Mar 23, 1988 - kinin are due to changes in receptor number rather than to direct coupling by p21r between the receptor and phospho- lipase C. The eukaryotic ...
Proc. Nail. Acad. Sci. USA Vol. 85, pp. 5774-5778, August 1988 Biochemistry
p21ras-induced responsiveness of phosphatidylinositol turnover to bradykinin is a receptor number effect (signal transduction/guanine nucleodde-binding protein)
JULIAN DOWNWARDt, JEAN DE GUNZBURG, REBECCA RIEHL, AND ROBERT A. WEINBERG Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142
Contributed by Robert A. Weinberg, March 23, 1988
Some recent reports have presented evidence that p2lras may indeed be involved in the direct control of this enzyme (14, 15). The strategy used to study p21s-mediated linkage between receptors and phospholipase C has involved creation of cell lines that overexpress one or another p2l"S species. As a result of p2lras overexpression, any physiological coupling between specific receptors and phospholipase C by p2iras could be enhanced, thus allowing identification of the interacting members of such a signal transduction pathway. In the work presented here, the role of p2lras in the regulation of phospholipase C is investigated. We have found that the marked enhancement of the PtdIns turnover response to a peptide mitogen in cells overexpressing both normal and transforming Ha-, Ki-, and N-ras proteins is not reflective of a role of p2lras in transducing signals from this receptor.
Proteins encoded by ras genes have recently ABSTRACT been reported to couple certain growth factor receptors to phospholipase C, the enzyme catalyzing phosphatidylinositol breakdown. To investigate this hypothesis, the normal and the transforming Ha-, Ki-, and N-ras genes were each transfected into Rat-i fibroblasts under the control of strong promoters. Several cell lines, both normal and transformed, were selected that expressed high levels of p2lr,. Phosphatidylinositol turnover was measured in these cells in response to a wide variety of peptide factors; bradykinin was found to have a greatly enhanced effect on the p21r overexpressors relative to the parental and control cells. Bradykinin receptor numbers were measured in these lines and found to be up to 40-fold higher in the p2lras overexpressors than in the parental cells. This was found to be the case for both normal and transforming forms of all three varieties of ras genes. Receptor number correlated well with the bradykinin-dependent phosphatidylinositol turnover response in all cases. These data indicate that the effects of p21r on cellular responses to the peptide hormone bradykinin are due to changes in receptor number rather than to direct coupling by p21r between the receptor and phospholipase C.
MATERIALS AND METHODS Cell Lines and Plasmids. Rat-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The plasmids used were pSV2-His (histidinol-resistance gene; S. Hartman and R. C. Mulligan, personal communication), pSVEPBL116 (normal human cHa-ras cDNA; ref. 16), ZnCK (normal human c-Ki-ras cDNA containing exon 4B; ref. 17), pCD1234B (transforming human Ki-ras cDNA containing exon 4B; ref. 18), pSV2-Neo (neomycin-resistance gene), pZIP.JGl.N-ras (normal human N-ras cDNA under the control of Moloney leukemia virus long terminal repeat). Rat-1 cells were transfected with these plasmids by calcium phosphate-mediated gene transfer. A ratio of 1 part selection plasmid to 50 parts ras construct was used. Cells transfected with pSV2-His were selected in 10% calf serum supplemented with DMEM without histidine and containing 250 ,uM histidinol. Cells transfected with pSV2Neo were selected in 10% calf serum-supplemented DMEM containing 500 ,ug of G418 per ml. Two previously created Rat-i-derived cell lines were used: Rat-l.N-ras*2 (transfected with pSV2-gpt and human transforming N-ras cDNA under simian virus 40 early promoter; J. Cunningham and R.A.W.) and Rat-i.H-ras*1 [transfected with pSV2-gpt and human transforming EJ (Ha-ras) gene; H. Land and R.A.W.]. A control pSV2-gpt-transfected Rat-1 line was also used (Rat-l.Gptl). The transformed lines Rat-l.N-myc* and Ratl.src* were generous gifts from J. M. Bishop (University of California, San Francisco); Rat-i.sis*1 was generated by Hartmut Land (Imperial Cancer Research Fund, London). Growth of cell lines in soft agar and on monolayers of parental cells was measured and classified as described in ref. 19.
The eukaryotic ras genes encode a family of proteins (M, 21,000) that appear to be important in control of cell growth. The acutely transforming Harvey and Kirsten murine sarcoma viruses contain activated mutants of Ha- and Ki-ras genes, and oncogenic forms of the Ha- and Ki-ras as well as of the related N-ras gene have been identified in a large number of human tumors (referred to as HRAS, KRAS, and NRAS in human gene nomenclature) (1-4). The mechanisms by which activated p2iras transforms cells and the ways in which normal p21ras may be involved in cellular growth control are currently unclear, although in recent years several clues have emerged. The ras proteins are peripheral membrane proteins (5) that bind and hydrolyze GTP; the activated proteins have reduced GTPase activity (6, 7). This points to a functional homology with members of the family of G proteins, GTP-binding proteins that are involved in the coupling of cell-surface receptor proteins to intracellular enzyme systems, most notably adenylate cyclase and cGMP phosphodiesterase (8). A limited sequence homology also exists between p2iras and the G proteins (9-12), so it is tempting to speculate that, despite their considerable differences, the ras proteins may also couple cell-surface receptors to intracellular enzymes. One enzyme known to be controlled by poorly characterized G proteins and to be of great importance in the regulation of cell growth is phospholipase C, the phosphodiesterase responsible for phosphatidylinositol (Ptdlns) breakdown (13).
Abbreviations: G protein, GTP-binding protein; PtdIns, phosphatidylinositol; GTP[y-35S], guanosine 5'-[y-[355]thio]triphosphate; [125I-Tyr8]bradykinin, bradykinin with l25l-labeled tyrosine at position 8. tTo whom reprint requests should be addressed.
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Proc. NatL. Acad. Sci. USA 85 (1988)
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'2MI-Labeled Bradykinin Binding. Cells were seeded at 2 x 105per 3-cm dish, grown for 24 hr in DMEM containing 10% calf serum and then serum starved for 12 hr prior to binding experiments on whole cells. The medium was removed and replaced by 0.69 ml of fresh DMEM to which was added bradykinin with 1251I-labeled tyrosine at position 8 ([125I1 Tyr8]bradykinin) (obtained from New England Nuclear) at the concentrations indicated. Background binding was measured by performing the reaction in the presence of 10 ,uM unlabeled bradykinin. The incubations were carried out for 30 min at 4°C; the medium was then removed and the cells were washed carefully four times with 2 ml of medium plus 1 mg ofbovine serum albumin per ml at 0°C. The washed cells were then solubilized by the addition of 1 ml of 1 M NaOH and counted for y radioactivity. All determinations are averages of triplicate experiments with background counts subtracted.
Immunoprecipitation and Quantitation of p21w. Dishes (10 cm) of cells were grown to confluence, labeled with [IS]methionine for 12 hr, and then lysed and p21 was immunopre4 cipitated with monoclonal antibody Y13-259 as described in ref. 20. Estimates of levels of p2lr expression were made by immunoprecipitation from unlabeled cells with monoclonal antibody Y13-259 as described above except that only the nonionic detergent Triton X-100 was used (1% in lysis buffer). The immunoprecipitates were washed four times with 1 ml each of a 20 mM Hepes buffer (pH 7.4) containing 500 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100. The washed pellets were incubated in 50 mM Hepes buffer (pH 7.4) containing 100 mM NaCl, 5 mM MgCl , and 1 AM guanosine 5'-[y-[35S]thio~triphosphate (GTP[y- 5S]) (150 Ci/ mmol; 1 Ci = 37 GBq) for 2 hr at 37°C. These conditions had previously been shown to give saturated binding of GTP[yS] to ras proteins. At the end of the incubation, the pellets were washed four times with 1 ml each of a 20 mM Hepes buffer (pH 7.4) containing 500 mM NaCl, 5 mM MgCl2, and 0.1% Triton X-100 at 4°C. Backgrounds were estimated in parallel incubations, which included 1 mM unlabeled GTP[yS]: they were in all cases
