THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 6, Issue of February 25, pp. 3102-3105.1990 Printed in U.S. A. Differential Degradation Rates of the G Protein (Y,, in Cultured Cardiac and Pituitary Cells* (Received for publication, September 29, 1989) Seth Silbert#z, Thomas Michel, Richard Lee, and Eva J. NeerQ From the DeDartment of Medicine. &FdiOvaSculaF Division, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Muskxhusetts d2115 Signal transduction in biological membranes is mod- ulated by a family of GTP-binding proteins termed G proteins. Differences in the tissue-specific expression of G protein subtypes suggest that the levels of individ- ual G proteins may be an important determinant of the hormonal response in a given cell type. We have used a polyclonal antibody raised against the purified G protein, a0 to study a0 in the rat pituitary cell line GH, and in primary rat cardiocytes in culture by quantita- tive immunoprecipitation. Biosynthetic labeling and specific immunoprecipitation of a0 in pulse-chase ex- periments demonstrated that the t% for a,, degradation is 28 2 7 h (n = 4) in GH, pituitary cells and is greater than 72 h (n = 4) in cardiocytes. The steady-state level of a,, protein is similar in both cell types as measured by Western blots. Northern blots of poly(A)-selected mRNA from these two cell types were probed with labeled a0 cDNA and showed they have similar a,, mRNA levels. The observation of different degradation rates, but similar steady-state protein levels, suggests that the rate of a, synthesis is different in GH, cells and cardiocytes. Since mRNA levels are approximately equal in both, our studies imply that protein translation controls may be important determinants of G protein a subunit concentrations in biological membranes. Receptors for many kinds of hormones and neurotransmit- ters are coupled to ion channels and intracellular enzymes by members of a closely related family of heterotrimeric guanine nucleotide-binding proteins (G proteins). At present, about a dozen different (Y subunits of the G proteins have been iden- tified by molecular cloning (reviewed by Neer and Clapham, 1988; Gilman, 1987; Stryer and Bourne, 1986). These multiple G protein (Y subunit isoforms exhibit varying levels of tissue- specific expression. At one extreme, the G protein LY subunits involved in phototransduction are segregated into different retinal cell types and are not found outside the retina (Lerea et al., 1986). In contrast, the adenylate cyclase stimulatory (Y, subunit is found in all normal cells (but not in some mutant cell lines). The ai family of G proteins has a variable pattern * This work was supported by Grants GM36259 and GM35417 (to E. J. N.), Physician Scientist Award HL01835 (to R. L.), and a Clinician-Scientist Award from the American Heart Association (to T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i Present address: Washintion Universitv of Medicine. St. Louis. Mb 63110. 5 To whom correspondence should be addressed: Cardiology Divi- sion, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. of expression. Although some cells may contain all three ai subtypes, they vary widely in the relative amounts, with no clear biochemical phenotype yet associated with the expres- sion of an individual isoform (Kim et al., 1988, Jones and Reed, 1987). The most striking differences in expression occur with (Ye, a protein first identifed in brain (Neer et al., 1984; Sternweis and Robishaw, 1984). a0 comprises about 0.5% of particulate protein in the central nervous system, is found at considerable lower levels in a wide variety of peripheral tis- sues, and is entirely lacking in some cells (Huff and Neer, 1986; Asano et al., 1988). The cellular function of (Y,, is not yet known, although it may regulate ion channels or enzymes in phosphoinositol metabolism (Worly et al., 1986; Hescheler et al., 1987; VanDongen et al., 1988). The ability of a cell to respond to a hormone or neurotrans- mitter depends not only on the amount of receptor and effector, but also on the amount and identity of G proteins present in the membrane. Regulation of G protein levels is a potential point of control which would determine the ability of a cell to respond to a particular hormone. Changes in G protein levels have been demonstrated to occur in cellular differentiation (Watkins et al., 1987) or organ development (Liang et al., 1986; Leutje et al., 1988) concomitantly with changes in hormone responsiveness. As a first step toward studying the mechanisms which regulate G protein LY subunit levels, we have compared the degradation rate of LYE, as well as steady-state protein and mRNA levels for LYE in two types of cells: rat GH, cells, a permanent dividing cell line derived from the anterior pituitary, and primary rat neonatal cardi- ocytes, which are fully differentiated but which do not divide in culture. MATERIALS AND METHODS Cell Culture and Biosynthetic Labeling-The rat pituitary cell line GH, (provided by A. Schonbrunn, Baylor University, Houston, TX) was maintained in Dulbecco’s modified Eagle media (GIBCO), sup- plemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin, and was grown at 37 “C in 5% CO,. Cells were split 2 days befire biosynthetii labeling. For biosynthetic labeling, the cells were washed with RPM1 medium supplemented with essential amino acids, except for methionine (RPMI-met) and starved for methionine for 1 h at 37 “C in RPMI-met supplemented with 5% fetal calf serum (dialyzed extensively against 150 mM NaCl). The medium was re- nlaced with RPMI-met, sunnlemented with 10% dialyzed fetal calf Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu- clear, final concentration, 0.1 WM [35S]methionine). Labeling was done for 6-20 h. Primary rat cardiocytes were isolated as previously de- scribed (Chien et al., 1985) and biosynthetic labeling with [35S] methionine was performed as described above. Following biosynthetic labeling, the medium was replaced with RPM1 medium containing 1 mM unlabeled methionine, and the cells were harvested at varying times as described below. Antibody Preparation and Zmmunoprecipitation-The polyclonal antibody against the bovine brain G protein 01, (o(& was previously characterized in our laboratory (Huff et al., 1985). The method of 3102 by guest on July 15, 2018 http://www.jbc.org/ Downloaded from
Transcript
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 265, No. 6, Issue of February 25, pp. 3102-3105.1990 Printed in U.S. A.
Differential Degradation Rates of the G Protein (Y,, in Cultured Cardiac and Pituitary Cells*
(Received for publication, September 29, 1989)
Seth Silbert#z, Thomas Michel, Richard Lee, and Eva J. NeerQ From the DeDartment of Medicine. &FdiOvaSculaF Division, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Muskxhusetts d2115
Signal transduction in biological membranes is mod- ulated by a family of GTP-binding proteins termed G proteins. Differences in the tissue-specific expression of G protein subtypes suggest that the levels of individ- ual G proteins may be an important determinant of the hormonal response in a given cell type. We have used a polyclonal antibody raised against the purified G protein, a0 to study a0 in the rat pituitary cell line GH, and in primary rat cardiocytes in culture by quantita- tive immunoprecipitation. Biosynthetic labeling and specific immunoprecipitation of a0 in pulse-chase ex- periments demonstrated that the t% for a,, degradation is 28 2 7 h (n = 4) in GH, pituitary cells and is greater than 72 h (n = 4) in cardiocytes. The steady-state level of a,, protein is similar in both cell types as measured by Western blots. Northern blots of poly(A)-selected mRNA from these two cell types were probed with labeled a0 cDNA and showed they have similar a,, mRNA levels. The observation of different degradation rates, but similar steady-state protein levels, suggests that the rate of a, synthesis is different in GH, cells and cardiocytes. Since mRNA levels are approximately equal in both, our studies imply that protein translation controls may be important determinants of G protein a subunit concentrations in biological membranes.
Receptors for many kinds of hormones and neurotransmit- ters are coupled to ion channels and intracellular enzymes by members of a closely related family of heterotrimeric guanine nucleotide-binding proteins (G proteins). At present, about a dozen different (Y subunits of the G proteins have been iden- tified by molecular cloning (reviewed by Neer and Clapham, 1988; Gilman, 1987; Stryer and Bourne, 1986). These multiple G protein (Y subunit isoforms exhibit varying levels of tissue- specific expression. At one extreme, the G protein LY subunits involved in phototransduction are segregated into different retinal cell types and are not found outside the retina (Lerea et al., 1986). In contrast, the adenylate cyclase stimulatory (Y, subunit is found in all normal cells (but not in some mutant cell lines). The ai family of G proteins has a variable pattern
* This work was supported by Grants GM36259 and GM35417 (to E. J. N.), Physician Scientist Award HL01835 (to R. L.), and a Clinician-Scientist Award from the American Heart Association (to T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
i Present address: Washintion Universitv of Medicine. St. Louis. Mb 63110.
5 To whom correspondence should be addressed: Cardiology Divi- sion, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115.
of expression. Although some cells may contain all three ai subtypes, they vary widely in the relative amounts, with no clear biochemical phenotype yet associated with the expres- sion of an individual isoform (Kim et al., 1988, Jones and Reed, 1987). The most striking differences in expression occur with (Ye, a protein first identifed in brain (Neer et al., 1984; Sternweis and Robishaw, 1984). a0 comprises about 0.5% of particulate protein in the central nervous system, is found at considerable lower levels in a wide variety of peripheral tis- sues, and is entirely lacking in some cells (Huff and Neer, 1986; Asano et al., 1988). The cellular function of (Y,, is not yet known, although it may regulate ion channels or enzymes in phosphoinositol metabolism (Worly et al., 1986; Hescheler et al., 1987; VanDongen et al., 1988).
The ability of a cell to respond to a hormone or neurotrans- mitter depends not only on the amount of receptor and effector, but also on the amount and identity of G proteins present in the membrane. Regulation of G protein levels is a potential point of control which would determine the ability of a cell to respond to a particular hormone. Changes in G protein levels have been demonstrated to occur in cellular differentiation (Watkins et al., 1987) or organ development (Liang et al., 1986; Leutje et al., 1988) concomitantly with changes in hormone responsiveness. As a first step toward studying the mechanisms which regulate G protein LY subunit levels, we have compared the degradation rate of LYE, as well as steady-state protein and mRNA levels for LYE in two types of cells: rat GH, cells, a permanent dividing cell line derived from the anterior pituitary, and primary rat neonatal cardi- ocytes, which are fully differentiated but which do not divide in culture.
MATERIALS AND METHODS
Cell Culture and Biosynthetic Labeling-The rat pituitary cell line GH, (provided by A. Schonbrunn, Baylor University, Houston, TX) was maintained in Dulbecco’s modified Eagle media (GIBCO), sup- plemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin, and was grown at 37 “C in 5% CO,. Cells were split 2 days befire biosynthetii labeling. For biosynthetic labeling, the cells were washed with RPM1 medium supplemented with essential amino acids, except for methionine (RPMI-met) and starved for methionine for 1 h at 37 “C in RPMI-met supplemented with 5% fetal calf serum (dialyzed extensively against 150 mM NaCl). The medium was re- nlaced with RPMI-met, sunnlemented with 10% dialyzed fetal calf Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu- clear, final concentration, 0.1 WM [35S]methionine). Labeling was done for 6-20 h. Primary rat cardiocytes were isolated as previously de- scribed (Chien et al., 1985) and biosynthetic labeling with [35S] methionine was performed as described above. Following biosynthetic labeling, the medium was replaced with RPM1 medium containing 1 mM unlabeled methionine, and the cells were harvested at varying times as described below.
Antibody Preparation and Zmmunoprecipitation-The polyclonal antibody against the bovine brain G protein 01, (o(& was previously characterized in our laboratory (Huff et al., 1985). The method of
Differential Degradation Rates of the G Protein cxO
immunoprecipitation was a modification of that described by Lau- dano and Buchanan (1986). Following biosynthetic labeling, the medium was aspirat.ed and cells washed three times with 150 mM NaCI, 10 mM Tris.HCl, pH 7.4. Lysis buffer (0.5% SDS,’ 10 mM Tris. HCl, pH 7.4, 1 mM EDTA) supplemented with 1 mM phenyl- methylsulfonyl fluoride and 10 units/ml aprotinin (Sigma) was added, and cells were harvested with a rubber policeman. Greater than 90% of the cells were adherent to the plate after 72 h in culture. The lysate was heated to 90% for 2 min, placed on ice, and Nonidet P-40 was added to a final concentration of 10%. Approximately 5 x lo” cells were harvested/well of a 6-well dish (Costar) and yielded 1 ml of Iysate. Antiserum was added and incubated for 4 h at 4 “C, and the lysate was centrifuged in a table-top microcentrifuge for 5 min. The supernatant was added to protein A-Sepharose CL4B (Sigma) under conditions of protein A excess (30 ~1 of protein A-Sepharose/ml lysate). The lysate was incubated with protein A-Sepharose for 2 h at room temperature, and then the protein A-Sepharose was washed three times in lysis buffer with Nonidet P-40. After a final wash in 150 mM NaCI, 10 mM Tris.HCl, pH 7.4, the protein A-Sepharose was heated at 90 “C for 2 min with SDS-PAGE sample buffer and the supernatant removed and analyzed by SDS-PAGE (Laemmli, 1970). The gels were fixed, soaked in Autofluor (Du Pont-New England Nuclear), dried and exposed on Kodak XAR film using intensifying screens as noted in the figure legends. Intensity of the band was quantitated using an LKB laser densitometer.
Western Blots-For analysis of Western blots, GH, cells, neonatal rat ventricles, or adult rat brain were homogenized in 50 M Tris-HCl, pH 7.6, 6 mM MgCl*, 75 mM sucrose, 1 mM dithiothreitol, and 3 mM benzamidine. The protein concentration was measured as described by Lowry (1951). Western blots were performed as described by Huff et al. (1985) using the same polyclonal antiserum to (Y, as was described in that paper.
Northern Blots-RNA was isolated using the guanidinium isothi- ocyanate method (Chirgwin et al., 1979) and poly(A)-containing RNA was prepared using oligo(dT)-cellulose. The RNA was separated on a denaturing 1% agarose gel and transferred to nitrocellulose filters (Maniatis et al., 1982). It was probed with rat (Y, cDNA (EcoRI fragment) comprising the whole cDNA insert. The rat (Y, clone was kindly provided by Dr. Randall Reed, John Hopkins University, Balt.imore, MD, and is described by Jones and Reed (1987).
RESULTS
The specificity of the immunoprecipitation reaction is shown in Fig. 1. The antibody precipitates a band with a molecular mass of 39 kDa. Immunoprecipitation is blocked by excess purified G,. Addition of the Pr subunit has no effect on immunoprecipitation (data not shown). It is important in studies comparing the amount of radiolabeled protein immu- noprecipitated at various times to be certain that, in each case, the experiments are performed in antibody excess. The data in Fig. 2 show that the amount of antiserum (10 ~1) used in the studies of protein degradation was indeed in excess. The amount of cellular protein used in this antiserum titration was equivalent to the largest amount present after several days of cell growth under “chase” conditions. Another control for adequacy of immunoprecipitation was to precipitate the supernatant from the first immunoprecipitation by adding another lo-~1 sample of antiserum. In such experiments, no more than lo-20% of the original immunoprecipitable mate- rial was left in the supernatant. The time course determined from this residual material left in the supernatant was the same as that for the first precipitation. Therefore, no sub- stantial error was introduced by incomplete immunoprecipi- tation.
Fig. 3, panel A, shows the time course of disappearance of pulse-labeled LY, in GH, cells. Data for cardiocytes are shown in panel B. The lower panels in Fig. 3 show the aggregate results of four individual experiments, while the upper panels show a radioautogram of a representative experiment with
’ The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
3103
12 3
ao- -.
FIG. 1. Specificity of a, immunoprecipitation. Lysates of bio- synthetically labeled GH, cells were incubated with a 1:lOO dilution of anti-n, immune serum (lanes I and 3) or nonimmune serum (lane 2), without (lanes 1 and 2) or with (lane 3) 4 pg of purified unlabeled G, protein (Neer et al., 1986). Immunoprecipitation, SDS-PAGE, and autofluorography were performed as described under “Materials and Methods.” Biosynthetic labeling with [‘“Slmethionine was for 20 h. Equivalent volumes of lysate were processed for each of the three treatments shown (1 ml of lysate from 5 x 10” cells in a 6-well tissue culture dish) and the experiment replicated twice with equivalent results. These data demonstrate a specific protein at the expected M, of 39,000 that is immunoprecipitated by immune, but not nonimmune, serum and blocked by purified G,,.
1
pl Antiserum
FIG. 2. Antiserum titration curve. Varying amounts of anti-a,, immune serum were incubated with a fixed volume (1 ml) of biosyn- thetically labeled GH, cell lysate. After precipitation, SDS-PAGE, and autofluorography. the intensity of the specific cy, band was quantitated by densitometry. Based upon these data, 10 ~1 of anti- serum was used in subsequent experiments precipitating similar or smaller quantities of cellular protein.
each time point determined in duplicate. From these data, a tl,* for LY,, degradation in GH, cells was calculated to be 28 f 7 h (n = 4). The half-life of the protein in cardiocytes was so long that we could not accurately measure Lo/,, which is greater than 72 h. There was no difference in degradation rates between atria1 and ventricular cardiocytes. The difference in the degradation rates is not simply due to dilution of the label as the GH, cells divide, since the experiments were performed under conditions of antibody excess where virtually all of the label initially present was immunoprecipitated. Therefore, the data reflects the degradation of that initial pool of metaboli- cally labeled (Ye and are independent of cell division.
The slow turnover observed in primary cardiocytes raises the question of whether those non-dividing cells have a gen- erally slower metabolism of protein. To evaluate this, we
3104 Differential Degradation Rates of the G Protein LX,
FIG. 3. Time course of (x, degra- dation. Panel A shows the time course for TV,, disappearance following pulse- labeling of GH., cells. Panel B reflects data similarly obtained in primary neo- natal rat cardiocytes; in this panel, the open a.vmbols represent atria1 cardiocytes and the closed symbols represent cardi- ocytes cultured from ventricular tissue. In each panel, the upper figure depicts a representative experiment, with dupli- cate determinations at each time point of the “chase.” Below, the results of these individual experiments are shown as ag- gregate data pooled from four similar experiments and quantitated by densi- tometry of the immunoprecipitated a,. Each type of symbol represents the mean of duplicate samples in separate experi- ments. Western blots of cells at the be- ginning and end of the pulse-chase ex- periments revealed no significant change in total amount of CY, protein present in the cells over the time course of these experiments.
followed the decay of radiolabel in several protein bands identified by radioautography following SDS-PAGE either of supernatant obtained after immunoprecipitation or of total cell lysates. Several bands were identified which decayed with half-lives of 20-30 h. This control indicates that the chase protocol is adequate.
Because of the marked difference in degradation rates of the N, protein in cardiocytes versus GH, cells, we wanted to determine whether there was also a difference in steady-state levels of the two proteins. Fig. 4 shows a Western blot of GH, and neonatal rat ventricular cells probed with the same 01” antibody used in the immunoprecipitation experiments de- scribed above. Although the antibody was raised against bo- vme No, cDNA analysis of bovine LY, and rat o(~ shows that these two proteins vary at only two amino acids (Jones and Reed, 1987; Van Meurs et al., 1987). It is, therefore, unlikely that there is an important immunological difference in 01, antigenicity between the two species. In any event, since both the neonatal rat ventricular cells and GH, cells are from the same species, the estimate of relative amounts of a, is valid regardless of the degree of antiserum cross-reactivity between bovine and rat (Y”. A Western blot shows similar overall levels of N,, in neonatal ventricular and GH, cells; a &fold greater
WESTERN BLOT
BG H
c -a0
FIG. 4. Western blots of GH, cells, neonatal rat ventricle, and adult rat brain probed with the a. antibody. Unfractionated homogenates of adult rat brain (lane B), GH, cells (lane G), or neonatal rat ventricle (lane H) were analyzed by Western blotting as described in the text. In the representative experiment shown, 75 pg of cellular protein was loaded in lanes G and H, and 81 pg of protein in lane B. Densitometry revealed that GH, cells and ventricular cells contained similar amounts of CY, and that rat brain contained 5-fold higher CY,, immunoreactivity.
GHB
Northern Blot
FIG. 5. Northern blots of GH, cells, primary rat cardiocytes, and rat brain probed with rat a, cDNA. 10 pg of poly(A)- containing RNA from GH, cells (lane G) or primary rat cardiocytes (lane H) and rat brain (lane B) were loaded/lane, transferred to nitrocellulose following denaturing agarose gel electrophoresis, and hybridized using the “P-labeled rat (Y, cDNA (EcoRI fragment) as probe using standard techniques. The autoradiograms shown were obtained following 48-h exposure on Kodak XAR film using two intensifying screens. Densitometry was performed as described in the text.
amount of CC, is present in rat brain (Fig. 4). The amount of LYE in neonatal rat heart is 8593% (range of two experiments) of that in GH, cells.
One of the puzzling features of the G proteins is that the levels of mRNA for the various LY subunits seem to bear no relationship to the quantity of (Y subunit protein present in the cell. For example, (Ye is by far the most abundant G protein (Y subunit in the brain, and yet the message is extremely difficult to detect (in contrast, the protein (Y, is one of the least abundant LY subunits in the brain membrane and its mRNA is relatively abundant) (Jones and Reed, 1987). We estimated the relative amount of mRNA for LY~ in adult rat brain, GH, cells, and neonatal rat cardiocytes by Northern blots (Fig. 5). Densitometry of the autoradiograms showed equivalent amounts of the 4.4-kilobase band which corre- sponds to the major species of (Ye mRNA (Jones and Reed, 1987; Leutje et al., 1988). The range of values was *lo% of the mean. The same poly(A)-selected mRNA Northern blot
Differential Degradation Rates of the G Protein a, 3105
probed with ai-2 showed that all three contained abundant aim2 message (not shown); these positive findings with an olie2 probe also act as a control for the quality of mRNA loaded on the Northern blot.
DISCUSSION
The mechanisms by which the quantity of individual G protein 01 subunits are controlled in the cell are not clearly understood. We have measured the degradation rates of 01, in two cell types in an effort to define the regulation of (Ye levels in intact cells. The half-time of (Ye degradation is very different in the two cell lines, with a tl,g of 28 + 7 hours (n = 4) in GH, cells and greater than 72 h in cardiocytes (n = 4).
Estimates of the stability of G protein-coupled receptors have been reported from several laboratories, with determi- nations made by different methods in different cells. Esti- mates for turnover of (Y receptors range from 18 to 46 hours (Bouhelal et al., 1987a; Schwartz et al., 1985; Taouis et al., 1986; Mahan et al., 1987). Turnover of fl-adrenergic receptors is more variable with reports ranging from 30 h in S49 cells (Mahan and Insel, 1986) to 8 days in C6 glioma and B&H1 cells (Bouhelal et al., 1987a). A single report of adenylate cyclase turnover in B&H1 cells gives a degradation half-time of 40 h (Bouhelal et al., 1987b). The regulation of protein turnover for these members of the G protein-coupled receptor system is a potential point of control which would determine the response of a given cell to a specific hormone.
We have found that the half-life of (Ye in cardiocytes is much longer than in GH, cells. If the rates of 01, synthesis were the same in these two cells, the steady-state level of LY, would be greater in cardiocytes than in GH, cells. Analysis of steady-state a, protein levels by Western blots show that this is not the case; rather, the steady-state levels of the (Y subunits are similar in the two cell types. Since the steady-state level reflects the balance of synthesis and degradation, we would conclude that the rate of synthesis of the 01, subunit in GH, cells is faster than the rate of synthesis in cardiocytes.
The rate of synthesis of a given protein in cells is usually proportional to the amount of its mRNA. Levels of mRNA for G protein (Y subunits have been shown to change during development, differentiation, and in response to hormones or lymphokines (Watkins et al., 1987; Lee et al., 1989; Leutje et al., 1989); it is not known whether these changes reflect differences in mRNA transcription or stability. However, in the case of (Ye, regulation appears to be more complex, possibly including additional mechanisms of control. The present stud- ies demonstrate that the steady-state level of cy, protein in rat brain is about five times higher than in ventricular or GH, cells; however, we have also shown that the mRNA level is approximately equal in brain, heart, and GH, cells. These results agree with those reported by Jones and Reed (1987) who found very small variations in (Y,, mRNA among different rat tissues despite the fact that protein levels vary dramati- cally (Huff et al., 1985). Our conclusion that GH, cells and cardiocytes synthesize (Y~ at different rates, together with our observation that they have equal levels of mRNA, suggests
that translational control mechanisms may be important de- terminants of the steady-state levels of G protein (Ye subunits.
Acknowledgments-We are grateful to Paula McColgan for expertly typing the manuscript and to Dr. Cynthia Tolman and Lawrence T. O’Connor for help with some of the experiments.
REFERENCES
Asano, J., Semba, R., Kamiva. N., Ogasawava. N.. and Kato, K. (1988) J. Neurochmi 50, i164-1169-
Bouhelal, R., Mermet-Bouvier, R., Mauger, J. P., Sladeczek, F., Hornburger, V., and Bockaert, J. (1987a) J. Recept. Res. 7, 299- 320
Bouhelal, R., Bockaert, J., Mermet-Bouvier, R., Guillon, G., and Homburger, V. (1987b) J. Biol. Chem. 262, 8470-8475
Chien, K. R., Sen, A., Reynolds, R., Cheng, A., Kim, Y., Gunn, M., Buia, L. M., and Willerson. J. T. (1985) J. Clin. Znuest. 75, 1770- 1780’
Chirgwin, J. M., Przybla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 8, 5294-5299
Gilman, A. G. (1987) Annu. Reu. Biochem. 56, 615-650 Heschler, J., Rosenthal, W., Trautwein, W., and Schultz, G. (1987)
Nature 325, 445-447 Huff, R. M., Axton, J. M., and Neer, E. J. (1985) J. Biol. Chem. 260,
10864-10871 Jones, D. T., and Reed, R. R. (1987) J. Biol. Chem. 262, 14241-
14249 Kim, S. Y., Ang, S.-L., Bloch, D., Bloch, K., Kawahara, Y., Lee, R.
L., Tolman, C., Seidman, J. G., and Neer, E. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85.4153-4157
Laemmli, U. K. (1970) I\jature 227, 680-685 Laudano, A. P., and Buchanan, J. M. (1986) Proc. Natl. Acad. Sci. U.
S. A. 83, 892-896 Lee, R. T., Brock, T. A., Tolman, C. A., Bloch, K. D., Seidman, J. G.,
and Neer, E. J. (1989) FEBS Lett. 249,139-142 Lerea, C. L., Somers, D. E., Hurley, J. B., Klock, I. B., and Bunt-
Milam, A. M. (1986) Science 234,77-80 Leutje, C. W., Tietje, K. M., Christian, J. L., and Nathanson, N. M.
(1988) J. Biol. Chem. 263,13357-13365 Liane. B. T.. Neer. E. J.. and Gainer. J. B. (1986) J. Biol. Chem. 261.
g($-go2i - Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)
J. Biol. Chem 193,265-275 Mahan, L. C., and Insel, P. A. (1986) Mol. Pharmacol. 29, 7-15 Mahan, L. C., McKernan, R. M., and Insel, P. A. (1987) Annu. Reu.
Pharmacol. Toxicol. 27.215-235 Maniatis, T., Fritsch, E..F., and Sambrook, J. (1982) in Molecular
Cloning: A Laboratory Manual. p. 545, Cold Snrina Harbor Labo- ratories, Cold Spring-Harbor, NY - -
Neer, E. J., and Clapham, D. E. (1988) Nature 333, 129-134 Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259,
14222-14229 Schwartz, K. R., Lanier, S. M., Carter, E. A., Homey, C. J., and
Graham, R. M. (1985) Mol. Pharmacol. 27, 200-209 Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259,
13806-13813 Stryer, L., and Bourne, H. R. (1986) Annu. Reu. Cell. Biol. 2, 391-
419 Taouis, M., Berlan, M., and Lafontan, M. (1986) Mol. Pharmacol.
31,89-96 VanDoneen. A. M.. Codina. J.. Olate. J.. Mattera. R.. Joho. R.. and
Birnbaumer, L. (1988) Science 242,1433-1437 ’ Van Meurs, K. P.. Angus. C. W.. Lavu. S.. Kuna. H.. Czarnecki. S.
K., Moss; J., and Vaughan, M.‘(1987) Proc. Natl. Acad. Sci. U: S. A. 84,3107-3111
Watkins, D. C., Northup, J. K., and Malbon, C. C. (1987) J. Biol. Chem. 262,10651-10657
Worley, P. F., Baraban, J. M., Van Dop, C., Neer, E. J., and Snyder, S. H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4561-4565
![Page 1: Differential Degradation Rates of the G Protein (Y,, in ... · Differential Degradation Rates of the G Protein ... Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu-](https://reader030.documents.pub/reader030/viewer/2022030902/5b3f40277f8b9aff118be75a/html5/page/1.jpg)
![Page 2: Differential Degradation Rates of the G Protein (Y,, in ... · Differential Degradation Rates of the G Protein ... Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu-](https://reader030.documents.pub/reader030/viewer/2022030902/5b3f40277f8b9aff118be75a/html5/page/2.jpg)
![Page 3: Differential Degradation Rates of the G Protein (Y,, in ... · Differential Degradation Rates of the G Protein ... Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu-](https://reader030.documents.pub/reader030/viewer/2022030902/5b3f40277f8b9aff118be75a/html5/page/3.jpg)
![Page 4: Differential Degradation Rates of the G Protein (Y,, in ... · Differential Degradation Rates of the G Protein ... Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu-](https://reader030.documents.pub/reader030/viewer/2022030902/5b3f40277f8b9aff118be75a/html5/page/4.jpg)
![Page 5: Differential Degradation Rates of the G Protein (Y,, in ... · Differential Degradation Rates of the G Protein ... Herum and 100 pCi of [‘5S]*iethionine (Du Pont-New England Nu-](https://reader030.documents.pub/reader030/viewer/2022030902/5b3f40277f8b9aff118be75a/html5/page/5.jpg)
![Differential N-end Rule Degradation of RIN4/NOI Fragments … · Differential N-end Rule Degradation of RIN4/NOI Fragments Generated by the AvrRpt2 Effector Protease1[OPEN] Kevin](https://static.documents.pub/doc/80x56/60c1572f5593fd29102a7d9d/differential-n-end-rule-degradation-of-rin4noi-fragments-differential-n-end-rule.jpg)
