Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation.

Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.

II To whom correspondence should be addressed Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. and ion channels by a set of heterotrimeric G proteins composed of a, b, and y subunits. The a subunits bind guanine nucleotides and are substrates for covalent modification by bacterial toxins. One structurally very similar set of G protein a subunits can be covalently modified by Bordetella pertussis toxin. This set includes the two transducins that are localized exclusively in the retina, as well as four proteins (a, and ai.l-3) that are found in many kinds of cells (reviewed by Neer and Clapham (1988)). The a, protein is most abundant in the central nervous system but is not located exclusively there (Neer et al., 1984;Sternweis and Robishaw, 1984;Huff et al., 1985). ai.2 and ai.3 have been found in all cells studied to date, whereas ai.' appears to have a more limited distribution, being absent in some cells of myelocytic origin, some fibroblast lines, endothelial cells (Kim et al., 1988;Lee et al., 1989), and GH4 cells.' The pertussis toxin substrates mediate inhibition of adenylylcyclase, activation of phospholipase C and phospholipase Az, and modulation of ion channels.
Within the family of nonretinal pertussis toxin substrates, the structural similarity is extremely close. The amino acid sequence of ai.l is 85% identical with ai.2 and 95% identical with ai.3. a, is about 70% identical with the ai group (reviewed by Lochrie and Simon (1988)). Given such a high degree of structural similarity, one important question is whether the different a subunits in this family preferentially couple particular plasma membrane receptors and effectors or whether the cell uses them interchangeably. If the functions of the subunits are entirely interchangeable, then hormonal responsiveness might be determined only by the total level of a subunits in this family and not by the subtype composition. One argument against the complete interchangeability of the a proteins is the extreme conservation of their structures across species. For example, ai.l is 98% identical among humans, rats, mice, and cows (Lochrie and Simon, 1988). This conservation argues that even the small difference in sequence must be important in order to have been conserved.
Analysis of the kinetics of GTP binding, hydrolysis, and release shows that there are subtle differences among the three ai isoforms (Carty et Linder et al., 1990). Although these differences in kinetics may be important for setting the rate of onset and duration of signal transmission, they do not reveal whether G protein a subunits are specific for particular receptors or effectors. To define the specificity of their interactions with receptors or effectors, different a i subtypes have been reconstituted with target proteins in vitro. The results of such reconstitution experiments have shown some specificity but also many examples of complete interchangeability (reviewed by Neer and Clapham, 1988).
We have tested the hypothesis that G protein subunit isoforms contribute differentially to hormonal responses by determining the consequences of altering a cell's complement of these proteins. To carry out the study, we needed a cell line with well defined plasma membrane receptors that does not express all three subtypes of ai. We found that Balb/c 3T3 cells lack ai.l and a, protein but express ai.2 and ai+ The growth of these cells and of other fibroblast cell lines is regulated by mitogens and growth factors that act through cell surface receptors, some of which are coupled to G proteins (Chen and Buchanan (1975); reviewed by Rozengurt, 1986;and Pouyssegur, 1990). Treating the cells with pertussis toxin blocks the mitogenic effect of such agonists, including thrombin, bombesin, vasopressin, or bradykinin, but does not affect growth stimulation by PDGF,2 fibroblast growth factor, or epidermal growth factor (Letterio et al., 1986;Chambard et al., 1987;Murayama and Ui, 1987;Hoshijima et al., 1988). The agonists that act through G proteins, as well as those that act through tyrosine kinase receptors, activate phospholipase C but do so by different mechanisms (Berridge and Irvine, 1984;Hasegawa-Sasaki et al., 1988;Hoshijima et al., 1988, Nanberg andRozengurt, 1988;Taylor et al., 1988). However, activation of phospholipase C is not obligatory for their stimulation of cell growth (Zachary et al., 1987;Taylor et al., 1988;Hill et aL, 1990). The exact pathway leading to increased cell growth in response to these agents is not known, although it appears to involve elevation of intracellular calcium and/or modulation of arachidonic acid metabolites and Na+/H+ exchange (Pouyssegur et al., 1982; Murayama and Ui, 1985; Raben et al., 1987;Nanberg and Rozengurt, 1988;Handler et al., 1990; also reviewed by Rozengurt, 1986).
In this paper we report the results of studies comparing control Balb/c 3T3 and cells expressing ai.l with respect to growth and to stimulation by thrombin and PDGF. We have previously shown that introduction of an additional a: subunit (ao) into Y1 adrenal cells slows their growth (Bloch et al., 1989). Expression of ai.l in a cell type that normally does not produce this subunit both slows its growth and alters its response to thrombin. Comparison of the thrombin response of the transfected cells to that of control cells suggests that the receptor for this mitogen may couple preferentially to the newly introduced a subunit.

Construction of the Expression Vector Containing Rat a,., cDNA
The rat a,.] cDNA was isolated from a rat olfactory neuroepithelium cDNA library by R. Reed and D. Jones (Johns Hopkins University School of Medicine) who kindly provided the clone (Jones and Reed, 1987). The EcoRI fragment of ai.l cDNA (representing the entire coding region) was inserted into the EcoRI site of the retroviral expression vector, pDOJ (Price et al., 1987); provided by C. Cepko, Harvard Medical School). The orientation of the insertion was confirmed by asymmetric digestion with restriction enzymes Hind111 and XbaI. In this vector (pDOJ-ai.]), the transcription of ai.] cDNA is directed by the Moloney murine leukemia virus long terminal repeat. The vector also contains the neomycin resistance gene under the control of the simian virus 40 early region promoter. In mammalian 'The abbreviations used are: GTPyS, guanosine 5'-3-0-(thio)triphosphate; PDGF, platelet-derived growth factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. cells, expression of the neomyocin resistance gene confers resistance to the antibiotic G418 sulfate.

Transfection of Balb/c 3T3 Cells
Balb/c 3T3 cells were transfected with pDOJ-ai.1 and pDOJ control vector by the calcium phosphate precipitation method (Wigler et al., 1978). Five 10-cm dishes of confluent Balb/c 3T3 cells were split 1:15 on the day before transfection. The cells were fed again with complete (10% serum) medium 4 h before transfection. 20 pg of plasmid DNA that had been purified twice by CsClz gradient centrifugation were used for each dish. DNA was precipitated in 0.5 ml of 250 p M CaCl', 5 mM Tris-OH, 0.5 mM EDTA (pH 7.6) and added to an equal volume of 2 X concentrated HEPES-buffered saline. The mixture was incubated at room temperature for 30 min to allow the DNA to precipitate before being added to 10-cm dishes of cells. The precipitates were left on the cells for 16 h, after which they were washed off with fresh culture medium containing 0.5 mg/ml G418. Control cells were transfected with the pDOJ vector without a cDNA insert and grown in G418.
Control cells and cells transfected with ~D O J -W .~ were selected in parallel. The cells were grown in 0.5 mg/ml G418, and approximately 40 resistant clones from pDOJ and P D O J -~~.~ tranfection were obtained after 12-14 days in culture. Analysis of the DNA from clones transfected with ~DoJ-cu,.~ by Southern blotting showed positive bands with an probe. However, none of these clones was positive on Northern analysis of their mRNA. Because of previous experience that Y1 adrenal cells expressing a, grew slowly (Bloch et al., 1989), we continued the selection to try to isolate slow growing colonies. Four such colonies were isolated after 3-4 weeks (as described below). These more slowly growing colonies were positive for mRNA (data not shown) and protein (data not shown) and were used in the subsequent studies. We did not see multinucleate cells such as were seen in Y1 adrenal cells transfected with a, (Bloch et al., 1989). To establish that all the functional changes that we observe are due to transfection and not to clonal variations within the Balb/c 3T3 cell line, three control and three transfected lines were characterized.

Analysis of ai Isoforms
Two-dimensional Electrophoresis of a Subunits-Cells from 10-cm culture dishes were collected by scraping them from the dishes. They were washed 3 times with phosphate-buffered saline, and the pellet taken up in a buffer containing 50 mM Tris, pH 7.6, 75 mM sucrose, 6 mM MgCl,, 1 mM EDTA, and 1 mM dithiothreitol. The cells were lysed by freeze/thawing three times and by repeated passage through a 23-gauge needle. To make a crude particulate fraction, the lysate was centrifuged at 10,000 rpm in a SS-34 rotor of a Sorvall centrifuge for 20 min. Protein was determined by the method of Lowry (1951), as modified by Bailey (1967), using bovine serum albumin as a standard.
30 pg of either total cellular protein or of the crude particulate fraction was incubated in a total of 40 p1 with 5 p~ NAD, 3 mM ATP, 12.5 mM isoniazid, 10 mM thymidine, 0.1 mM GTP, 50 mM Tris (pH 7.6), 0.5 pCi [32P]NAD, 0.1% Lubrol PX, and 10 pg/ml activated pertussis toxin (List Biochemicals). The toxin was activated in 20 mM dithiothreitol at 30 "C for 15 min. The ADP-ribosylation reaction was carried out at 37 "C for 30 min. For one-dimensional SDS-PAGE, the reaction was stopped by adding 3 X concentrated Laemmli sample buffer (Laemmli, 1970). For two-dimensional gel electrophoresis, the reaction was stopped with 5 mM KZCOS and 0.2% deoxycholate. Twodimensional gel electrophoresis was carried out as described by O' Far-re11 (1975). The gel was dried between cellophane membranes and used to expose Kodak XAR film at -70 'C.
Western Blots-50-75 pg of total cell lysate or crude particulate fraction was applied to lanes of an 11% SDS-PAGE gel. The proteins were transferred to nitrocellulose, as described by Towbin et al. (1979). The filters were blocked with 3% bovine serum albumin or with 3% bovine serum albumin and 3% goat serum for 1-2 h at room temperature or overnight at 4 "C. They were incubated with antibody diluted in 3% bovine serum albumin or 3% bovine serum albumin, with 10 mM Tris, 0.9% NaC1, pH 7.4 (TBS), once with TBS plus 3% goat serum overnight at 4 "C. The nitrocellulose was washed twice 0.05% Nonidet P-40 and again with TBS before incubating with Iz5Ilabeled goat anti-rabbit IgG (ICN, Inc.). The radioactive IgG was washed from the membrane as described above, and the membrane was used to expose Kodak XAR film with two screens at -70 "C. The antibodies used were LD, an antibody raised against peptide corresponding to ai.] sequence 159-168 (a kind gift of Dr. Allen Spiegel, Washington. D. C. (Spiegel et al., 1990)); R7, an antibody against bovine brain p; R4, an antibody against brain a, (Huff et al., 1985); AS/7, an antibody raised against a peptide corresponding to the carboxyl terminus of transducin, which recognizes ai.] and (Y;.~ equally (Du Pont-New England Nuclear; NE1, 801 (Spiegel, 1990)); NE1, 803 (Du Pont-New England Nuclear), an antibody to the carboxylterminal peptide of a8.:,, which recognizes and a". To quantitate the amounts of ai.] and (yi+ the radioautograms were scanned with an LKB laser densitometer that resolved the two peaks. The ratio of (Y,.] to ei.2 was estimated in two ways that are described below under "Results."

Adenylylcyclnse Assay
The adenylylcyclase assay was performed as described by Salomon (1979). The standard assay contained 0.5 mM ATP, 1 pCi of ['"PI ATP (Amersham Corp.), 50 pM CAMP, 5 mM MgCI?, 50 mM Tris, pH 7.6, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 10 mM creatine phosphate, 5 units/ml creatine phosphokinase, and 20-30 pg of membrane protein in a total volume of -50 pl. The reaction was carried out at 30 "C for 20 min and stopped with a solution containing 8,000-15,000 cpm of ['HICAMP to monitor recovery of ['"PICAMP from the columns. Under these conditions, the reaction was proportional to the amount of protein (not shown).

Analysis of Cell Growth
Cells were plated in 24-well plates and allowed to grow for 24 h. They were trypsinized to remove them from the dish, stained with trypan blue (final concentration, 0.2%), and counted in a hemocytometer.
['HIThymidine Labeling of DNA Approximately 2-5 X lo" cells were plated into each well of a 24well culture dish (Nunc, Inc.) and grown to confluence (2 days) in 2 ml of Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 mg/ml streptomycin. The concentration of fetal calf serum was then lowered to 1% for 4 days. Prior to the experiment, the cells were placed in serumfree medium for 24 h to make cells quiescent and to remove them from serum mitogens. The cells were washed once with serum-free medium, then incubated in serum-free medium containing 1 pCi of [:'H]thymidine/ml, and the indicated amounts of mitogen and/or pertussis toxin. Bovine a-thrombin was from Sigma. For stimulation through the PDGF receptor, we used recombinant c-sis (R-B homodimer), the generous gift of Dr. T. Deuel, Washington University, St. Louis, MO. It was diluted into 0.1% bovine serum albumin in phosphate-buffered saline. Pertussis toxin, when it was used, was added 3 h prior to addition of the the mitogen and the ["Hlthymidine. Labeling was carried out for 36 h after addition of ['Hlthymidine. Labeled cells were treated with trypsin and resuspended in 0.5 ml of phosphate-buffered saline. The cells were transferred to microcentrifuge tubes and precipitated with 5% trichloroacetic acid. The pellets were washed twice with 75% ethanol and dissolved in 0.5 ml of 0.1 M NaOH, 2% Na2CO:3, and the radioactivity was determined by liquid scintillation counting.

Expression of
Protein by Transfected Cells-We used two methods to show that the ai.l-transfected cells, but not the control cells, expressed ai.l protein. Fig. 1 shows the results of a Western blot probed with antisera that recognize .] and ei.2 recognized by this antibody are identical. IAnes I and 2, two different control clones. Lanes 3-5, three different %.,-transfected clones. Bound antibody was detected with 1151-laheled anti-rabhit IgG. The radioautogram was exposed for 66 h without screens. with ai.I and ai.p, which are identical with each other in this region (Spiegel, 1990).
The immunologic studies show that ai.l-transfected cells contain a new immunoreactive protein that is absent from control cells, confirming the results described above. Because the antibody recognizes and equally, the ratio of immunoreactivity in the two bands should reflect the proportion of to in the transfected cells. We used two methods to estimate the ratio of ai.I to ai.p. The first was to average the area of the peaks of ~yi.~ in the controls and subtract this area from the total of ai., and ai.p in the transfected cells. By this method, we estimate that the ai.l band is 30 st 4% (range, n = 3) of the endogenous ai.p. The second method was to cut the peaks out and separate them along a line drawn perpendicular to the trough between them. The ratio of to cyl.? was calculated from the weights of the two peaks. By this method, we estimate that is 44 f 2% ( n = 3) of ai.p. Since (~i .~ is more abundant than this method would tend to overestimate the amount of ai.l. Fig. 3 shows a two-dimensional gel analysis of pertussis toxin ADP-ribosylated proteins in membranes from control cells and cells transfected with ai.I. The pertussis toxin substrates in control cells are resolved into one major and one minor spot. The cyi.l-transfected cells have an additional ADPribosylated protein with a more positive isoelectric point. This pertussis toxin-labeled protein has a PI very similar to that which we have found for purified ADP-ribosylated ai., from bovine brain (data not shown). By densitometry, the spot was 80 & 20% ( n = 2) of the endogenous spot. Balb/c 3T3 cells were also transfected with pDOJ containing rat CY, cDNA. Although G418-resistant colonies were obtained that were positive on Southern blots using rat CY, cDNA as a probe, none of these produced CY, protein (data not shown).
Adenylylcyclase Activity in cui.l-trunsfected Cells-The CYI proteins were first described as inhibitors of adenylylcyclase (Murayama and Ui, 1985). Therefore, changes in the complement of ai subtype might be expected to change the activity of that enzyme. Fig. 4 shows a comparison of GTPyS-stimulated adenylylcyclase activity in membranes from control and ai., transfected cells. In cyi.l-transfected cells, the level of adenylylcyclase activity achieved a t maximal concentrations of GTPyS is approximately 60% of that in the control cells.
In addition, the ECso for GTPyS apparently increased from 33 k 4 to 115 f 15 nM ( n = 4). The maximal level of GTPstimulated activity is also diminished in the ni.,-transfected cells, although there seems to be no difference in the apparent ECso (data not shown).
Thrombin inhibits adenylylcyclase in several cell types (Murayama and Ui, 1985; and does so both in ai.,-transfected and control cells (Fig. 5). Although all activities are blunted in the e.,-transfected cell, there is no dramatic difference in the ability of thrombin to inhibit adenylylcyclase, especially when the activities are calculated relative to the basal activity.
Effect of Transfection of on Cell Growth-Cells expressing protein grow more slowly than cells transfected with the pDOJ plasmid without insert. The slowness of growth created some difficulties in the isolation of cells expressing (see "Materials and Methods"). Fig. 6 shows a comparison of growth rates of three independent cui.,-transfected and control clones. The doubling time is 11 k 1 ( n = 3) h for control cells and 24 f 1 ( n = 3) h for cYi.,-transfected cells.
Stimulation of DNA Synthesis by Thrombin in a;.,-trunsfected and Control Cells- Fig. 7 shows the response of quiescent Balb/c 3T3 cells to stimulation by thrombin at the concentrations indicated on the figure. The ECso for thrombin is 8 nM for both control and ai.,-transfected cells, although the response is greater in cells expressing (~i -~ than in controls. Thymidine uptake is increased 2-fold in control cells, but 5fold in ai.l-transfected cells. The response of the cells to 1 and 10% serum was measured in the same experiments. The absolute response of control cells and ai.l-transfected cells was similar, although the increase over unstimulated thymidine incorporation was greater in control than in ai.l-transfected cells (1% serum, 8and 6-fold increases, respectively; 10% serum, 15-and 10-fold increases, respectively).
The stimulation of DNA synthesis by thrombin is inhibitable by pertussis toxin (Fig. 8). The ai.l-transfected cells are more sensitive than the controls to pertussis toxin, with an ECso of 0.9 f 0.1 ng/ml (n = 4) compared with 5 f 0.6 ng/ml ( n = 4) for the controls. Furthermore, the inhibition of DNA synthesis is more complete for the a!i.l-transfected than the control cells.
Effect of PDGF (rc-sis) on the Growth of Transfected and Control Cells-Control and ai.l-transfected cells respond to PDGF (rc-sis) in exactly the same way (Fig. 9). Pertussis toxin inhibits the growth response to PDGF, although it does so at higher concentrations and to a lesser extent than the inhibition of response to thrombin (Fig. 10). The significance of growth inhibition at high concentrations of pertussis toxin is not clear.

DISCUSSION
Transfection of BALB c/3T3 cells with pDOJ-ai.l results in expression of ai.l protein. Immunologic analysis confirmed the presence of ai.l protein in ai.l-transfected cells but not in control cells. One antibody we used recognizes a sequence that is identical in ai.l and ai.2. The ratio of immunoreactive bands allows us to estimate the relative concentration of these two a subunits. By this analysis, the amount of ai.l is between 30 and 44% of the amount of ai.2. There was no detectable change in the amount of /3 in a!i.l-transfected cells. These calculations are important for the interpretation of subsequent experiments. A massive overproduction of an a subunit could reveal low affinity associations or could significantly shift the equilibrium between a and /3 subunits by sequestering 0-y subunits. Our estimate of the increase in a subunits caused by transfection is an overestimate since we are relating it only to ai.2, whereas in fact the cell also has as, (~i .~ (by immunoblot, data not shown) and probably other, as yet unidentified, a! subunits that might participate in an equilibrium with /3r subunits. The expression of in the transfected cells was also shown by the appearance of a new radiolabeled spot on two-dimensional gel electrophoresis of pertussis toxin-catalyzed ["PI ADP-ribosylation of a subunits. Densitometry of the radioautograms shows that the new a! subunit is present in an amount that is approximately 80% of the endogenous a subunit(s). Susceptibility to ADP-ribosylation is a complex process. It requires interaction of a with 0-y subunits (Neer et al., 1984) and may reflect the affinity of different a subunits for /3r subunits (Huff et al., 1985), as well as factors intrinsic to the different a! structures. ADP-ribosylation of appears to be more efficient than that of ai.2, since ADP-ribose in is 80% of that in ai.2, whereas by immunologic analysis, the ai.1 protein is 30% of ai.2. This observation is consistent with our findings that the thrombin responses of wl-transfected cells are more sensitive to pertussis toxin than control (see below).
A major goal of this study was to determine whether a change in the a subunit isoform composition affected coupling of receptors. To this end, we analyzed the effects of thrombin on adenylylcyclase and on DNA synthesis. Expression of blunted basal activity, as well as stimulation of adenylylcyclase by GTPyS, GTP, or forskolin, as if the adenylylcyclase inhibitory "tone" were generally higher. However, inhibition of adenylylcyclase by thrombin was not substantially changed.
Since the changes were small and generalized, we could not interpret them unambiguously.
In contrast, transfection of the cells with had a marked effect on thrombin stimulation of DNA synthesis. Thymidine uptake in response to thrombin was greater in transfected than in control cells. The growth response to thrombin was inhibited by pertussis toxin, and the ai.l-transfected cells were now 5-fold more sensitive to pertussis toxin than the control cells. Taken together, these results suggest that the thrombin receptor may couple preferentially to the newly introduced ai.l, simultaneously giving a greater response to thrombin and a greater sensitivity of that response to inhibition by pertussis toxin. We were not able to determine how thrombin affects proliferation. Thrombin responses are blocked by serum, and we were not successful in supporting proliferation of either the control or q1-transfected cells in 0.5% serum or less.
The effects of transfection are not the same for all agonists that stimulate DNA synthesis in these cells. There was no difference in the dose-response curve to PDGF (rc-sis) between control and ai.l-transfected cells nor in the effect of pertussis toxin on PDGF (rc-sis)-stimulated DNA synthesis. Pertussis toxin does cause a partial inhibition of DNA synthesis, but this happens at rather high concentrations of the toxin. Similar effects have been observed in other cells (for example, Taylor et al., 1988), but the significance of this observation is not clear.
Selection of transfectants expressing ai.l was hampered by the fact that the cells expressing grew more slowly than the controls. A similar decrease in growth rate was seen in Y-1 adrenal cells transfected with a . (Bloch et al., 1989). In addition to decreased growth rates, the Y-1 cells became multinucleate. We did not see multinucleate ai.l-transfected or control BALB c/3T3 cells. The stimulation of DNA synthesis in quiescent wtransfected and control cells by 1 or 10% serum is similar, although the response relative to unstimulated values is greater in control than in ai.,-transfected cells. It is possible that ai.l-transfected cells are unable to respond normally to a growth factor in serum. Such a defect would explain their slower growth. However, the ai.l-transfected cells are not intrinsically defective in DNA synthesis nor in the ability to go from Go to S phase since their response to thrombin is greater than control. We propose, therefore, that steps distal to DNA synthesis are affected by transfection of Future studies to define the precise step in the cell cycle that is affected may suggest the mechanism by which this effect takes place.
The pathway by which thrombin and other mitogens stimulate cell growth is not yet known. Activation of phospholipase C and initiation of a cascade of intracellular changes in Ca2+ and protein kinase C may be involved (reviewed by Rozengurt, 1986 andPouyssegur, 1990). Thrombin does activate this pathway in 3T3 cells and other fibroblasts (Dicker and Rozengurt, 1980;Carney et al., 1985;Magnaldo et al., 1987;Burch and Axelrod, 1987;. However, in several instances, receptor activation of phospholipases has been uncoupled from the mitogenic response (Zachary et al., 1987;Taylor et al., 1988;Hill et aL, 1990). The modest effects on adenylylcyclase do not suggest a central role of cyclic nucleotide metabolism in mediating the effects of ai.1 on cell growth.
These studies suggest that the ai subunit composition of the cell can have profound effects on its response to stimulation through cell surface receptors. Further analysis of the specificity of actions will require transfecting these cells with low levels of other a subunits. However, in the present studies, it is important that ai.l is a quantitatively minor addition to the a subunits already present in Balb/c 3T3 cells. The difference between ai-transfected and control cells in the sensitivity of thrombin-stimulated DNA synthesis to inhibition by pertussis toxin suggests that the newly introduced a subunit effectively competes with the endogenous proteins for receptor and/or effectors. Although the difference in amino acid sequence between ai.2, the major endogenous G protein ai subunit in BALB c/3T3 cells, and C X .~ is only 12%, that difference appears to have important functional consequences for the growth of intact cells.