Differential Tissue Expression and Developmental Regulation of ...

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Charles W. Luetje, Kathleen M. Tietje, Jan L. Christian, and Neil M. NathansonS. From the ..... hybridized with nick-translated cDNA probe (1 X lo6 to 1 X lo' cpm/.
Vol. 263, No . 26, Issue of September 15, pp. 13357-13365,1988 Printed in U.S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential Tissue Expression and Developmental Regulation of Guanine Nucleotide Binding Regulatory Proteins and Their Messenger RNAs in Rat Heart* (Received for publication, October 20, 1987)

Charles W. Luetje, KathleenM. Tietje, Jan L. Christian, andNeil M. NathansonS From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195

The expression and developmental regulation of the Cockcroft and Gomperts, 1985; Blackmore et al., 1985; Pfafand @ subunitsoftheguaninenucleotidebinding finger et al., 1985; Breitwieser and Szabo, 1985). G proteins regulatory proteins, Gi and Go,were examined in rat are heterotrimers consisting of an a, fi, and y subunit. The a atria and ventricles. Protein levels were determined subunits range in size from 39,000 to 52,000 Da and are unique by quantitative immunoblot analysis using affinity pu- to each G protein. Each a subunit has an intrinsic GTPase rified monospecific antibodies. Northern blot and dot activity and a bacterial toxinADP-ribosylation site (Gilman, blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein 1984,1987; Sternweis and Robishaw, 1984; Mumby et al., subunits. The concentrations Goa, of Gia, and (3 subunit 1986). Molecular cloning has revealed multiple forms of the protein were found to begreater in adultatrial than in a subunits of G., Gi, and transducin (Brayet al., 1986; Itoh et adult ventricular membranes(5.2-, 1.5-,and 2.8-fold, al., 1986; Nukada et al., 1986; Didsbury and Snyderman, 1987; respectively). A corresponding 3.4-fold difference in Lerea et al., 1986; Jones and Reed, 1987). Each a subunit is Goa mRNA level was also observed, as well as a 1.3- associated with a common fi subunit (Gilman, 1984; Manning fold difference in Gia-3 mRNA level. No difference and Gilman, 1983). Although 35,000- and 36,000-Da forms of @, Gia-1, Gia-2mRNA fi have been observed (Sternweis andRobishaw, 1984; Woolkwas seen between the amount of in adultatria and adultventricles. Comparison ofneo- alis and Manning, 1987) and distinct fi subunits have been natal and adult tissues revealed a developmental de- identified by molecular cloning (Fong et al., 1987; Gao et al., crease inventricular Gia proteinandGia-2 mRNA 1987), the functional significance of these observations is not levels (70 and 47%,respectively). Developmental de- known. Each G protein also contains a y subunit of approxicreases were also observed in the amount of mRNA mately 8,000 Da (Hildebrandt et al., 1984), about which little encoding @ and Goa in ventricles (47 and 61%,respectively), and /3 and Gia-2 in atria (40 and 36%,respec- is known, although the y subunit of transducin is structurally tively), while a developmental increase in atrial Gia-3 and immunologically distinct from the y subunits of other G mRNA levels was observed (57%).These results dem- proteins (Hildebrandt et al., 1985; Gierschik et al., 1985; Roof onstrate differences intheexpressionof G protein et al., 1985). Receptors which inhibit adenylate cyclase activity act subunits in ratatria and ventricles, as well as regulation of the levels of these subunits during cardiac de- through Gi, and roles for both the a subunit and fiy complex velopment. have been postulated (Katada et al., 1984, 1986). Recently, the existence of three related forms of Gia has been demonstrated by molecular cloning. Gia-1 has been cloned from a bovine brain cDNA library (Nukada et al., 1986), Gia-2 from Guanine nucleotide binding regulatory proteins (G proteins)’ couple membrane receptors to a variety of effector a rat C6 glioma cDNA library (Itoh et al., 1986), and Gia-3 mechanisms including stimulation and inhibition of adenylate from a human HL-60 cDNA library (Didsbury and Snydercyclase (G, and Gi, respectively), a cGMP phosphodiesterase man, 1987). Gia-l, Gia-2, and Gia-3 have also been cloned (transducin), phosphatidylinositol 4,5-bisphosphate hydroly- from a rat olfactory cDNA library (Jones and Reed, 1987). sis, and ion channels (Gilman, 1984, 1987;Stryer et al., 1981; An additional G protein, Go, has been demonstrated in brain (Sternweis and Robishaw, 1984; Neer et al., 1984), heart *This workwas supportedin part by agrant-in-aid from the (Halvorsen and Nathanson, 1984; Luetje et al., 1987), and American Heart Association and by National Institutes of Health other tissues (Mumby et al., 1986; Huff et al., 1985). Go i s Grants HL 30639 and HL07312. The costs of publication of this structurally homologous to Gi, and the a subunit of Go, like article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accord- that of Gi, serves as a substratefor ADP ribosylation by islet activating protein (IAP), a toxin of Bordatella pertussis. An ance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Established Investigator of the American Heart Association. To IAP substrate couples the cardiac muscarinic acetylcholine whom correspondence should be addressed Dept. of Pharmacology, receptor (mAChR) to a K+ channel (Pfaffinger et al., 1985; SJ-30, University of Washington School of Medicine, Seattle, WA Martin et al., 1985; Endoh et al., 1985; Sorota et al., 1985), 98195. The abbreviations used are: G protein, guanine nucleotide binding and a Gi-like protein, but not Go, has been shown to activate regulatory protein; G.a, a subunit of the stimulatory G protein of the cardiac muscarinic K+ channel aswell as a K+ channel in adenylate cyclase; Gia, a subunit of the inhibitory G protein of GH3 pituitary cells (Yatani et al., 1987; Codina et al., 1987a, adenylate cyclase; Goa,39,000-Da a subunit; Ta, LY subunit of trans- 1987b; Logothetis et al., 1987). Both the a subunit and fir ducin; TP, /3 subunit of transducin; mAChR, muscarinic acetylcholine receptor(s); IAP, islet activating protein, a toxin of B. pertussis; SDS, complex have been suggested to regulate KC channelfunction (Codina et al., 1987a, 198713; Logothetis et al., 1987),while the sodium dodecyl sulfate; PBS, phosphate-buffered saline; MOPS, 4morpholinepropanesulfonic acid kb, kilobase(s). a subunit of Gohas been implicated in the functional coupling a

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of opiate receptors to neuronal voltage-sensitive Ca2+channels (Hescheler et al., 1987). The presence of at least two IAP substrateshas been demonstrated in bovine heart (Sternweis and Robishaw, 1984), embryonic chicken heart (Halvorsen and Nathanson, 1984),and rabbitventricle (Malbon et al., 1985).The presence of both Gi and Gohas also been demonstrated immunologically in bovine heart (Huff et al., 1985; Mumby et al., 1986, 1988) and embryonic chicken atria and ventricles (Luetje et al., 1987). Recently several laboratories have reported that rat ventricle contains a single 41,000-Da IAP substrate (Murakami and Yasuda, 1986; Steinberg et al., 1985). It was assumed that, because of its molecular weight, the IAP substrate was Gia. It was also suggested that a developmental increase in the amount of IAP substrate occurred during rat ventricular development (Steinberg et al., 1985). In this study, rat atria andventricles were analyzed for the presence of Goa and Gia, as well as the common ,f3 subunit. The presence and amount of these polypeptides was determined by IAP labeling and quantitative immunoblot analysis, while the presence and relative amounts of mRNA encoding these subunits were determined by Northern and dot blot analyses. While Gia, Goa, and p were present in both atria and ventricles, large differences were observed between the levels in ventricles. In the levels of Goaand p in atria and the case of Goa, the difference in protein level correlated with a difference in mRNA level. In contrast to what was reported by Steinberg et al. (1985), a developmental decrease in the level of Gia protein, as well as the level of Gia-2 mRNA, was observed. The level of Gia-3 mRNA increased in atria during development but did not change in ventricles. EXPERIMENTALPROCEDURES

Materials-Sprague-Dawley rats were obtained from Tyler Labs (Bellevue, WA). For neonatal rat studies, time-mated females were obtained and neonatal rats were sacrificed 2-3 days afterbirth. Phenylmethylsulphonyl fluoride, 1,lO-phenanthroline, iodoacetamide, pepstatin A, Tween 20, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt,p-nitro blue tetrazolium chloride, and p-nitrophenyl phosphate disodium salt were from Sigma. Nitrocellulose was from Schleicher & Schuell. Alkaline phosphatase-conjugated goal antirabbit IgG was from Cappel, CooperBiomedical, Malvern, PA. IAP was a gift from Jennifer Martin (University of Washington) and was purified from the 24-h culture supernatant of B. pertussis (Tahoma phase I) as described (Sekura et al., 1983). [w3'P]NAD was synthesized from [cx-~'P]ATPas described (Cassel and Pfeuffer, 1978). [a-32P]dCTP(3000 Ci/mmol) was from Du Pont-New England Nuclear. Chemicals used for electrophoresis were of electrophoresis grade; other chemicals were of reagent grade. Antisera-Rabbit antiserum RV3, produced by immunization with partially purified Gproteins (Gierschik et al., 1986b), and rabbit antiserum AS7, produced by immunization with the Cterminus decapeptide of the a subunit of bovine transducin (Goldsmith et al., 1987), were obtained from Dr. Allen Spiegel, National Institutes of Health. Membrane Preparation-Adult and neonatal rat hearts were removed and separated into atria and ventricles in ice-cold PBS (20 mM NaH2P04,150 mM NaC1, pH 7.4). The tissue was frozen on Dry Ice and stored at -70 "C until use. Tissue was thawed in ice-cold PBS containing protease inhibitors (0.4 mM phenylmethylsulfonyl fluoride, 1mM 1,lO-phenanthroline, 1 mM iodoacetamide, 1pM pepstatin A), homogenized with a Brinkman Polytron at setting 6 for 20 s, and centrifuged a t 80 X g for 15 min. Membranes were then pelleted from the supernatant by centrifugation a t 17,600 X g for 15 min, washed, and recentrifuged. The membranes were then resuspended and stored at -70 'C until use, Bovine cerebral cortex membranes were prepared as described (Hurko, 1978). Partially purified G proteins were prepared as described (Milligan and Klee, 1985), and bovine transducin was purified as described (Gierschik et al., 1985). ZAP Labeling-Cholate extracts of adult rat atrial andventricular membranes were prepared and IAP-catalyzed ADP ribosylation was performed as described (Pobiner et al., 1985), except that 0.45 pg of

purified IAP was used in each reaction. Autoradiography was performed using Kodak X-Omat film a t -70 "C with a Cronex intensifying screen. SDS Gel Electrophoresis-SDS-PAGE was performed using a modification (Nathanson andHall, 1979) of the discontinuous system of Laemmli (Laemmli, 1970). The following proteins were used as molecular weight (in parentheses) standards: @-galactosidase(116,000), bovine serum albumin (68,000), pyruvate kinase (57,000), fumarase (49,000),aldolase (40,000),and glyceraldehyde-3-phosphate dehydrogenase (36,000). Quantitative Zmmumblot Analysis-Proteins were electrophoretically transferred to nitrocellulose as described (Towbin et al., 1979). The nitrocellulose was then eitherstained with Amido Black or incubated with PBS containing 10% bovine hemoglobin for 1 h at room temperature and rinsed with distilled water. The nitrocellulose was then incubated with affinity purified antibodies diluted in PBS, 0.5% Tween 20 (TPBS) for 12-20 h a t room temperature. Following several rinses in TPBS, thenitrocellulose was incubated with alki line phosphatase-conjugated goat anti-rabbit IgG diluted in TPBS for 2 h at room temperature. Bound antibodies were then visualized as described (Smith and Fisher, 1984) by incubating the nitrocellulose in 50 mM sodium glycinate, pH 9.6,O.l mglmlp-nitro blue tetrazolium chloride, 0.05 mg/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, 4 mM MgClz, 0.5% Tween 20. The reaction was stopped after 5-10 min by rinsing with TPBS. Monospecific antibodies were affinity purified from the polyclonal antisera using the method of Smith andFisher (1984) as described previously (Luetje et al., 1987). Quantitation of immunostained bands was performed as described (Luetje et al., 1987), using affinity purified antibodies a t dilutions that were determined to be saturating in control experiments (data not shown). Briefly, each immunostained band was cut from the nitrocellulose, placed in a wellof a 96-well microtiter plate, and incubated with 100 pl of p-nitropbenyl phosphate. After 10-20 min, the reaction was stopped by the addition of 100 pl of 13% KZHPOI, andthe absorbance of the solution was determined a t 410 nm. Comparison of sample values with a standard curve generated using known amounts of G protein subunits allowed the concentration of G proteinsubunits in the samples to be determined. Standard curves used in these experiments were prepared using samples of partially purified G proteins whose subunit concentrations were determined by densitometry. cDNA Clones-cDNA clones I13 and 123, isolated from a rat C6 glioma cDNA library and encoding Gia-2 and Goa, respectively, were obtained from Drs. Hiroshi Itoh and Yoshito Kaziro, University of Tokyo (Itoh et al., 1986). cDNA clones G27 and G16, isolated from a rat olfactory cDNA library encoding Gia-1 and Gia-3, respectively, were obtained from Drs. David T. Jones andRandall R. Reed (Johns Hopkins University) (Jones and Reed, 1987). A 600-base pair XbaI fragment from Gia-1 and a 620-base pair EcoRV fragment from Gia3 were used in Northern and dot blot analyses to ensure specificity of hybridization, as described by Jones andReed, (1987). TO, a cDNA clone encoding the @ subunit of bovine transducin, was obtained from Dr. James Hurley, University of Washington (Fong et al., 1986). 4A, a cDNA clone encoding human 28 S RNA, was obtained from Dr. Randall Moon, University of Washington. Plasmid preparation was performed as described (Maniatis et al., 1982). Gel-purified cDNA inserts were nick-translated to specific activities of 1to 2 X lo9 cpm/ pg essentially as described (Rigby et al., 1977). Northern Blot Analysis-Adult and neonatal rat hearts andbrains were removed, the hearts separated into atria and ventricles in icecold PBS, the tissue frozen in liquid nitrogen, and then stored at -70 "C until use. Total cellular RNA was then prepared as described (Cathala et al., 1983). RNA for size markers (1-kb ladder, Bethesda Research Laboratories) were denatured in 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 0.5 mM EDTA, 2.2 M formaldehyde, 50% formamide at 55 "C for 55 min, size-fractionated on agarose gels in the same buffer without formamide, and hybridized to cDNA probes essentially as described (Uhler et al., 1986).Briefly, the gel was blotted to nitrocellulose, the nitrocellulose baked a t 80 "C for 2 h and then incubated in prehybridization buffer (50% formamide, 0.9 M NaCl, 60 mM NaH2P04, pH 7.4, 6 mM EDTA, 0.1% SDS, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% Ficoll, 100 pg/ml salmon sperm DNA) for 4-16 h at 39 "C. The nitrocellulose was then hybridized with nick-translated cDNA probe (1X lo6 to 1 X lo' cpm/ ml) in hybridization buffer (50% formamide, 0.9 M NaCl, 60 mM NaHzP04, pH 7.4, 6 mM EDTA, 0.1% SDS, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 100 pg/ml salmon sperm DNA) for 16-72 h at 42 "C. The nitrocellulose was then washed

Regulation of Cardiac GTP Binding Proteins in 0.3 M NaCl, 20 mM sodium citrate, pH 7.0, 0.2% SDS at room temperature, and then in 75 mM NaC1, 5 mM sodium citrate, pH 7.0, 0.2% SDS a t room temperature and then a t 50 “C. Autoradiography was a t -70 “C for 16-114 h. The nitrocellulose was reprobed after stripping with 5 mM Tris-HC1, pH 8.0, 0.2 mM EDTA, 0.05% NaPPi, 0.001% bovine serum albumin, 0.001% polyvinylpyrrolidone, 0.001% Ficoll for 5 h a t 70 “C. Dot Blot Analysis-Dot blot analysis was performed as described (Thomas, 1983). Briefly, RNA samples were denatured in 1M glyoxal, 10 mM NaP04, pH6.6, a t 50 “C for 1 h. The RNA was then spotted onto nitrocellulose disks, baked for 2 h a t 80 “C, and thenprehybridized and hybridized with nick-translated cDNA probes as for Northern blot analysis. The amountof 32P-labeledprobe hybridized to the nitrocellulose was determined by Cerenkov counting. Protein Assay-Protein concentration was determined as described previously (Halvorsen and Nathanson,1981) by the method of Lowry et al. (1951). Statistical Anulyses-Statistical significance was determined by using a two sample t test following an F-test to ensure equality of variance. For samples with unequal variance ( p > 0.05), statistical significance was determined by using a two sample t test for samples with unequal variance (Cochran’s method). RESULTS

IAP-mediated ADP Ribosylation of a 40,000-Da Polypeptide in Both Atrial and Ventricular Membranes-The presence of G protein a subunits was first investigated by IAP-catalyzed [32P]ADPribosylation. Cholate extracts of membranes prepared from adult rat atria and ventricles were incubated.with [cY-~*P]NAD in the presence and absence of IAP as described under “Experimental Procedures.’’ Fig. 1 shows that a polypeptide of approximately 40,000 Da was specifically labeled in extracts prepared from both atrial or ventricular membranes. The labeled band could represent asingle polypeptide; however, the diffuse nature of the band suggests that it may consist of more than one IAP substrate. Specific Recognition of G Protein Subunits by Affinity Purified Antibodies-Immunological methods were employed as a more reliable method of determining the presence and identity of G protein a subunits in rat atria and ventricles. Antiserum RV3 was raised against purified G proteins from bovine cerebral cortex and has been previously shown to contain antibodies which recognize the 39,000-Da a subunit

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ofGo (but not the 41,000-Da a subunit ofGi) and the p subunit of bovine Go, Gi, G., and transducin (Gierschik et al., 1986b). Antiserum RV3 clearly recognizes the 36,000-Da p subunit; however, it is unclear whether the 35,000-Da p subunit is also recognized (Sternweis and Robishaw, 1984; Roof et al., 1985).Antiserum AS7 wasraised against the C terminus decapaptide of the a subunit of bovine transducin and recognizes the transducin a subunit, as well as thea subunit of Gi, but not thea subunit of Go (Goldsmith et al., 1987). Monospecific antibodies were affinity purified from antisera RV3 and AS7 as described under “Experimental Procedures.” The antibodies exhibited a high degree of specificity for the detection of G protein subunits in purified samples and crude membrane preparations (Fig. 2). The affinity purified anti-G,a antibodies recognized the a subunit of Go,but not Gi, in a sample of partially purified Go and Gi (Fig. 2B, lane 2). The a subunit of purified bovine transducin was not recognized bythese antibodies (Fig. 2B, lane 1). The anti-G,a antibodies also recognized a 40,000-Da polypeptide in both atrial and ventricular membrane samples (Fig. 2B, lanes 3 .and 4). The amount of this polypeptide in atrial membranes is much greater than the amountin ventricular membranes. The affinity purified anti-Ta antibodies recognized the a subunit of purified bovine transducin and the a subunit of Gi, but not Go, in a sample of partially purified Gi and Go (Fig. 2C, lanes 1 and 2). The anti-Ta antibodies also recognized a 40,000-Da polypeptide in both atrial and ventricular membrane samples (Fig. 2C, lanes 3 and 4 ) . In contrast to the polypeptide recognized by the anti-Goa antibodies, similar amounts of theanti-Ta immunoreactive polypeptide are present in the atrial and ventricular membrane samples. The affinity purified anti-@ antibodies recognized the /3

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FIG. 1. IAP catalyzed [a-S2P]ADPribosylation of G protein a subunits in rat atria and ventricle membranes. Cholate extracts of adult rat atrial (lanes 1 and 2) andventricular (lanes 3 and

4) membranes were prepared, incubated with [cY-~’P]NADinthe presence (lanes 1 and 3 ) and absence (lanes 2 and 4)of IAP, subjected to SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography as described under“Experimental Procedures.’’ Each lane contains 26 pg of protein.

FIG. 2. Immunoblot analysis of G protein subunits in rat cardiac membranes using affinity purified antibodies. Samples were subjected to SDS polyacrylamide gel electrophoresis on an 8% separation gel and transferred to nitrocellulose as described in “Experimental Procedures.’’ The nitrocellulose was then stained with amido black or incubated with affinity purified antibodies. Bound antibody was visualized as described under “Experimental Procedures.” A, nitrocellulose stained with Amido Black. Lane 1, 1 pg of bovine transducin. Lane 2, 3 pg of partially purified G proteins. Lane 3, 30pgof atrial membrane protein. Lane 4, 30 pgof ventricular membrane protein. B, nitrocellulose incubated with anti-Goa antibodies. Lane 1,200 ng of bovine transducin. Lane 2,300 ng of partially purified G proteins. Lane 3, 300 pg of atrial membrane protein. Lane 4,300 pg of ventricular membrane protein. C, nitrocellulose incubated with anti-Ta antibodies. Lane 1, 200 ng of bovine transducin. Lane 2, 300 ng of partially purified G proteins. Lane 3, 100 pg of atrial membrane protein. Lane 4, 100 pg of ventricular membrane protein. D, nitrocellulose incubated with anti-/3 antibodies. Lane 1, 200 ng of bovine transducin. Lane 2, 300 ng of partially purified G proteins. Lane 3, 300 pg of atrial membrane protein. Lane 4, 300 pg of ventricular membrane protein.

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subunit of purified bovine transducin and the p subunit in a sample of partially purified Gi and Go (Fig. 20, lanes 1 and 2 ) . The anti-/3 antibodies also recognized a 35,000-Da polypeptide in both atrial and ventricular membrane samples (Fig. 20, lanes 3 and 4 ) . A greater amount of this polypeptide is present in atrial membranesthan in ventricular membranes. Determination of G Protein Subunit Levels by Quantitative Immunoblot Analysis-Fig. 2 suggests that the levels of Goa and p in atrialmembranesaregreater than the levels in ventricular membranes. In order to accurately measure levels of the Goa, Gia, andsubunits,quantitative immunoblot analysis was performed using the affinity purified anti-Goa, anti-To, and anti$ antibodies, respectively. The results of quantitative immunoblot analysis are shown in Table I. The level of each G protein subunit was found to be greater in atrial membranes than in ventricular membranes. The Goa subunit level in atrial membranes was found to be 5.2-fold greater than that of ventricular membranes. In contrast, the atrial membrane Gia level was only 1.5-fold greater than the ventricular membrane level. The p subunit level inatrial membranes was 2.8-fold greater than the level in ventricular membranes. A recent report suggests a developmental increase in the concentration of the ventricular IAP substrate (Steinberg et al., 1985). The data presented in Table I show Goa to be a minor fraction of the sum of the a subunits ofGi and Go present in adult ventricle and therefore unable to account for the reported increase in IAP substrate. The level of Gia was examined in neonatal ventricular membranes and compared to thelevel in adult ventricular membranes. Fig. 3 shows the results of immunoblot analysis of neonatal and adultventricular membranes. There appears to be more Gia in neonatal than adult ventricular membranes. Quantitative immunoblot analysis (Table I) showed the level of Gia in neonatal ventricular membranes to be 3.3-fold greater than the level in adult ventricularmembranes. Thus, adevelopmental decrease occurs in the concentration of ventricular Gia. Northern Blot Analysis of Gia, Goa, and p mRNA-The molecular basis for differences in G protein subunit expression between atria and ventricles was investigated by Northern blot analysis. Total cellular RNA was prepared from adult and neonatal rat atria,ventricles, and brain, size-fractionated under denaturing conditions on agarose/formaldehyde gels, and transferred to nitrocellulose filters. The filters were then probed with nick-translated [32P]cDNAprobes 123,113, G27, G16, and Tg, which encode rat Goa and Gia-2 (Itoh et al., 1986), Gia-1 and Gia-3 (Jones and Reed, 1987), and the p subunit of bovine transducin (Fong et al., 1986), respectively (Figs. 4 and 5). Filters were then washed free of probe and reprobed with 4A, a cDNA clone encoding human 28 S RNA, to ensure that similar amounts of RNA were present in the atrial andventricular RNA samples. When the Goa probe was used, 4 bands were detected in TABLE I Quantitative immunoblot analysis of G protein subunits in rat heart Atrial and ventricular membranes were prepared and subjected to quantitative immunoblot analysis as described under “Experimental Procedures.” Data are presented as the mean picomoles/mg membraneprotein f S.D. of six to ninedeterminations.Statistically significant differences from the corresponding adult ventricle sample are denoted by an asterisk (*, p < 0.001). NA, not assayed. Adult Neonatal ventricles ventricles Adult atria subunit 13.4 k 2.2* 2.6 ? 0.3 NA Goa 22.5 2.5* 14.6 f 1.4 48.2 f 13.3* GC iY P 6.8 f 1.0* 2.5 f 0.4 NA

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FIG.3. Immunoblot analysis of Gla in neonatal and adult ventricular membranes. Samples were subjected to SDS-polyacrylamide gel electrophoresis on a 9% separation gel and transferred to nitrocellulose as described under “Experimental Procedures.“ The nitrocellulose was then stained with Amido Black (lanes 1 and 2 ) or incubated with affinity purified anti-Tcu antibodies (lanes 3 and 4 ) . Bound antibody was visualized as described under “Experimental Procedures.” Lane 1 contains 50 pg and lane 3 contains 100 pgof neonatal rat ventricular membrane protein. Lane 2 contains 50 pg and lane 4 contains 100 pg of adult rat ventricular membrane protein.

FIG.4. Northern blot analysis oftotal cellular RNA isolated from adult and neonatal atria, ventricles,and brain. Adult ( A ) and neonatal ( B ) atrial (lanes I and 4), ventricular (lanes 2 and 5), and brain (lanes 3 and 6 ) RNA samples, isolated and subjected to Northernblot analysis as described under“ExperimentalProcedures:” were hybridized to nick-translated cDNA probes encoding Goa (123; lanes 1 3 ) or TB (lanes 4-6) and exposed to film. At the bottom of each blot a portion of the same blot probed with nicktranslated 4A (encoding 28 S RNA) is shown. The dash marks to the left of each blot denote the chain lengthsof size markers: 5.1,4.1,3.1, 2.0, 1.6, and 1.0 kb. Each Northern blot was repeated an additional one to five times with similar results.

adult atrial, ventricular, and brain RNA samples (Fig. 4 A , lanes 1 - 3 ) with chain lengths of 4.0, 3.4, 3.0, and 1.7 kb. It is clear that a greater amount of probe hybridized to the atrial RNA sample than to the ventricular RNA sample. This result suggests that thedifference in Goa proteinlevel between atria and ventricles is due to a diference in thelevel of Goa mRNA. The Goa probe also hybridized to 4 bands in neonatal RNA samples (Fig. 4B, lanes 1-3). Agreateramount of probe hybridized to the neonatal atrial RNA sample than to the ventricular RNA sample. The T g probe detected 2 bands in adult atrial, ventricular

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concentrations was used. Relative RNA levels were compared on a counts/min/pg RNA basis (Table 11) and by measuring 123 456 789 123 456 789 ~the slope of the lines in Fig. 6 (data not shown). Similar results were obtained with bothmethods. Fig. 6A shows the results obtained with the Goa probe. Clear differences can be seen between the amount of hybridization to atrial as compared to ventricular RNA samples. When compared on a counts/min/pg RNA basis, the adult atrial RNA level was 3.4-fold greater than the adultventricular RNA level. Similarly, the neonatal atrial RNA level was 1.7-fold greater than the neonatalventricular RNA level. While the neonatal and adult atrial RNA levels were similar, the neonatal ventricular RNA level was 2.5-fold greater than that of adult ventricle. The TBprobe revealed little difference between atrial andventricular RNA levels in adult or neonatal samples (Fig. 6B). However, the neonatal atrial RNA level was 1.7-fold greater than the level in adult atria and the neonatal ventricular RNA level was 1.9-fold greater than the FIG.5. Northern blot analysis of total cellular RNA isolated level in adult ventricles. Results obtained using the Gia-2 from adult andneonatal atria, ventricles, and brain. Adult ( A ) probe are shown in Fig. 6C. While little difference between and neonatal ( B ) atrial (lunes I, 4 , and 7 ) , ventricular (lanes 2, 5 , the RNA levels in atrial and ventricular samples of the same and 8). and brain (lanes 3, 6, and 9) RNA samples, isolated and subjected to Northern blot analysis as described under “Experimental age was seen, statistically significant differences were seen were Procedures,” were hybridized to nick-translated cDNA probes encod- when the RNA levels inneonatalandadulttissues compared. The level in neonatal atria was1.5-fold greater ing Gin-1 (G27; lanes 1-3), Gioc-2 (113; lunes 4-6), or Gin-3(G16; lunes 7-9)and exposed to film. A t the bottom of each blot a portion than the level in adult atria. Thelevel in neonatal ventricles of the same blot probed with nick translated 4A (encoding 28 S RNA) was 1.9-fold greater than the level inadult ventricles. In is shown. The dash marks to the left of each blot denote the chain contrast, use of the Gia-3 probe indicated statistically signif2.0, 1.6, and 1.0 kb. Each lengths of size markers: 5.1,4.1,3.1, Northern blot was repeated an additional one to five times with icant changes in the levels of atrial and ventricular mRNA levels at each age (Fig. 6D). The level in adult atria was 1.3similar results. fold greater than the level in adult ventricle, while the level in neonatal ventricle was 2.0-fold greater than the level in and brain RNA with chain lengthsof 3.2 and 1.8 kb (Fig. 4A, neonatal atria. Also, a 2.3-fold increase in the adult atrial lanes 4-6). The amount of hybridization to the atrial and level was seen as compared to theneonatal atrial level. When ventricular RNA samples was approximately equal. Similar the Gia-1 probe was used, no signal was detectable above results were obtained with neonatal RNA samples (Fig. 4B, background by this method of analysis (data not shown). lanes 4-6). Fig. 6E shows results obtainedwhen selected nitrocellulose The Gia-1 probe detected one band with a chain length of disks were washed free of probe, then reprobed with 4A, a 3.5 kb in adult andneonatal brain RNA (Fig. 5,A and B, lane cDNA clone encoding human 28 S RNA. No significant 3 ) . Hybridization to adult andneonatal atrial and ventricular differences were observed between adult or neonatal atrial or RNA samples was not detected, consistent with the absence ventricular samples. This demonstrates that similar amounts of Gia-1 in rat heart reported by Jones and Reed (1987). of RNA were present in each of these samples. When the Gia-2 probe was used, one major and one minor band were detectedinadultatrial,ventricular, and brain DISCUSSION RNA samples (Fig. 5, lanes 4-6). The major band had a chain The results presentedin this study demonstrate differential length of 2.2 kb and the minor band was 1.6 kb in length. In tissue expression and developmental regulation of G protein contrast to Goa, the amountof hybridization to the atrial and ventricular samples was approximately equal. Similar results subunit levels in the rat heart. Measurement of subunit prowere obtained with neonatal RNA samples (Fig. 5B, lanes 4- tein levels using quantitative immunoblot analysis showed 6 ) . The Gia-3 probe detected one bandwith a chain length of the levels of Goa, Gia, and p to be higher in adult atria than 3.5 kb in adult atria, ventricle, and brain RNA samples. The in adult ventricles (5.2-, 1.5, and 2.8-fold, respectively). The amount of hybridization to these adult samples was approxi- difference in Goa level between atria and ventricles appears mately equal (Fig. 5A, lunes 7-9). In contrast, a 3.5-kb band to be due to a difference in mRNA level as determined was detected in neonatal ventricularand brain RNA samples, qualitatively by Northern blotanalysis, and quantitatively by dot blot analysis. The levels of mRNA encoding Gia-2 and p but not inneonatal atrial samples (Fig. 5B,lanes 7-9). Filters were then reprobed with 4A, a cDNA clone encoding were approximately equal in atria andventricular RNA samhuman 28 S RNA. This is shown at the bottom of each blot ples, and thus, cannotaccount for the observed differences in in Figs. 4 and 5. The amount of hybridization to the atrial protein levels. The levels of Gia-3 mRNA were,however, and ventricular samples on each blot was approximately equal. slightly higher in atria than in ventricles and thus may be at Quantitation of mRNA Levels-Dot blot analysis was per- leastpartially responsible for the observed differences in formed to quantitatethe relative differences between the protein levels. TheGiasubunit protein level in neonatal atrial and ventricularlevels of mRNA encoding Goa, Gia, and ventricle was found to be 3.3-fold greater than the level in p (Fig. 6). Total cellular RNA was used in these experiments adult ventricle. This 70% decrease in the Gia level appears to to eliminate problems arising from the variability of the be due, a t least in part,to a 46% decrease in theGia-2 mRNA level. No significant developmental change in the levels of recovery of poly(A+) RNA that inevitably resultsafter oligo(dT)-cellulose chromatography. Glyoxal denatured RNA ventricular Gia-1 or Gia-3 mRNA was observed. A developwas spotted onto nitrocellulose disks and probed with each of mental decrease was also observed in the Goa and @ mRNA the [“PIcDNA probes. For each tissue,a range of RNA levels in ventricles and in the Gia-2 and p mRNA levels in ~

Regulation of Cardiac GTP Proteins Binding

13362

4w

I

0

IO0 80

20

I

a

0 4

0

2

4

6

8

w

Pg RNA

1

0

1

2

RNA

FIG. 6. Dot blot analysis of rat cardiac G protein subunit mRNA level. Increasing amounts of total cellular RNA prepared from adult atria (O), adult ventricles (m), neonatal atria (A), and neonatal ventricles (A) were subjected to dot blot analysis as described under “Experimental Procedures.” The 32P-labeledcDNA probes used were 123, encoding rat Goa ( A ) , TO, encoding the @ subunit of transducin ( B ) , 113, encoding rat Gia-2 ( C ) , G16, encoding rat Gia-3 (D), and 4A, encoding human 28 S RNA ( E ) .Each point representsthe mean of duplicate determinations which usually differed from the mean by less than 10%.An additional experiment yielded similar results. TABLEI1 Dot blot analysis of G protein subunit mRNA levels in rat heart Adult and neonatal rat atrial and ventricular total cellular RNA samples were subjected to dot blot analysis as described in “Experimental Procedures.” Data from Fig. 5 are presented as the mean countslminlgg RNA f S.E. of the 3-5 duplicate determinations which make up each line. (Statistically significant differences from the ventricular value of the same age (superscripts a and b ) ; statistically significant differences from the corresponding adult sample (superscripts c-f).) Probe Age Atria Ventricles Adult Neonatal

wmlgg RNA 21.9 f 1.9” 6.5 f 1.1 16.6 f 4.2‘ 28.1 f 5.6b

Adult Neonatal

6.4 f 0.4 10.7 f 1.8‘

5.8 1.1 11.0 f 1.5d

Adult Neonatal

20.0 f 3.3 31.1 5 5.1d

22.2 f 3.9 41.5 f 12.1e

Adult Neonatal

7.9 f 0.4b 3.5 +- 0.7‘*’

Adult Neonatal

662.5 113.0 677.0 f 125.4

*

*

6.1 f 0.4

1.0 f 0.9

623.7 f 133.0 692.9 f 174.4

“ p< 0.001. b p < 0.02. < p< 0.01. d p < 0.02. ‘ p < 0.05. ’ p < 0.001.

atria. In contrast,the levels of Gia-3 mRNA were found to be 2.3 times greater in adult atria than in neonatal atria, indicating adevelopmental increase in thissubunit’s mRNA level. While we cannot eliminate the possibility that some of the

differences in mRNA levels between atria and ventricles and between neonatal and adult tissuesmay be due to differences in nonmyocardial cell types, the independent nature of the differences of the RNA levels for the individual subtypes demonstrates thatthe developmental and tissue-specific changes in G protein mRNAs cannot be due solely to differences in the proportions of myocardial and non-myocardial cells. Recent reports have suggested that adult ratventricle contains a single IAP substrate (Murakami and Yasuda, 1986; Steinberg et aZ., 1985). This IAP substrate was suggested to be the a subunit of Gi because of its apparent molecular weight of 41,000 Da. However, the a subunits of Gi and Go are often difficult to distinguish by IAP-catalyzed ADP ribosylation and SDS-polyacrylamide gel electrophoresis, making the use of specific antibodiesa more reliable method for determining the presence and identity of G protein subunits. Affinity purified antibodies specific for the a subunits of Gi and Go were used in this study to demonstrate that the a subunits of bothGproteinsarepresent in rat atria and ventricles. Quantitative immunoblot analysis employing these antibodies, and affinity purified antibodies specific for the common @ subunit, revealed large differences between the levels of theseGproteinsubunitsin atria and ventricles (Table I). The Goalevel was found to be 5.2-fold greater, the Gia level was 1.5-fold greater, and the @ level was 2.8-fold greater, in atria than thelevel in ventricles. The results presentedhere provide two possible reasons for the inability of others to distinguish both a subunits by IAPcatalyzed ADP ribosylation. First, and most importantly, the a subunits of Gi and Go in rat heart have similar mobilities on SDS-polyacrylamide gels (Fig. 2). Second, while the level

Regulution of Cardiac GTP Binding Proteins of Goa is 60% that of Gia in atria, the level of Goa is only 18%that of Gia in ventricles; the ventricular Goa level is SO low as to be barely detectable by immunoblot analysis (Table I). For these reasons, IAP labeling reveals only one band in rat cardiac membranes. Recently, it was suggested that the level of Gia in rat ventricle was regulated during development (Steinberg et al., 1985). A 16-fold increase in IAP substrate in adult ventricle membranes compared to neonatal ventricle membranes was reported. Since functional adrenergic innervation of the rat ventricle begins at 1-2 weeks after birth (Bareis and Slotkin, 1980; Bartolome et al., 1980), it was suggested that thedevelopmental increase in IAP substrate level may be due to the onset of sympathetic innervation. In addition, coculture of neonatal rat ventricle cells with sympathetic neurons was shown to cause an increase in the level of IAP substrate. This increase correlated with the acquisition of an a-adrenergic negative chronotropic response in the nerve-muscle cultures. In contrast, the results presented in this study demonstrate that the level of Goa in adult ventricle is too low to account for the increase in IAP substrate and that thelevel of Gia in neonatal ventricles was actually 3.3-fold greater than thelevel in adult ventricles. A combination of reasons prevented the quantitation of Goa and p in neonatal ventricles or the quantitation of any G protein subunitsin neonatal atria, including the relative weakness of the anti-Goa and anti+ antibodies, the low levels of Goa andp present in ventricular tissue, and the limited amount of neonatal atrial tissue available. The levels of mRNA encoding the G protein subunitswere examined by both Northern blot and dot blot analysis. Northern blot analysis of total cellular RNA with G27, a cDNA clone encoding rat olfactory Gia-1, was unable to detect hybridizing bands in adult or neonatal atria and ventricles, anddotblot analysis yielded no significant hybridization above background. These observationsare consistent with the work of Jones and Reed (1987), who reported the absence of Gia-1 mRNA from whole rat heart. Northern blot analysis of total cellular RNA with 113, a cDNA clone encoding rat C6 glioma Gia-2, revealed two hybridizing bands of 2.2 and 1.6 kb, with similar hybridization patterns being observed in neonatal and adult atrial, ventricular, and brain RNA samples. Others have observed Gia-2 messages of 2.1 and 2.6 kb in the U937 monocyte-like cell line (Didsbury et al., 1987) and theS49 lymphoma cell line (Sullivan et al., 1986), respectively. Dot blot analysis showed the Gia-2 mRNA levels to be similar between atrial and ventricular RNA samples of the same age. However,comparison of neonatal and adult samples showed a developmental decrease in the Gia-2 mRNA level. The developmental decrease in the ventricular level of Gia-2 mRNA was consistent with the observed decrease in the Gia protein level. When a cDNA clone encoding rat olfactory Gia-3 (G16) was used in Northern analysis, one hybridizing band of 3.5 kb was observed in adult atrial and ventricularsamples, and in neonatal ventricularsamples, while no analogous band was observed in neonatal atrial samples. Dot blot analysis demmRNA onstrated a developmental increase in the atrial Gia-3 levels. TWO bands also hybridized with the TBcDNA probe. Bands of 3.2 and 1.8 kb were observed in neonatal and adult atrial, ventricular, and brain RNA samples. Similar hybridization patterns have been reported using RNA samples prepared from retina, brain, liver, heart, and a variety of other tissues from several different species (Sugimoto et al., 1985; Fong et al., 1986, 1987). Little difference was observed between the levels of /3 mRNA in atria and ventricles of the same age.

13363

However, similar to what was observed for Gia, a developmental decrease in the p mRNA level occurred in both tissues. Northern blot analysis revealed bands of 4.0, 3.4, 3.0, and 1.7 kb which hybridized to 123, a cDNA clone encoding Goa, in samples of adult and neonatal atrial, ventricular, and brain RNA. The level of Goa mRNA was 3.4-fold greater in adult atriaas compared to adult ventricles. Thus,the 5.2-fold difference in Goa protein level between adult atrial and ventricular membranes appears to be the result of differences in the atrial andventricular Goa mRNA levels. The level of Goa mRNA was 1.7-fold greater in neonatal atria as compared to neonatal ventricles. Similar to what was observed for Gia and p, a developmental decrease in the Goa mRNA levelwas observed in ventricles. The existence of three forms of Gia complicates the interpretation of the immunoblot quantitationpresented here. Antiserum AS7, from which theanti-Ta antibodies were affinity purified, was prepared against the C-terminal decapeptide of the a subunit of bovine transducin (Goldsmith et al., 1987). The C-terminal decapeptides of Gia-1 and Gia-2 are identical, differing by only one amino acid from the Ta C-terminal decapeptide. The C-terminal decapeptide of the a subunit of Go differs from that of Ta by five amino acids and is not recognized by the anti-Ta antibodies. The Gia-3 Cterminal decapeptide differs from that of Gia-1 andGia-2, as well as Ta, by twoadditional aminoacid changes at positions 350 and 354. Changes at these positions also occur in the Cterminal decapeptide of Goa and may contribute to the inability of anti-Ta antibodies to recognize Goa. Although a novel IAP substrate is present in human neutrophils (Gierschik et al., 1986a) and antiserum AS7 recognizes a protein in neutrophils and the HL-60 neutrophil-like cell line, the major IAP substrate in these cells is Gia-2 (Goldsmith et a i , 1987). For these reasons it is unclear whether, in addition to Gia-l andGia-2, the anti-Ta antibodies also recognize Gia-3. The results of the Northern and dot blot analyses indicate that there is no developmental increase in the ventricular levels of mRNA encoding Goa, Gia-1, Gia-2, or Gia-3. Thus, while we cannot formally exclude the possibility that the developmental increase in IAP substrate reported by Steinberg et al. (1985) may be due to an increase in the level of Gia-3 protein, it seems most likely that it is due either to an as yet uncharacterized a subunit, or a change in the extent to which the a subunitsare susceptible to ADP-ribosylation without a change in protein level. Comparison of the p subunit protein level with the sum of the Gia and Goa protein levels reveals an a/P ratio of 5.3 in adult atria and 6.9 in adult ventricles. A smaller a subunit excess ( a / p = 1.4) has been reported in rat brain (Milligan et al., 1987). These resultssuggest that free a subunits may exist in rat cardiac membranes, although it is also possible that additional p subunits are present that are not recognized by the anti+ antibodies. While antiserum RV3 has been shown to recognize the 36,000-Da p subunit (Gierschik et al., 1986b), it is unclear whether the 35,000-Da p subunit is also recognized (Sternweis and Robishaw, 1984; Roofet al., 1985).While it is also possible that a selective loss of fl subunits occurred during preparation of the membranes, this was shown not to be the case for rat brain (Milligan et aZ., 1987). In contrast to rat heart, the a/fl ratio in chick heart ranges from 0.28 to 0.67 during development, suggesting the presence of free p subunits (Luetje et al., 1987). Previously, we examined the regulation of subunits of Go and Gi and the mAChR during embryonic chick cardiac development (Luetje et al., 1987). The mAChR has been shown to interact with Goand Gi (Florio and Sternweis,1985; Kurose

13364

Regulation of Cardiac GTP Binding Proteins

et al., 1986; Haga et al., 1986). Atrial-specific changes in the concentration of Goa,Gia, (3 subunit, andmAChR wereshown to occur at times which coincided with or closely followedthe onset of functional parasympathetic innervation of the chick heart. These changes were associated with changes in the regulation of adenylate cyclase activity. These results suggest a role for the process of innervation in the regulation of both the number and function of the mAChR and the G proteins with which it interacts. In thepresent study, differences were observed between the levels of Goa, Gia, and p in rat atria and the levels in rat ventricle. Developmental regulation of subunit protein and mRNA levels was also observed. Interestingly, similar tissue distribution and developmental regulation has been reported for the mAChR (Wei and Sulakhe, 1978; Nedoma et al., 1986). In neonatal rats, the atrialmAChR number was %fold higher than that of ventricles. The atrial mAChR number then decreased to a value in adult atria thatwas 2-foldhigher than the adult ventricular mAChR number. Thus, as has been shown in the embryonic chick heart, subunits of Gi and Go, and the mAChR with which they interact, are regulated in a developmental and tissue specific manner during rat cardiac development. Acknowledgments-We would like to thank Dr. Allen Spiegel for his generous gift of antisera RV3 and AS7. We would also like to thank Drs. Hiroshi Itoh, Yoshito Kaziro, Randall Reed, James Hurley, and Randall Moon for their gifts of the cDNA clones used in this work. Note Added in Proof-Antiserum AS7 has recently been shown to exhibit weak reactivity with Gia-3 (Goldsmith et al., 1988). REFERENCES Bareis, D.L., and Slotkin, T. A. (1980) J. Phurmacol. Exp. Ther. 2 1 2 , 120-125 Bartolome, J., Mills, E., Lau, C., and Slotkin, T. A. (1980) J. Phurmacol. Exp. Ther. 2 1 6 , 596-600 Blackmore, P. F., Bocckino, S. B., Waynick, L. E., and Exton, J. H. (1985) J. Bid. Chem. 260,14477-14483 Bray, P., Carter, A., Guo, V., Puckett, C., Kamholz, J., Spiegel, A., and Nirenberg, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,51155119 Britwieser, G. E., and Szabo, G. (1985) Nature 317, 538-540 Cassel, D., and Pfeuffer, T. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2669-2673 Cathala, G., Savouret, J. F., Mendez, B., West, B.L., Karin, M., Martial, J. A., and Baxter, J. D. (1983) DNA (N.Y.) 2 , 329-335 Cockcroft, S., and Gomperts, B. D. (1985) Nature 314,534-536 Codina, J., Grenet, D., Yatani, A., Birnbaumer, L., and Brown, A. M. (1987a). FEBS Lett. 2 1 6 , 104-106 Codina, J., Yatani, A., Grenet, D., Brown, A. M., and Birnbaumer, L. (1987b) Science 236,442-445 Didsbury, J. R., and Snyderman, R. (1987) FEBS Lett. 219,259-263 Didsbury, J. R., Ho, Y.-S., and Snyderman, R. (1987) FEBS Lett. 2 11,160-164 Endoh, M., Maruyama, M., and Iijima, T. (1985) Am. J. Physiol. 2 4 9 , H309-H320 Florio, V. A., and Sternweis, P. C. (1985) J. Biol. Chem. 2 6 0 , 34773483 Fong, H. K. W., Hurlet, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F., and Simon, M. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,2162-2166 Fong, H. K. W., Amatruda, T. T., Birren, B. W., and Simon, M. I. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 3792-3796 Gao, B., Gilman, A. G., and Robishaw, J. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 6122-6125 Gierschik, P., Codina, J., Simons, C., Birnbaumer, L., and Spiegel, A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 727-731 Gierschik, P., Falloon, J., Milligan, G., Pines, M., Gallin, J. I., and Spiegel, A. (1986a) J. Biol. Chem. 261,8058-8062 Giershick, P., Milligan, G., Pines, M., Goldsmith, P., Codina, J., Klee,

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,

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and Birnbaumer, L. (1987)