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• W2079136503 abstract "The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor β receptor (PDGFβR). The E5/PDGFβR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys499 at the extracellular juxtamembrane position and Thr513 within the transmembrane domain of the PDGFβR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFβR was performed to further characterize the nature of the E5/PDGFβR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr513 as well as its positioning along the transmembrane α-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile506 and Leu520 as additional requirements for the interaction. Because Lys499, Thr513, Ile506, and Leu520 all align along the same face of the predicted PDGFβR transmembrane α-helix, our data support the model that the PDGFβR contacts the E5 protein via multiple amino acids along a single α-helical interface. The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor β receptor (PDGFβR). The E5/PDGFβR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys499 at the extracellular juxtamembrane position and Thr513 within the transmembrane domain of the PDGFβR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFβR was performed to further characterize the nature of the E5/PDGFβR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr513 as well as its positioning along the transmembrane α-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile506 and Leu520 as additional requirements for the interaction. Because Lys499, Thr513, Ile506, and Leu520 all align along the same face of the predicted PDGFβR transmembrane α-helix, our data support the model that the PDGFβR contacts the E5 protein via multiple amino acids along a single α-helical interface. Bovine papillomavirus type 1 (BPV-1) 1The abbreviations used are: BPV, bovine papillomavirus type 1; PDGF, platelet-derived growth factor; PDGFβR, PDGF β receptor; IL-3, interleukin-3; TBS, Tris-buffered saline; WGL, wheat germ lectin 1The abbreviations used are: BPV, bovine papillomavirus type 1; PDGF, platelet-derived growth factor; PDGFβR, PDGF β receptor; IL-3, interleukin-3; TBS, Tris-buffered saline; WGL, wheat germ lectin induces fibropapillomas in cattle and can tumorigenically transform cultured rodent fibroblast lines (1Dvoretzky I. Shober R. Chattopadhyay S.K. Lowy D.R. Virology. 1980; 103: 369-375Google Scholar, 2Lancaster W.D. Olson C. Microbiol. Rev. 1982; 46: 191-207Google Scholar). The E5 open reading frame of BPV-1 is primarily responsible for the transforming activity of BPV-1 (3Bergman P. Ustav M. Sedman J. Moreno-Lopez J. Vennstrom B. Pettersson U. Oncogene. 1988; 2: 453-459Google Scholar, 4Burkhardt A. DiMaio D. Schlegel R. EMBO J. 1987; 6: 2381-2385Google Scholar, 5DiMaio D. Guralski D. Schiller J.T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1797-1801Google Scholar, 6Schiller J.T. Vass W.C. Vousden K.H. Lowy D.R. J. Virol. 1986; 57: 1-6Google Scholar). E5 encodes a small, 44-amino acid transmembrane protein that exists as a dimer and localizes mostly to cytoplasmic membranes (7Burkhardt A. Willingham M. Gay C. Jeang K.T. Schlegel R. Virology. 1989; 170: 334-339Google Scholar, 8Schlegel R. Wade-Glass M. Rabson M.S. Yang Y.C. Science. 1986; 233: 464-467Google Scholar). The manner by which E5 functions to achieve transformation has been studied rather extensively. Previous studies have shown that the platelet-derived growth factor (PDGF) β receptor (PDGFβR), a transmembrane receptor tyrosine kinase, is the primary cellular target of the E5 protein. Specifically, the E5 protein forms a complex with and constitutively activates the PDGFβR (9Petti L. Nilson L.A. DiMaio D. EMBO J. 1991; 10: 845-855Google Scholar, 10Petti L. DiMaio D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6736-6740Google Scholar), and activation of this receptor by E5 is required for E5-mediated transformation (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar, 12Nilson L.A. DiMaio D. Mol. Cell. Biol. 1993; 13: 4137-4145Google Scholar). Evidence suggests that the E5 protein binds as a dimer to two PDGFβR molecules and thereby promotes receptor dimerization, resulting in receptor autophosphorylation and stimulation of its intrinsic kinase activity (13Lai C.C. Henningson C. DiMaio D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15241-15246Google Scholar). Once the receptor is tyrosine phosphorylated, key cytoplasmic substrates can bind to the receptor and transmit signaling cascades eventuating in cell proliferation (14Heldin C.H. Cell. 1995; 80: 213-223Google Scholar). Activation of the PDGFβR by the E5 protein occurs independent of the native ligand, PDGFBB (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar). There have been several reports that the E5 protein can complex with other cellular transmembrane proteins such as other growth factor receptors (15Martin P. Vass W.C. Schiller J.T. Lowy D.R. Velu T.J. Cell. 1989; 59: 21-32Google Scholar, 16Petti L. DiMaio D. J. Virol. 1994; 68: 3582-3592Google Scholar), α-adaptin (17Cohen B.D. Lowy D.R. Schiller J.T. Mol. Cell. Biol. 1993; 13: 6462-6468Google Scholar), and the 16-kDa subunit of the vacuolar H+-ATPase (18Goldstein D.J. Schlegel R. EMBO J. 1990; 9: 137-145Google Scholar, 19Goldstein D.J. Finbow M.E. Andresson T. McLean P. Smith K. Bubb V. Schlegel R. Nature. 1991; 352: 347-349Google Scholar, 20Goldstein D.J. Andresson T. Sparkowski J.J. Schlegel R. EMBO J. 1992; 11: 4851-4859Google Scholar). However, in many of these studies, an interaction with E5 was demonstrated under conditions of overexpression, which may artificially enhance nonspecific interactions. Indeed, it has been shown that although the E5 protein can interact with several different growth factor receptors under conditions of transient overexpression in COS cells (16Petti L. DiMaio D. J. Virol. 1994; 68: 3582-3592Google Scholar), it can interact only with the PDGFβR when stably expressed at normal levels (16Petti L. DiMaio D. J. Virol. 1994; 68: 3582-3592Google Scholar, 21Goldstein D.J. Li W. Wang L.M. Heidaran M.A. Aaronson S. Shinn R. Schlegel R. Pierce J.H. J. Virol. 1994; 68: 4432-4441Google Scholar). Furthermore, since the ability of E5 to interact with these other proteins does not correlate with its transforming activity, the biological significance of such interactions has not been established. Therefore, the PDGFβR appears to be the most specific target of the E5 protein, and complex formation with this receptor is clearly related to a biochemical (receptor activation) and biological (cellular transformation) response (9Petti L. Nilson L.A. DiMaio D. EMBO J. 1991; 10: 845-855Google Scholar, 10Petti L. DiMaio D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6736-6740Google Scholar, 11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar, 12Nilson L.A. DiMaio D. Mol. Cell. Biol. 1993; 13: 4137-4145Google Scholar). In attempts to characterize the E5-PDGFβR complex, mutagenic analysis of both proteins has been performed. Initial studies indicated that the E5 protein and the PDGFβR contact each other via transmembrane domain interactions, suggesting a novel mechanism of activation for this receptor (22Cohen B.D. Goldstein D.J. Rutledge L. Vass W.C. Lowy D.R. Schlegel R. Schiller J.T. J. Virol. 1993; 67: 5303-5311Google Scholar, 23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar, 24Staebler A. Pierce J.H. Brazinski S. Heidaran M.A. Li W. Schlegel R. Goldstein D.J. J. Virol. 1995; 69: 6507-6517Google Scholar). Subsequent studies identified two potential sites of contact between the transmembrane regions of these two proteins. Specifically, it was shown that Lys499at the outer juxtamembrane position and Thr513 at a central transmembrane position within the receptor were required for stable complex formation with the E5 protein (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar). Interestingly, the analogously positioned Asp33 and Gln17, respectively, in the E5 protein, were found to be necessary for the transforming activity of E5 (25Horwitz B.H. Burkhardt A.L. Schlegel R. DiMaio D. Mol. Cell. Biol. 1988; 8: 4071-4078Google Scholar, 26Kulke R. Horwitz B.H. Zibello T. DiMaio D. J. Virol. 1992; 66: 505-511Google Scholar) as well as its ability to form a complex with and activate the PDGFβR (27Nilson L.A. Gottlieb R.L. Polack G.W. DiMaio D. J. Virol. 1995; 69: 5869-5874Google Scholar). Replacing Lys499 in the receptor with Asp, Glu, or Ala hindered an interaction with E5, whereas an Arg substitution was tolerated, suggesting a requirement for a positive charge at this position (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar). Similar studies revealed a requirement for a negative charge at the corresponding juxtamembrane position (Asp33) of the E5 protein (27Nilson L.A. Gottlieb R.L. Polack G.W. DiMaio D. J. Virol. 1995; 69: 5869-5874Google Scholar, 28Klein O. Kegler-Ebo D. Su J. Smith S. DiMaio D. J. Virol. 1999; 73: 3264-3272Google Scholar, 29Meyer A.N. Xu Y.F. Webster M.K. Smith A.E. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4634-4638Google Scholar). Furthermore, it was shown that only amino acids with side groups capable of hydrogen bond formation could functionally substitute for Gln17 in E5 and permit an interaction with the PDGFβR (20Goldstein D.J. Andresson T. Sparkowski J.J. Schlegel R. EMBO J. 1992; 11: 4851-4859Google Scholar). Thus, it was proposed from these studies that complex formation between the PDGFβR and the E5 protein involves an electrostatic interaction between Asp33 of E5 and Lys499 of PDGFβR and hydrogen bond formation between Gln17 of E5 and Thr513 of the receptor (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar,28Klein O. Kegler-Ebo D. Su J. Smith S. DiMaio D. J. Virol. 1999; 73: 3264-3272Google Scholar, 29Meyer A.N. Xu Y.F. Webster M.K. Smith A.E. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4634-4638Google Scholar, 30Klein O. Polack G.W. Surti T. Kegler-Ebo D. Smith S.O. DiMaio D. J. Virol. 1998; 72: 8921-8932Google Scholar). It was also found that dimerization of E5, which is mediated by two extracellular cysteines, is necessary for transformation and stable complex formation with the PDGFβR (25Horwitz B.H. Burkhardt A.L. Schlegel R. DiMaio D. Mol. Cell. Biol. 1988; 8: 4071-4078Google Scholar, 27Nilson L.A. Gottlieb R.L. Polack G.W. DiMaio D. J. Virol. 1995; 69: 5869-5874Google Scholar). This implies that dimerization of E5 promotes a conformation suitable for making contacts with the PDGFβR. We recently obtained data suggesting that several other amino acids within the PDGFβR transmembrane domain besides Lys499 and Thr513 may be required for a stable interaction with the E5 protein (31Nappi V.M. Petti L.M. J. Virol. 2002; 76: 7976-7986Google Scholar). Here, additional mutagenesis analysis of the PDGFβR was performed to further identify and characterize the PDGFβR requirements for this interaction. First, we showed that a minimal segment of the PDGFβR consisting primarily of the transmembrane domain is capable of forming a complex with the E5 protein. We also provide evidence that Thr513 is important for the interaction by virtue of its polar nature and its position along the transmembrane α-helix. Finally, we identify Ile506 and Leu520 as additional receptor transmembrane amino acids that play a role in the interaction. This stands to reason because Ile506 and Leu520 are predicted to align with Lys499 and Thr513 along the same face of the PDGFβR transmembrane α-helix when in a left-handed coiled coil complex with another transmembrane α-helix (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar). Taken together, these data suggest that the PDGFβR contacts the E5 protein via multiple amino acids aligned along a single α-helical interface. A doubly truncated receptor construct containing primarily the transmembrane domain of the PDGFβR was created by ligating the truncated portion of a receptor construct lacking the extracellular domain with that of one lacking the intracellular domain at a common transmembrane site. The first step was to attach the COOH-terminal 13 amino acids of the human PDGFβR (the epitope recognized by the PDGF receptor-specific antibody used in these studies) to the COOH terminus of the truncated receptor construct lacking most of the intracellular domain, pECTM (gift from C. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden; Ref.32Severinsson L. Claesson-Welsh L. Heldin C.H. Eur. J. Biochem. 1989; 182: 679-686Google Scholar). A double-stranded oligonucleotide linker with the sense sequence, 5′-GCCCTGCGCCTCGAGCGGAAGCAGAGGATAGCTTCCTGTAAGCT-3′, encoding this epitope and containing SacI compatible ends (gift from D. DiMaio, Yale University) was inserted in-frame into the SacI site located at the stop codon of the receptor open reading frame in pECTM. The resulting construct pECTM-epi was subcloned into the pLXSN retrovirus vector by standard methods, generating pECTM-epi-LXSN. Another truncated receptor construct, TPR (gift from C. Heldin), lacking most of the extracellular domain, was also subcloned into the pLXSN retrovirus vector (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar), generating pTPR-LXSN. We exploited the XcmI site located in the transmembrane domain of the human PDGFβR and the SacII site located in pLXSN, and ligated the XcmI-SacII fragment of pECTM-epi-LXSN to the SacII-XcmI fragment of pTPR-LXSN to generate pTMPR. Sequence analysis confirmed that this TMPR lacks extracellular amino acids 38–530 and intracellular amino acids 574–1060, corresponding to the published sequence of the human receptor (33Claesson-Welsh L. Eriksson A. Moren A. Severinsson L. Ek B. Ostman A. Betsholtz C. Heldin C.H. Mol. Cell. Biol. 1988; 8: 3476-3486Google Scholar). Site-directed mutagenesis was performed using the QuikChange procedure (Stratagene) as described by the manufacturer to introduce single or double amino acid substitutions into the transmembrane domain of the PDGFβR. For each mutation, complimentary oligonucleotides were designed to contain the appropriate base pair mismatches with respect to the wild type receptor sequence (33Claesson-Welsh L. Eriksson A. Moren A. Severinsson L. Ek B. Ostman A. Betsholtz C. Heldin C.H. Mol. Cell. Biol. 1988; 8: 3476-3486Google Scholar, 34Yarden Y. Escobedo J.A. Kuang W.J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Williams L.T. Nature. 1986; 323: 226-232Google Scholar) that would achieve the desired mutation(s). In the case of I506A, a SpeI site was established by introduction of a silent mutation concomitantly with the I506A substitution, which allowed for screening of potential mutants by SpeI digestion. Templates included the murine or human (either wild type or mutant) PDGFβR cDNA cloned into the LXSN retroviral vector, which also contains the G418 resistance gene as a selectable marker. The plasmid DNA products from site-directed mutagenesis were sequenced to confirm the presence of the desired mutations. The Phoenix ecotropic retrovirus producer cell line was obtained from the ATCC with permission from Dr. Gary Nolan (Stanford University) and maintained in Dulbecco's minimal essential medium with high glucose supplemented with 10% fetal bovine serum. Ba/F3 murine hematopoetic cells were maintained as previously described (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar) in RPMI 1640 medium supplemented with 10% fetal bovine serum, antibiotics, 50 μm β-mercaptoethanol, and 10% WEHI conditioned medium as a source of IL-3 (RPMI/IL-3). The various PDGFβR constructs, E5, and v-sis were introduced into Ba/F3 cells by retroviral mediated gene transfer. The recombinant retroviral vectors used were the receptor-LXSN constructs described above and E5 or v-sissubcloned into the pBabepuro retroviral vector, which contains the puromycin resistance marker. High titer ecotropic retrovirus was obtained from these retroviral vectors as described previously (35Petti L.M. Ray F.A. Cell Growth Differ. 2000; 11: 395-408Google Scholar). Briefly, Phoenix ecotropic cells grown to 70–80% confluence in 60-mm dishes were transfected with 10 μg of plasmid DNA using the calcium phosphate method. The next day the media was replaced, and 24 h later the virus-containing supernatant from each dish was collected and filtered through a 0.45-μm syringe filter. Retroviral infection of Ba/F3 cells was performed as described previously with some modifications (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar). First, Ba/F3 cells stably expressing E5 or v-sis were established by infecting ∼5 × 106 cells with ∼1–2 × 105 colony forming units of pBabepuro-E5 or pBabepuro-v-sis ecotropic retrovirus in 10 ml of RPMI/IL-3 supplemented with 4 μg/ml Polybrene. Cells expressing no viral oncogene were generated in parallel by infection with retrovirus derived from the pBabepuro vector alone. 48 h post-infection, 2 ml of the infected cells was added to 8 ml of selective media (RPMI/IL-3 containing 1 μg/ml puromycin (Sigma)). After reaching a density of ∼106 cells/ml, cells were passaged again under selection conditions. Selection was repeated through 2–4 additional passages until 100% of a mock-infected culture died, thus establishing stable cell lines. The resulting cells expressing E5, v-sis, or no viral oncogene (Puro) were then infected with recombinant ecotropic retroviruses containing the various LXSN-receptor constructs as described above. Stable cell lines were generated using selective media containing 1 mg/ml G418 (Gemini) as well as puromycin. Ba/F3 cells expressing a receptor construct without (Puro) or with E5 or v-sis were grown to an approximate density of 1 × 106 cells/ml, washed twice with phosphate-buffered saline, and resuspended in an equal volume of RPMI lacking IL-3 (RPMI/−IL-3; RPMI 1640 medium supplemented with only 1% fetal bovine serum, antibiotics, 50 μm β-mercaptoethanol, and without WEHI conditioned medium). Approximately 5 × 105 of these cells were inoculated into 10 ml of RPMI/−IL-3, incubated at 37 °C, and monitored for growth. Total or viable cells were counted using a hemacytometer at various times after seeding. For experiments testing the murine PDGFβR, Ba/F3 cells that proliferated at least 20-fold during a 10-day period were considered IL-3-independent. For experiments involving the human PDGFβR, cells that proliferated at least 10-fold during a 10-day period were considered IL-3-independent. Multiple independently derived cell lines of each genotype were tested to the confirm results. Ba/F3 cells were lysed by incubation in cold EBC buffer (50 mm Tris-HCl, pH 8.0, 120 mm NaCl, 0.5% Nonidet P-40) supplemented with 1 mm phenylmethylsulfonyl fluoride, 2 mm sodium orthovanadate, 20 mg/ml leupeptin, and 20 mg/ml aprotinin on ice for 15 min. Lysates were cleared of nuclei and cell debris by centrifugation in a microcentrifuge at 15,000 rpm for 10 min at 4 °C and the supernatant extracts were used for immunoprecipitation. To immunoprecipitate the E5 protein and any associated protein, 750–1100 μg of extracted protein was incubated overnight at 4 °C with ∼10 μl of a rabbit polyclonal antibody directed against the 16 COOH-terminal amino acids of the E5 protein (gift from D. DiMaio). To immunoprecipitate the wild type and mutant forms of the PDGFβR, 500–1200 μg of extracted protein was incubated overnight at 4 °C with 5–12 μl of a rabbit polyclonal antibody directed against the COOH-terminal 13 amino acids of the human PDGFβR (gift from D. DiMaio). Following incubation with primary antibody, extracts were incubated with 60 μl of a 1:1 slurry of protein-A-Sepharose CL-4B beads (Amersham Biosciences) in Tris-buffered saline (TBS) containing 10% bovine serum albumin for 60 min at 4 °C. Beads were then washed 3–5 times with cold EBC buffer. For the experiments presented in Figs. 3 A and 5 A, the PDGFβR was precipitated from cell extracts through affinity purification of glycosylated proteins with wheat germ lectin (WGL)-Sepharose beads (Amersham Biosciences). Approximately 100 μl of a 1:1 slurry of WGL beads was incubated with 800–1000 μg of EBC extracts overnight at 4 °C and then washed as described above. Protein complexes were dissociated from beads by boiling in 2× Laemmli protein sample buffer.Figure 5Biochemical and functional analysis of the T513L/I514T receptor mutant. Ba/F3 cells expressing the wild type human PDGFβR (WT), the T513L/I514T mutant receptor, or no exogenous receptor (LXSN) with (+) or without (−) E5 or v-sis were established as described under “Experimental Procedures.” The PDGFβR (A) or the E5 protein (B) was precipitated from cell extracts as in Fig. 3.A, WGL were subjected to anti-phosphotyrosine (PY) immunoblot analysis for assessment of PDGFβR activation (upper panel) or anti-PDGFβR (PR) immunoblotting to determine the receptor expression levels (lower panel). B, E5 immunoprecipitates (E5IP) were subjected to PDGFβR immunoblotting to assess the presence of a physical complex between the receptor and E5 (upper panel), or anti-E5 (E5) immunoblotting to detect E5 protein expression levels (lower panel). Each lane represents 670 (upper panel of A), 170 (lower panel ofA), or 750 μg (B) of extracted protein. Thearrows on the right denote the mature (m) and precursor (p) forms of the PDGFβR (PR) and the E5 protein. C, IL-3-dependent phenotype of Ba/F3 cells co-expressing the T513L/I514T mutant and E5. Ba/F3 cells expressing the indicated receptor construct with E5, v-sis, or the empty retroviral vector (Puro) were seeded into medium lacking IL-3 at a density of 5 × 105 cells per 10 ml and counted 11 days later. Cells expressing no receptor (LXSN) with v-siswere counted after 9 days. The graph shown is representative of several different sets of independently derived cell lines.View Large Image Figure ViewerDownload (PPT) SDS-PAGE and immunoblot analysis was performed as described previously (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar). Briefly, immunoprecipitates were either electrophoresed on an SDS-7.5% polyacrylamide gel and transferred to nitrocellulose (for PDGF receptor or phosphotyrosine imunoblotting) or electrophoresed on an SDS-15% polyacrylamide gel and transferred to Immobilon (Millipore) (for E5 immunoblotting). Blots were blocked for 2 h in milk buffer (5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 7.4, 167 mm NaCl, 1% Tween 20)), then incubated overnight with either a 1:2000 or 1:1000 dilution of monoclonal anti-phosphotyrosine antibody P-Tyr-100 (Cell Signaling) or 4G10 (Upstate Biotechnology), respectively, or a 1:500–1:1000 dilution of the anti-PDGFβR or anti-E5 antiserum described above. Following incubation with primary antibody, immunoblots were washed 5 times, 10 min each, in either TBST buffer for phosphotyrosine and E5 immunoblots or TNET buffer (10 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1% Tween 20) for PDGF receptor immunoblots. Each blot was then incubated for 1 h with a 1:5000 dilution of a protein A (Pierce; for PDGFβR or E5 blots) or goat anti-mouse IgG (Pierce; for anti-phosphotyrosine blots) horseradish peroxidase conjugate, washed as above, and subjected to enhanced chemiluminescence (ECL) detection (Amersham Biosciences) as described by the manufacturer. For the experiment presented in Fig.7 A, after ECL detection of the phosphotyrosine immunoblot, primary and secondary antibodies were removed according to the stripping protocol provided by the manufacturer. In brief, membranes were incubated in stripping buffer (100 mmβ-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7) for 30 min at 60 °C. The stripped blot was then washed and subjected to ECL detection to ensure that the stripping process was effective. The membrane was then washed in TBST several times, blocked in 5% milk-TBST, and subjected to anti-PDGFβR immunoblotting as described above. Previous work identified two specific amino acids of the PDGFβR, Lys499 at the extracellular juxtamembrane position and Thr513 within the transmembrane domain, that are required for an interaction with the BPV E5 protein (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar). To further elucidate the nature of the E5/PDGFβR interaction and to determine whether other amino acids in the receptor are required for the interaction, additional mutagenesis analysis of the receptor was performed. Receptor mutants were examined for an interaction with the E5 protein in the mouse hematopoietic Ba/F3 cell line because these cells do not express endogenous PDGF receptors, which might otherwise obscure the analysis of mutant receptors. Furthermore, these cells provide a convenient assay for the functional cooperation of receptor mutants with E5 because they normally depend on IL-3 for survival and proliferation (36Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Google Scholar). Co-expression of a growth factor receptor and its cognate ligand in these cells can alleviate this requirement for IL-3 (37D'Andrea A.D. Yoshimura A. Youssoufian H. Zon L.I. Koo J.W. Lodish H.F. Mol. Cell. Biol. 1991; 11: 1980-1987Google Scholar) apparently because activation of the receptor by the ligand results in a compensatory proliferative signal. Hence, expression of the PDGFβR with v-sis, which encodes the viral homologue of PDGF BB (38Waterfield M.D. Scrace G.T. Whittle N. Stroobant P. Johnsson A. Wasteson A. Westermark B. Heldin C.H. Huang J.S. Deuel T.F. Nature. 1983; 304: 35-39Google Scholar), or the BPV E5 protein in these cells allows for IL-3-independent growth (11Drummond-Barbosa D.A. Vaillancourt R.R. Kazlauskas A. DiMaio D. Mol. Cell. Biol. 1995; 15: 2570-2581Google Scholar). Therefore, after co-expressing E5 with various PDGFβR mutants in Ba/F3 cells, we were able to assess the ability of receptor mutants to form a complex with the E5 protein, to undergo activation by E5, and to cooperate with E5 to induce a proliferative response. The functional integrity of each receptor mutant was ascertained by determining the ligand-induced responsiveness to v-sis. Previous work established that the transmembrane domain of the PDGFβR is required for complex formation with the E5 protein and implicated that the transmembrane domains of the two proteins may contact each other directly (23Petti L.M. Reddy V. Smith S.O. DiMaio D. J. Virol. 1997; 71: 7318-7327Google Scholar, 28Klein O. Kegler-Ebo D. Su J. Smith S. DiMaio D. J. Virol. 1999; 73: 3264-3272Google Scholar,30Klein O. Polack G.W. Surti T. Kegler-Ebo D. Smith S.O. DiMaio D. J. Virol. 1998; 72: 8921-8932Google Scholar). Here, we asked if the transmembrane domain of the receptor by itself is sufficient to interact with the E5 protein. To address this question we constructed a truncated receptor consisting primarily of the PDGFβR transmembrane domain with only 6 and 30 amino acids derived from the extracellular and intracellular domains, respectively, and tested its ability to form a stable complex with the E5 protein. In this truncated receptor construct a segment of the human PDGFβR containing the juxtamembrane lysine, transmembrane domain, and 17 adjacent cytoplasmic amino acids is linked to the COOH-terminal 13 amino acids, the epitope recognized by the PDGFβR antiserum used in these studies (Fig. 1 A). A recombinant retrovirus carrying the construct for the truncated receptor was introduced by retroviral infection into Ba/F3 cells expressing either the BPV E5 gene or no exogenous gene. Cells stably expressing the truncated receptor were established after selection for a G418 resistance marker present in the retroviral vector. Cells were first analyzed for expression of the truncated receptor by immunoblot analysis with the PDGFβR antiserum. As expected, the truncated receptor was expressed as a small protein with an apparent molecular mass of ∼14.5 kilodaltons (Fig. 2,top, left two lanes). Similar amounts of the truncated receptor were expressed regardless of whether or not E5 was co-expressed. To assess the ability of the truncated receptor to form a stable complex with the E5 protein, cell extracts were immunoprecipitated with an E5 antiserum, and E5 immunoprecipitates were subjected to immunoblotting for the PDGFβR. Fig. 2 (top, fourth lane) shows that a significant amount of the truncated receptor could be co-immunoprecipitated with the E5 protein. This co-immunoprecipit" @default.
• W2079136503 created "2016-06-24" @default.
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• W2079136503 creator A5018752104 @default.
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• W2079136503 date "2002-12-01" @default.
• W2079136503 modified "2023-09-30" @default.
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