©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Coupling of m2 and m4 Muscarinic Receptors to Inhibition of Adenylyl Cyclase by G and G Subunits (*)

Jacques C. Migeon , Sarabeth L. Thomas , Neil M. Nathanson (§)

From the (1)Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We compared the G-protein requirements for coupling of human and chicken m2 and m4 muscarinic acetylcholine receptors (mAChRs) to inhibition of adenylyl cyclase, using a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of intracellular cAMP levels. Previously, we used this system to demonstrate that the chick m4 receptor preferentially coupled to G-2 and G over G-1 and G-3. We found that both the chick and human m2 mAChRs can couple to G-1, G-2, G-3, and G, while the human m4 mAChR preferentially couples to G-2 and G. Both the G and G forms of the G subunit were effective in reconstituting coupling of the m2 and m4 mAChRs to inhibit adenylyl cyclase activity. The m2 and m4 mAChRs thus couple to inhibition of adenylyl cyclase by overlapping but different sets of G-protein subunits.


INTRODUCTION

Muscarinic acetylcholine receptors (mAChR)()are members of the superfamily of G-protein-coupled receptors characterized by seven putative transmembrane domains. These receptors regulate intracellular effectors such as ion channels and the enzymes adenylyl cyclase (AC) and phospholipase C. Five subtypes of mAChR have been identified; m1, m3, and m5 couple preferentially to stimulation of phospholipase C, and m2 and m4 couple preferentially to inhibition of AC. However, the specificity of mAChR functional coupling is dependent both on levels of receptor expression and on the cell type in which a given receptor subtype is expressed (Kubo et al., 1986a, 1986b; Peralta et al., 1987a, 1987b; Bonner et al., 1987, 1988; Shapiro et al., 1988; Ashkenazi et al., 1989; Tietje et al., 1990; Tietje and Nathanson, 1991).

Heterotrimeric GTP-binding regulatory proteins (G-proteins) couple mAChR to their intracellular effectors and consist of , , and subunits. The subunit is unique for each G-protein and contains the site of GTP binding and hydrolysis as well as sites for receptor and effector interaction. There are several classes of G-proteins, defined by their subunits. The best characterized are G and G, named for their abilities to stimulate and inhibit AC, respectively. Several forms of and subunits have also been identified. Both the and subunits can regulate the activity of various effector proteins (see Simon et al.(1991) and Spiegel et al.(1992) for review).

Previously, we carried out studies using a luciferase reporter gene under the transcriptional control of a cAMP response element (CRE) as a sensitive monitor of intracellular cAMP levels and cAMP-regulated gene expression. We have used this system to examine both the functional responses of mAChRs (Migeon and Nathanson, 1994) and the function of various wild type and mutated G-protein subunits (Migeon et al., 1994). We found that the chick m4 (cm4) receptor had a surprising specificity for G-protein coupling: it could use G-2 and G but not G-1 or G-3 to mediate inhibition of AC activity. In this study, we have compared the G-protein-coupling requirements of human and chicken m2 and m4 subtype mAChRs. As m2 and m4 muscarinic receptors both couple to inhibition of AC, we wanted to determine whether m2 and m4 mAChRs required different G-protein subunits in order to mediate inhibition of AC. Because only the m2 subtype of mAChR is expressed in the human heart while both m2 and m4 are expressed in the chicken heart, and because species-specific differences in the coupling of receptors to G subtypes have been observed (Jockers et al., 1994), we also compared the chicken and human receptors. It has been shown that muscarinic and somatostatin receptor coupling to a Ca current in GH cells is exquisitely sensitive to G-protein , , and subunit composition (Kleuss et al., 1991, 1992, 1993). The mAChR and the somatostatin receptors use G and G, respectively, to mediate inhibition of the Ca current. In this report, we also examined G-mediated inhibition of AC by mAChR to determine whether individual mAChR subtypes require specific G subunits.


MATERIALS AND METHODS

DNA and Expression Vectors

The chick m4 mAChR genomic clone (Tietje et al., 1990), the chicken m2 mAChR (Tietje and Nathanson, 1991), the human m2 mAChR (Bonner et al., 1988), the human m4 (Bonner et al., 1988), the rat G-1 (Jones and Reed, 1987), the rat G-2 (Jones and Reed, 1987), the rat G (Jones and Reed, 1987), the mouse G-1 (Strathmann et al., 1989), and the mouse G-2 (Strathmann et al., 1990). G-protein subunit cDNAs were expressed in the expression vector pCD-PS (Bonner et al., 1988). The human G-3 (Beals et al., 1987) cDNA was expressed in the expression vector pNUT (Palmiter et al., 1987). The -inhibin CRE-luciferase plasmid consisted of a CRE containing 74-base pair BstXI(-170) to NcoI(-96) fragment from the -inhibin promoter (Pei et al., 1991) blunt end ligated into the TK-105 luciferase plasmid. The constitutively active RSV--galactosidase construct (Edlund et al., 1985) has been described previously (Day et al., 1989).

Cell Transfection and Culture

JEG-3 cells were obtained from the American Type Culture Collection and were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Transient transfection of JEG-3 cells and subsequent assays of luciferase and -galactosidase activity were carried out as described elsewhere (Migeon and Nathanson, 1994). Cells seeded at 2.5 10 cells/15-mm well were transiently transfected with between 200 and 300 ng total DNA by calcium phosphate precipitation 72 h after plating. The transfection mixes consisted of 15 ng/well of the -inhibin CRE-luciferase gene construct, 40 ng/well of RSV--galactosidase, to normalize for transfection efficiency, and the indicated amounts of receptor or G-protein cDNAs. Carrier DNA was used to ensure that all transfections within a given experiment have a constant amount of total DNA. The medium was replaced 24 h after transfection and cells were treated with the appropriate drug another 24 h later. Triplicate wells were treated with various drugs for 5 h before harvesting of cells.

Assay of Luciferase Activity

After removal of media, transfected cells were harvested by solubilization in 100 µl of extraction buffer (100 mM KPO, 4 mM ATP, 1.5 mM MgSO, 1 mM dithiothreitol, 0.1% Triton X-100). 25 µl of cell extract were added to 350 µl of luciferase assay buffer (100 mM KPO, 4 mM ATP, 1.5 mM MgSO) in luminometer cuvettes. The luminometer injects 100 µl of D-luciferin (1 mM; Analytical Luminescence Laboratories, Inc.) into each sample and determines luminescence over 30 s. Luciferase counts were divided by -galactosidase values (see below) to determine normalized luciferase activity.

Assay of -Galactosidase Activity

25 µl of cell extract were added to microtiter plate wells containing 200 µl of -galactosidase assay buffer (60 mM NaPO, 10 mM KCl, 1 mM MgCl, 50 mM -mercaptoethanol). 40 µl of the -galactosidase substrate o-nitrophenyl--galactopyranoside (2 mg/ml; Calbiochem) were added to each well, and plates were colorimetrically assayed for -galactosidase activity.

Determination of mAChR Expression Levels in Transient Transfections

The level of mAChR expression in transiently transfected cells was determined as described previously (Migeon and Nathanson, 1994). In brief, the number of mAChR binding sites was determined by the binding of the membrane-impermeable ligand [H]NMS to intact cells, and the fraction of transfected cells in the culture was estimated by in situ staining for -galactosidase activity with X-gal. The transfection-specific level of [H]NMS binding was divided by the percentage of transfected cells to yield the level of extrapolated level of mAChR expression.

Immunoblot Analysis of G-protein Expression

Immunoblot analysis of G-protein expression was carried out as described previously (Migeon and Nathanson, 1994; Migeon et al., 1994).


RESULTS AND DISCUSSION

Expression of mAChR and G-proteins in Transfected Cells

The expression of mAChR and G-proteins in transiently transfected JEG-3 cells has been described previously (Migeon and Nathanson, 1994; Migeon et al., 1994). Transfection with the G-protein expression vectors has been previously shown to result in similar increases in the levels of each of the subunits (Migeon and Nathanson, 1994; Migeon et al., 1994). The levels of cm2, cm4, human m2 (hm2), and hm4 were quantitated by the binding of [H]NMS to intact cells, and dividing the level of specifically bound [H]NMS by the transfection efficiency, as determined by cytochemical staining for -galactosidase activity due to the cotransfected -galactosidase expression vector. The extrapolated levels of receptor expression were similar for all four receptors, in the range of 5-12 pmol/mg of protein. As discussed previously (Migeon and Nathanson, 1994), because the cytochemical staining probably underestimates the actual fraction of transfected cells in the cultures, these calculations probably overestimate the true levels of receptor expression.

Chicken m2 and m4 mAChR Preferentially Couple to Different G-proteins Subunits to Inhibit AC

We used a luciferase reporter gene under the transcriptional control of a CRE to measure intracellular cAMP levels and cAMP-regulated gene expression in transiently transfected JEG-3 cells. This system allows detection of physiologically relevant changes in intracellular cAMP levels without requiring the presence of phosphodiesterase inhibitors or supraphysiological concentrations of forskolin. In addition, it allows measurement of responses following cotransfection of clones encoding receptors and G-proteins in transiently transfected cells without confounding effects due to the presence of untransfected cells in the cultures. Previously we have used the JEG-3 cell CRE luciferase expression assay to characterize cm4 mAChR-mediated inhibition of forskolin-stimulated AC activity. We found that in JEG-3 cells, which express G-1 and G-3 but not G-2 or G (Montmayeur et al., 1993), transiently transfected cm4 mAChR required coexpression of either G-2 (Migeon and Nathanson, 1994) or G (Migeon et al., 1994) in order to see mAChR-mediated inhibition of CRE-driven luciferase expression. As both the cm2 and cm4 can couple to inhibition of AC, we wanted to determine which G-proteins couple the cm2 mAChR to this response. We found that, while the cm4 mAChR preferentially couples to G-2 and G (Fig. 1B), the cm2 receptor can couple to G-1, G-2, G-3, and G (Fig. 1A). Thus, the cm2 receptor has a more promiscuous pattern of G-protein coupling specificity than the cm4 receptor. The optimal EC values for carbachol for the cm2 and cm4 receptors are 1-2 10M and 4-5 10M, respectively. This is consistent with previous work which demonstrated that, when stably expressed in either CHO or Y1 cells, the cm4 receptor exhibited a higher sensitivity for carbachol than the cm2 receptor (Tietje et al., 1990; Tietje and Nathanson, 1991).


Figure 1: Chicken m2 and m4 preferentially couple to different sets of G-protein subunits for inhibition of forskolin-stimulated AC activity. Control (open circles), G-1 (filled circles), G-2 (open squares), G-3 (filled squares), and G (filled triangles) expression vectors (100 ng/well) were cotransfected with chicken m2 (A) (15 ng/well) or m4 (B) mAChR expression vector (10 ng/well), -inhibin luciferase reporter gene (15 ng/well), and RSV--galactosidase gene (40 ng/well). Transfected cells were treated with 0.316 µM forskolin and varying concentrations of carbachol. Data are shown as -fold increases in normalized luciferase activity and values are means ± S.E., n = 3.



Human m2 and m4 mAChR Exhibit Different G-protein Coupling Specificities for Inhibition of AC-The m2 mAChR is the only subtype expressed to a significant extent in mammalian heart (Luetje et al., 1987; Peralta et al., 1987a), while both the m2 and m4 mAChR subtypes are present in chicken heart (Tietje and Nathanson, 1991). Species-specific differences in the coupling of the human and bovine A1 adenosine receptors to G-proteins have been recently reported (Jockers et al., 1994). We compared the G-protein coupling patterns of the human and chicken m2 and m4 mAChR in order to determine whether species differences between the receptor subtypes may account for the differing patterns of receptor expression in mammalian and avian heart. We found no differences in the G-protein coupling requirements between the human and chicken receptors. G-1, G-2, G-3, and G can all couple the hm2 mAChR to inhibition of AC (Fig. 2A), while the human m4 receptor requires G-2 or G (Fig. 2B). Thus, the differences in mAChR expression in human and chicken heart do not result from different patterns of G-protein coupling. The optimal EC values for carbachol for the hm2 and hm4 receptors are 3-4 10M and 3-4 10M, respectively. The human and chick receptors are therefore also similar in the increased functional sensitivity of the m4 receptor compared to the m2 receptor.


Figure 2: Human m2 and m4 preferentially couple to different sets of G-protein subunits for inhibition of forskolin-stimulated AC activity. Control (open circle), G-1 (filled circles), G-2 (open squares), G-3 (filled squares), and G (filled triangles) expression vectors (100 ng/well) were cotransfected with human m2 (A) or m4 (B) mAChR expression vector (10 ng/well), -inhibin luciferase reporter gene (15 ng/well), and RSV--galactosidase gene (40 ng/well). Transfected cells were treated with 0.316 µM forkolin and varying concentrations of carbachol. Data are shown as -fold increases in normalized luciferase activity and values are means ± S.E., n = 3.



We have previously demonstrated that, in both transiently transfected and stably transfected cells, the cm4 receptor can increase intracellular cAMP due to ectopic coupling to the stimulatory G-protein G and subsequent activation of adenylyl cyclase (Migeon and Nathanson, 1994; Dittman et al., 1994). In the absence of transfected G-proteins, cm4, hm2, and hm4 all mediate an increase in luciferase expression ( Fig. 1and Fig. 2); as demonstrated previously, expression of the appropriate G-protein subunits converts this stimulation to inhibition. Interestingly, the cm2 receptor does exhibit little if any stimulation of CRE-luciferase when expressed at a similar level as the other receptors (Fig. 1A and 3A).

Both the G-1 and G-2 Forms of the G Subunit Can Couple m2 and m4 mAChR to Inhibition of AC

There are two forms of G, G-1 and G-2, which arise from differentially splicing of a single gene. Inhibition of a Ca current in GH3 cells by the m4 receptor has been reported to be blocked by antisense oligonucleotides corresponding to G-1 but not G-2 (see below). The experiments using G in Fig. 1and Fig. 2used rat G-1. In order to determine whether mAChR inhibition of AC in JEG-3 cells requires a specific G form, cells were transiently transfected with combinations of human and chicken mAChR and either of the two G subtypes (Fig. 3A-D). Because rat G-2 is not available, we used cDNA clones corresponding to murine G-1 and G-2. Both the human and chick m2 and m4 receptors couple equally well to both subtypes of G, thus demonstrating that both forms of G can couple to either the m2 or the m4 receptor to mediate inhibition of AC in JEG-3 cells.


Figure 3: m2 and m4 mAChR can couple to both G1 and G2 forms of G for inhibition of forskolin-stimulated AC activity. Control DNA (open circles), G-1 (filled circles), and G-2 (filled squares) expression vectors (100 ng/well) were cotransfected with chicken m2 (A), chicken m4 (B), human m2 (C), or human m4 (D) mAChR expression vector (A, 15 ng/well; B-D, 10 ng/well), the -inhibin luciferase reporter gene (15 ng/well), and the RSV--galactosidase gene (40 ng/well). Transfected cells were treated with 0.316 µM forkolin and varying concentrations of carbachol. Data are shown as -fold increases in normalized luciferase activity and values are means ± S.E., n = 3.



Diverse methods have been used to examine muscarinic receptor G-protein coupling. These techniques include reconstitution of purified proteins in lipid vesicles and expression of exogenous receptors and G-proteins in well characterized cell lines. Pertussis toxin (PTX) treatment, subunit antisense oligonucleotides, and agonist-dependent subunit labeling have also been used for determination of in situ G-protein coupling. PTX was one of the first tools used to study the G-proteins involved in mAChR-mediated responses, particularly inhibition of AC. While PTX treatment abolishes m2 and m4 mAChR inhibition of AC (Shapiro et al., 1988; Peralta et al., 1988; Jones et al., 1991), it does not distinguish between the individual PTX-sensitive G-proteins. Transfected m2 mAChRs were shown to couple to both G-2 and G-3 in CHO cells (Dell'Acqua et al., 1993) and to G-1, G-2, and G-3 in transfected 293 cells (Offermanns et al., 1994).

Reconstitution studies have provided information on in vitro mAChR-G-protein coupling capabilities. Chick cardiac (predominantly cm2) mAChR receptors can activate G (Richardson et al., 1991), and activation of reconstituted mammalian recombinant m2 receptors stimulates the binding of the nonhydrolyzable GTP analog GTPS to G-1, G-3, G, and G subunits (Tota et al. 1990; Parker et al., 1991). Similarly, G-1, G-2, and G-3 allow reconstitution of coupling of the m2 mAChR to activation of the atrial inwardly rectifying K current. (Yatani et al., 1988). Thus, our results are consistent with a variety of results demonstrating that the m2 receptor can activate the entire PTX-sensitive G-protein family.

In our previous work, we demonstrated that the chicken m4 mAChR preferentially coupled to G-2 and G (Migeon and Nathanson, 1994; Migeon et al., 1994). Here we expand on those studies by examining coupling of the chicken m2 mAChR to inhibition of AC. We found that the m2 mAChR can couple to a larger set of G-protein subunits, using G-1, G-2, G-3, and G to inhibit AC. Thus while m2 and m4 mAChR can couple to inhibition of AC, they can do so by coupling to overlapping but different sets of G-protein subunits. While the JEG-3 cells express endogenous G-1 and G-3, they appear not to express enough of these G-proteins to allow m2 mAChR-mediated inhibition of forskolin-stimulated CRE-luciferase without cotransfection with G-protein cDNA. In contrast to the difference in G-protein coupling seen between the human and bovine A1-adenosine receptors (Jockers et al., 1994), both the m2 and m4 receptors exhibit identical G-protein requirements between the human and chick homologues. While the reason for the difference in subtype expression between mammalian and avian hearts remains unclear, the presence of multiple subtypes in chick heart raises the possibility that there may be differential control of expression of receptor subtypes that would allow a higher level of regulatory complexity in chick compared to mammalian heart.

We previously showed that G-1 could couple the m4 receptor to inhibition of AC in JEG-3 cells (Migeon et al., 1994). Before our report, the only direct evidence of mAChR functional coupling through G was in GH cells where mAChRs mediate inhibition of a Ca current. Injection of GH cells with antisense oligonucleotides against the G-1 form of G abolished mAChR-mediated inhibition of a Ca current (Kleuss et al., 1991), while injection of antisense oligonucleotides against the G-2 form did not. Specific forms of and subunits are also required (Kleuss et al., 1992, 1993). We tested m2 and m4 coupling to G-1 and G-2. We found that chicken and human m2 and m4 receptors could couple equally well to the G-1 and G-2 forms of G to inhibit forskolin-stimulated CRE-luciferase activity. Thus, in contrast to the mAChR-mediated Ca current in GH cells, there is no subtype specificity for mAChR inhibition of AC through G in JEG-3 cells. These differences in coupling specificities may be related to differences either in the subunit composition between the two cell types or the regulatory properties of calcium channels compared to adenylyl cyclase.

In summary, we have shown that the G-protein coupling specificity of the m2 mAChR is different from that of the m4 receptor. Thus, we have shown that their apparent functional redundancy is in fact more complicated and that the two receptors can couple to AC through overlapping but different sets of G-protein subunits. We have also shown that mAChR-G-protein coupling is conserved across species and that different patterns of G-protein coupling cannot explain differences in mAChR expression in chicken and human cardiac tissue. Finally, we have shown that m2 and m4 inhibition of AC in JEG-3 cells can utilize either G-1 or G-2. The use of the CRE-luciferase reporter gene system in JEG-3 cells has proved very useful for examination of mAChR-G-protein coupling capabilities and has given a greater understanding of possible receptor subtype function.


FOOTNOTES

*
This research was supported by grants from the National Institutes of Health (to N. M. N.) and by National Institutes of Health Training Grants GM07108, GM07750, and NS07332. 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.: 206-543-9457; Fax: 206-616-4230.

The abbreviations used are: mAChR, muscarinic acetylcholine receptor; AC, adenylyl cyclase; CRE, cAMP response element; G-protein, guanine nucleotide-binding regulatory protein; NMS, N-methyl scopolamine; PTX, pertussis toxin; X-gal, 5-bromo-4-chloro-3-indolyl--galactopyranoside; RSV, Rous sarcoma virus; CHO, Chinese hamster ovary; GTPS, guanosine 5`-3-O-(thio)triphosphate.


ACKNOWLEDGEMENTS

We are grateful to Dr. Randy Reed (Johns Hopkins University) for the gift of the rat G-1, G-2, and G G-protein subunit cDNAs, Drs. Roger Perlmutter and Chan Beals (University of Washington) for the gift of the human G-3 cDNA, Dr. Kelly Mayo (Northwestern University) for the gift of the -inhibin CRE-luciferase reporter gene construct, Dr. Melvin Simon (University of California, San Francisco) for the gift of the murine G-1 and G-2. Dr. Tom Bonner (National Institute of Mental Health) for the gift of the human m2 and m4 mAChR clones and the expression vector pCD-PS. We also thank Michael Schlador for his help in setting up some of these experiments and Dr. Stan McKnight for his kind help and advice.


REFERENCES
  1. Ashkenazi, A., Winslow, J. W., and Capon, D. (1989) Nature340, 146-150 [CrossRef][Medline] [Order article via Infotrieve]
  2. Beals, C. R., Wilson, C. B., and Perlmutter, R. M. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 7886-7890 [Abstract]
  3. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. (1987) Science237, 527-532 [Medline] [Order article via Infotrieve]
  4. Bonner, T. I., Young, A. C., Brann, M. R., and Buckley, N. J., (1988) Neuron1, 403-410 [Medline] [Order article via Infotrieve]
  5. Day, R. N., Walder, J. A., and Maurer, R. A. (1989) J. Biol. Chem.264, 431-436 [Abstract/Free Full Text]
  6. Dell'Acqua, M. L., Carroll, R. C., and Peralta, E. G. (1993) J. Biol. Chem.268, 5676-5685 [Abstract/Free Full Text]
  7. Dittman, A. H., Weber, J. P., Hinds, T. J., Choi, E.-J., Migeon, J. C., Nathanson, N. M., and Storm, D. R. (1994) Biochemistry33, 943-951 [Medline] [Order article via Infotrieve]
  8. Edlund, T., Walker, M. D., Barr, P. J., and Rutter, W. J. (1985) Science230, 912-916 [Medline] [Order article via Infotrieve]
  9. Jockers, R., Linder, M. E., Hohenegger, M., Nanoff, C., Bertin, B., Strosberg, A. D., Marullo, S., and Freissmuth, M. (1994) J. Biol. Chem.269, 32077-32084 [Abstract/Free Full Text]
  10. Jones, D. T., and Reed, R. R. (1987) J. Biol. Chem.262, 14241-14249 [Abstract/Free Full Text]
  11. Jones, S. V. P., Heilman, C., J., and Brann, M. R. (1991) Mol. Pharmacol.40, 242-247 [Abstract]
  12. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (1991) Nature353, 43-48 [CrossRef][Medline] [Order article via Infotrieve]
  13. Kleuss, C., Scherübl, H., Hescheler, J., Schultz, G., and Wittig, B. (1992) Nature358, 424-426 [CrossRef][Medline] [Order article via Infotrieve]
  14. Kleuss, C., Scherübl, H., Hescheler, J., Schultz, G., and Wittig, B. (1993) Science259, 832-834 [Medline] [Order article via Infotrieve]
  15. Kubo, T., Maeda, K., Sugimoto, K., Akiba, I., Mikami, A., Takahashi, H., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Hirose, T., and Numa, S. (1986a) FEBS Lett.209, 367-372 [CrossRef][Medline] [Order article via Infotrieve]
  16. Kubo, T., Fukuda, K., Mikami, A., Maeda, K. Takahashi, H., Mishina, T., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1986b) Nature323, 411-416 [Medline] [Order article via Infotrieve]
  17. Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry26, 6892-6896 [Medline] [Order article via Infotrieve]
  18. Migeon, J. C., and Nathanson, N. M. (1994) J. Biol. Chem.269, 9767-9773 [Abstract/Free Full Text]
  19. Migeon, J. C., Thomas, S. L., and Nathanson, N. M. (1994) J. Biol. Chem.269, 29146-29152 [Abstract/Free Full Text]
  20. Montmayeur, J. P., Guiramand, J., and Borelli, E. (1993) Mol. Endocrinol.7, 161-170 [Abstract]
  21. Novotny, E., and Brann, M. R. (1989) Trends Pharmacol. Sci.4, (suppl) (Abstr. 66)
  22. Offermanns, S., Wieland, T., Homann, D., Sandmann, J., Bombien, E., Spicher, K., Schultz, and Jakobs, K. H. (1994) Mol. Pharmacol.45, 890-898 [Abstract]
  23. Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., and Maxwell, I. H. (1987) Cell50, 435-443 [Medline] [Order article via Infotrieve]
  24. Parker, E. M., Kameyama, K., Higashijima, T., and Ross, E. M. (1991) J. Biol. Chem.266, 519-527 [Abstract/Free Full Text]
  25. Pei, L., Dodson, R., Schoderbek, W. E., Maurer, R. A., and Mayo, K. E. (1991) Mol. Endocrinol.5, 521-534 [Abstract]
  26. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J., and Capon, D. J. (1987a) EMBO J.6, 3923-3929 [Abstract]
  27. Peralta, E. G., Winslow, J. W., Peterson, B. L., Smith, D. L., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I., and Capon, D. J. (1987b) Science236, 600-605 [Medline] [Order article via Infotrieve]
  28. Peralta, E. G., Askenazi, A., Winslow, J. W., Smith, O. H., Ramachandran, J., and Capon, D. J. (1988) Nature334, 434-437 [CrossRef][Medline] [Order article via Infotrieve]
  29. Richardson, R. M., Mayanil, C. S. K., and Hosey, M. M. (1991) Mol. Pharmacol.40, 908-914 [Abstract]
  30. Shapiro, R. A., Scherer, N. M., Habecker, B. A., Subers, E. M., and Nathanson, N. M. (1988) J. Biol. Chem.263, 18397-18403 [Abstract/Free Full Text]
  31. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science252, 802-808 [Medline] [Order article via Infotrieve]
  32. Spiegel, A. M., Shenker, A., and Weinstein, L. S. (1992) Endocr. Rev.13, 536-565 [Medline] [Order article via Infotrieve]
  33. Strathmann, M., Wilkie, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A.86, 7407-7409 [Abstract]
  34. Strathmann, M., Wilkie, T. M. and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 6477-6481 [Abstract]
  35. Tietje, K. M., and Nathanson, N. M. (1991) J. Biol. Chem.266, 17382-17387 [Abstract/Free Full Text]
  36. Tietje, K. M., Goldman, P. S., and Nathanson, N. M. (1990) J. Biol. Chem.265, 2828-2834 [Abstract/Free Full Text]
  37. Tota, M. R., Xia, Z., Storm, D. R., and Schimerlik, M. I. (1990) Mol. Pharmacol.37, 950-957 [Abstract]
  38. Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature336, 680-682 [CrossRef][Medline] [Order article via Infotrieve]

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