be related, in part, to the sharp decline of the levels of G proteins (36, 37) and the decrease of ADP-ribosylated G(38) .


Figure 5: RNA blot analysis of type II ( A) and type IV ( B) adenylyl cyclase expression. Poly(A) RNA (10 µg) isolated from brain ( B), myometrium ( M), lung ( L), and kidney ( K) were hybridized to a P-labeled adenylyl cyclase II or IV probe, as described under ``Experimental Procedures''. Position of co-electrophoresed RNA size markers (in kb) are shown.



In conclusion, our data present the first detailed analysis of the functionality and dual coupling of the -AR subtypes in a physiological model. Interestingly, our data clearly prove the observations made in vitro on transfected cell lines. These findings could have important implications in the understanding of the routing of signals that depends, for one tissue, on the molecular diversity in receptors, G proteins, and effector systems and on distinct pattern of their regulation. Further attempts are now necessary to identify which factor(s) of pregnancy underly the molecular mechanisms leading to the differential regulation of myometrial adenylyl cyclase activity at mid-pregnancy versus term.

  
Table: Affinities of -AR agonists for inhibiting specific [H]rauwolscine binding in pregnant rat myometrium

The values are mean ± S.E. of four independent determinations. Binding data were analyzed using an iterative nonlinear least squares technique. The Kvalue of [H]rauwolscine determined by Scatchard analysis was 3.7 ± 0.7 nM.


  
Table: Effects of GTP and isoproterenol on adenylyl cyclase activity in myometrial plasma membranes of pregnant rat

Adenylyl cyclase activity was measured as described under ``Experimental Procedures'' in the presence of isoproterenol 0.1 mM, propranolol 10 µM, GTP 0.1 mM; RM/1 antiserum was used at 1:50 dilution. Adenylyl cyclase activities are expressed as total pmol of cAMP/mg of protein/10 min. Results are presented as the mean ± S.E. of the number of determinations indicated in parentheses. * indicates a significant difference between membranes from mid-pregnancy and term ( p < 0.05; nonpaired Student's t test).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-16-1-44-27-36-99; Fax: 33-16-1-44-27-26-50.

The abbreviations used are: AR, adrenoreceptor; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank S. Cotecchia for helpful discussion and critical reading of the manuscript. We thank D. E. Handy and C. S. Flordellis for providing the cDNA for rat - and -ARs, A. G. Gilman, R. R. Reed, and W. J. Tang for the rat adenylyl cyclases II and IV cDNA. We also thank M. T. Robin for expert assistance in Northern blotting analysis and illustration of the manuscript.


REFERENCES
  1. Papka, R. E., Cotton, J. P., and Traurig, H. H. (1985) Cell Tissue Res. 242, 475-490 [Medline] [Order article via Infotrieve]
  2. Marshall, J. M. (1981) Am. J. Physiol. 240, C165-C174
  3. Maltier, J. P., Benghan-Eyéné, Y., and Legrand, C. (1989) Biol. Reprod. 40, 531-540 [Abstract]
  4. Vivat, V., Cohen-Tannoudji, J., Revelli, J. P., Muzzin, P., Giacobino, J. P., Maltier, J. P., and Legrand, C. (1992) J. Biol. Chem. 267, 7975-7978 [Abstract/Free Full Text]
  5. Cohen-Tannoudji, J., Vivat, V., Heilmann, J., Legrand, C., and Maltier, J. P. (1991) J. Mol. Endocrinol. 6, 137-145 [Abstract]
  6. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238, 650-656 [Medline] [Order article via Infotrieve]
  7. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6301-6305 [Abstract]
  8. Lomasney, J. W., Lorenz, W., Allen, L. F., King, K., Regan, J. W., Yang-Feng, T. L., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5094-5098 [Abstract]
  9. Weinshank, R. L., Zgombick, J. M., Macchi, M., Adham, N., Lichtblau, H., Branchek, T. A., and Hartig, P. R. (1990) Mol. Pharmacol. 38, 681-688 [Abstract]
  10. Zeng, D., Harrison, J. K., D'Angelo, D. D., Barber, C. M., Tucker, A. L., Lu, Z., and Lynch K. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3102-3106 [Abstract]
  11. Lanier, S. M., Downing, S., Duzic, E., and Homcy, C. J. (1991) J. Biol. Chem. 266, 10470-10478 [Abstract/Free Full Text]
  12. Voigt, M. M., McCune, S. K., Kanterman, R. Y., and Felder, C. C. (1991) FEBS Lett. 278, 45-50 [CrossRef][Medline] [Order article via Infotrieve]
  13. Flordellis, C. S., Handy, D. E., Bresnahan, M. R., Zannis, V. I., and Gavras, H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1019-1023 [Abstract]
  14. Bylund, D. B., Ray-Prenger, C., and Murphy, T. J. (1988) J. Pharmacol. Exp. Ther. 245, 600-607 [Abstract]
  15. Bylund, D. B., Blaxall, H. S., Iversen, L. J., Caron, M. G., Lefkowitz, R. J., and Lomasney, J. W. (1992) Mol. Pharmacol. 42, 1-5 [Abstract]
  16. Legrand, C., Mhaouty, S., Rigolot, C., and Maltier, J. P. (1993) J. Pharmacol. Exp. Ther. 266, 439-449 [Abstract]
  17. Jones, S. B., and Bylund, D. B. (1988) J. Biol. Chem. 263, 14236-14244 [Abstract/Free Full Text]
  18. Duzic, E., and Lanier, S. M. (1992) J. Biol. Chem. 267, 24045-24052 [Abstract/Free Full Text]
  19. Fraser, C. M., Arakawa, S., Mc Combie, W. R., and Venter, J. C. (1989) J. Biol. Chem. 264, 11754-11761 [Abstract/Free Full Text]
  20. Jones, S. B., Halenda, S. P., and Bylund, D. B. (1991) Mol. Pharmacol. 39, 239-245 [Abstract]
  21. Eason, M. G., Kurose, H., Holt, B. D., Raymond, J. R., and Liggett, S. B. (1992) J. Biol. Chem. 267, 15795-15801 [Abstract/Free Full Text]
  22. Pepperl, D. J., and Regan, J. W. (1993) Mol. Pharmacol. 44, 802-809 [Abstract]
  23. Ruffolo, R. R., Nichols, A. J., and Hieble, J. P. (1988) The Alpha Adrenergic Receptors (Limbird, L. E., ed) pp. 187-280, The Humana Press Inc., Clifton, NJ
  24. Lomasney, J. W., Cotecchia, S., Lefkowitz, R. J., and Caron, M. G. (1991) Biochim. Biophys. Acta 1095, 127-139 [CrossRef][Medline] [Order article via Infotrieve]
  25. Cheng, Y., and Prussoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108 [CrossRef][Medline] [Order article via Infotrieve]
  26. Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokata, T., and Atai, K. (1987) Methods Enzymol. 154, 3-28 [Medline] [Order article via Infotrieve]
  27. Lehbarch, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16, 4743-4751 [Medline] [Order article via Infotrieve]
  28. Handy, D. E., Flordellis, C. S., Bogdanova, N. N., Bresnahan, M. R., and Gavras, H. (1993) Hypertension 21, 861-865 [Abstract]
  29. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 21-52, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  30. Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W. J., Krupinski, J., Gilman, A. G., and Reed, R. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10173-10177 [Abstract]
  31. Tang, W. J., and Gilman, A. G. (1991) Science 254, 1500-1503 [Medline] [Order article via Infotrieve]
  32. Lorenz, W., Losmaney, J. W., Collins, S., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1990) Mol. Pharmacol. 38, 599-603 [Abstract]
  33. Flordellis, C. S., Castellano, M., Franco, R., Zannis, V. I., and Gavras, H. (1990) Hypertension 15, 881-887 [Abstract]
  34. Legrand, C., Vivat, V., Rigolot, C., and Maltier, J. P. (1991) J. Pharmacol. Exp. Ther. 256, 767-772 [Abstract]
  35. Harrison, J. K., D'Angelo, D. D., Zeng, D., and Lynch, K. R. (1991) Mol. Pharmacol. 40, 407-412 [Abstract]
  36. Tanfin, Z., Goureau, O., Milligan, G., and Harbon, S. (1991) FEBS Lett. 278, 4-8 [CrossRef][Medline] [Order article via Infotrieve]
  37. Cohen-Tannoudji, J., Mhaouty, S., Elwardy-Mérézak, J., Lécrivain, J. L., Robin, M. T., Legrand, C., and Maltier, J. P. (1995) Biol. Reprod. 182, in press
  38. Elwardy-Merezak, J., Maltier, J. P., Cohen-Tannoudji, J., Lecrivain, J. L., Vivat, V., and Legrand, C. (1994) J. Mol. Endocrinol. 13, 23-37 [Abstract]
  39. Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D., Lapetina, E. Y., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1990) J. Biol. Chem. 265, 63-69 [Abstract/Free Full Text]
  40. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D., and Bourne, H. R. (1992) J. Biol. Chem. 267, 31-34 [Abstract/Free Full Text]
  41. Lustig, K. D., Conklin, B. R., Herzmark, P., Taussig, R., and Bourne, H. R. (1993) J. Biol. Chem. 268, 13900-13905 [Abstract/Free Full Text]
  42. Morimoto, B. H., and Koshland, D. E., Jr. (1994) J. Biol. Chem. 269, 4065-4069 [Abstract/Free Full Text]
  43. Gao, B., and Gilman, A. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10178-10182 [Abstract]
  44. Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161 [CrossRef][Medline] [Order article via Infotrieve]

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