v-fps causes transformation by inducing tyrosine phosphorylation and activation of the PDGFb receptor

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1 v-fps cuses trnsformtion y inducing tyrosine phosphoryltion nd ctivtion of the PDGF receptor Deorh H Anderson nd Preeti M Ismil Oncogene (1998) 16, 2321 ± Stockton Press All rights reserved 95 ± 9232/98 $12. Ssktoon Cncer Centre Reserch Unit, 2 Cmpus Drive, Ssktoon, Ssktchewn, S7N 4H4 Cnd The v-fps oncogene encodes n ctivted tyrosine kinse which is cple of trnsforming rolsts. In this report, we provide evidence tht within few minutes of ctivtion of the tyrosine kinse ctivity of v-fps, tyrosine phosphoryltion of the pltelet derived growth fctor (PDGF) receptor is oserved. Further, sustined expression of ctivted v-fps results in downregultion of the PDGF receptor oth t the level of the mrna (*4 ± 8-fold), ut even more mrkedly t the level of the receptor protein (41-fold). The kinse ctivity of the v-fps oncoprotein ws found to e required for oth the induction of PDGF receptor tyrosine phosphoryltion nd ultimtely the reduced receptor protein levels. Tyrosine phosphoryltion of kinse inctive PDGF receptor ws lso demonstrted in cells which lso express v-fps, ut this ws not su cient to induce trnsformtion. Only cells expressing oth v-fps nd wild type PDGF receptor were le to form colonies in soft gr. These ndings suggest tht wild type v-fps my use tyrosine phosphoryltion of the PDGF receptor to constitutively ctivte the kinse ctivity of the receptor, resulting in sustined prolifertive signl nd rolst trnsformtion. Keywords: Fps/Fes; PDGF receptor; down-regultion; tyrosine phosphoryltion Introduction Fps/Fes is 92 kd cytoplsmic tyrosine kinse with unique N-terminl domin (N-Fps), n SH2 domin nd C-terminl kinse domin. More recently, leucine zipper-like domin hs lso een identi ed etween the N-Fps nd SH2 domin of the protein (Prk nd Seo, 1995). The oncogenic form of the fps proto-oncogene, v-fps, encodes 13 kd protein with virlly derived gg sequence fused N-terminlly to the Fps sequences. The tyrosine kinse ctivity of the resulting gg-fps (v-fps) fusion protein is constitutively ctive nd is cple of cusing cellulr trnsformtion (Foster et l., 1985; Sodroski et l., 1984). Both the utophosphoryltion nd trnsformton ctivities of the protein re reduced y muttion of Leu-746 in the leucine zipper-like domin (Prk nd Seo, 1995). The SH2 domin is lso positive regultor for oth the kinse ctivity nd trnsforming ility of Fps/Fes, nd hs een suggested to provide Correspondence: DH Anderson Received 28 July 1997; revised 9 Decemer 1997; ccepted 9 Decemer 1997 inding sites for sustrtes nd/or regultors of the djcent kinse domin (DeClue et l., 1987; Hjermstd et l., 1993; Koch et l., 1989; Sdowski et l., 1986). In fct, the SH2 domins of mny signling proteins hve een shown to medite protein-protein interctions y inding to phosphotyrosine-contining sequences (reviewed in Cohen et l., 1995; Pwson, 1995; Schlessinger, 1994). Activtion of the Fps/Fes tyrosine kinse induces phosphoryltion of vrious cytoplsmic signling proteins including SHC (McGlde et l., 1992), the GTPse ctivting protein of p21 rs (rsgap) (Ellis et l., 199), nd phosphtidylinositol 3-kinse (PI3 kinse) (Fukui et l., 1991). Fps/Fes hs een shown to ind to nd phosphorylte BCR on tyrosine, inducing the ssocition of GRB2/SOS with BCR, nd providing mechnism y which to ctivte the Rs pthwy (Mru et l., 1995). In this report, we provide evidence tht ctivtion of the tyrosine kinse ctivity of v-fps, leds to rpid tyrosine phosphoryltion of the PDGF receptor. Our experiments lso demonstrte tht the ctlytic ctivity of the receptor is solutely required for v-fps trnsformtion. These results suggest tht the PDGF receptor plys n ctive role in the trnsformtion of v- fps expressing cells, likely y recruiting vrious SH2 contining signling proteins including GRB2 nd the ssocited SOS, resulting in the ctivtion of the Rs pthwy. Results Expression of v-fps down regultes the PDGF receptor During our studies of v-fps trnsformed Rt-2 rolst cells, we noted tht for two independently generted v-fps expressing Rt-2 cell lines, NW16 nd Cl-1, the levels of PDGF receptor protein were t lest 1-fold lower when compred to receptor levels in the prentl Rt-2 cells, s judged using Western lot nlysis (Figure 1). Both NW16 nd Cl-1 cells expressed similr levels of the v-fps protein (Figure 1). Further studies were crried out with the NW16 cells (herefter referred to s Rt-2 v-fps). Down-regultion of the PDGF receptor hs een reported in murine 3T3 rolsts which express trnsforming src or rs oncogenes, or which hve een continully stimulted with mitogenic growth fctors (Vziri nd Fller, 1995). This reduced receptor protein expression ws found to result solely from similr reduction in the PDGF receptor mrna levels. In the cse of the src nd rs trnsformed cells, this reduction ws estimted t 5 ± 1-fold less PDGF receptor mrna nd protein thn ws found in the norml prentl cell line (Vziri nd Fller, 1995).

2 2322 whole cell lystes NW16 CI 1 Figure 1 Expression of v-fps results in severely reduced levels of the PDGF receptor protein. Rt-2 rolsts (Rt-2) or two independently derived Rt-2 cell lines expressing v-fps (NW16, Cl-1) were serum strved in.5% fetl ovine serum for 2 dys to deplete the medi of growth fctors. Whole cell lystes (1 mg totl protein) were resolved y SDS ± PAGE (7.5%) nd lotted onto nitrocellulose. The lot ws proed with nti-pdgf receptor ntiodies (PDGFR) (), detected using secondry ntiody conjugted to horserdish peroxidse nd visulized y chemiluminescence. The lot ws then stripped nd reproed with nti-gg ntiodies () (to detect v-fps i.e. P13 gg-fps ) nd detected s in In order to ssess whether v-fps expression ws cusing down-regultion in PDGF receptor expression t the level of trnscription, we mesured the stedy stte levels of PDGF receptor mrna. A Northern lot nlysis proved inconclusive, likely due to nucleotide sequence di erences etween the humn PDGF receptor cdna proe nd the rt mrna on the lot. As result, we hve used n RNse protection nlysis to ddress this question. RNse protection requires homologous RNA proe which hs sequence exctly complementry to tht of the trget mrna. Since we were working with rt cells, nd only mouse nd humn PDGF receptor cdnas hd een cloned nd sequenced previously, this pproch required tht we otin t lest prtil cdna for the rt PDGF receptor. We used reverse trnscription polymerse chin rection (RT ± PCR) to otin prtil cdna for the rt PDGF receptor (Figure 2). This prtil cdna (lunt, with phosphorylted ends) ws cloned into the SmI site of pgex2t nd sequenced (Figure 2). A portion of this cdna (.35 k HincII ± EcoRI) ws sucloned into pbluescript KS + nd ws used s templte for T7 RNA polymerse to synthesize rdioleled RNA proe corresponding to the noncoding strnd of the rt PDGF cdna (Figure 2c). The proe ws hyridized to totl RNA prepred from Rt-2 nd Rt-2 v-fps cells, digested with single-strnd speci c RNse nd resolved on n crylmide-ure gel (Figure 2d). The results show 4 ± 8-fold reduction in the mounts of PDGF receptor mrna in Rt-2 v-fps cells s compred to Rt-2 cells, suggesting tht cells trnsformed y v-fps do down-regulte their PDGF receptor mrna levels to some extent. If we ssume tht the trnsltion of the PDGF receptor mrna into protein is the sme in oth Rt-2 nd Rt- 2 v-fps cells, we would expect to see 4 ± 8-fold less receptor protein in v-fps expressing cells. The fct tht we oserve PDGF receptor protein levels t lest two orders of mgnitude (1-fold) less in the v-fps expressing Rt-2 cells, rises the possiility tht v-fps expression my e cusing the turnover of receptor protein. One mechnism y which v-fps expression could ccomplish this would e to induce these cells, which lredy express PDGF receptor, to lso express PDGF BB. This would estlish n utocrine loop nd drive cell prolifertion. The reduction in the level of PDGF receptor protein in v-fps trnsformed cells ws not due to the utocrine production of PDGF BB y these cells PDGF receptors which hve een stimulted with PDGF BB re internlized nd degrded in the lysosome (Nilsson et l., 1983; Rosenfeld et l., 1984; Sorkin et l., 1991). Therefore, it is possile tht the depressed numer of PDGF receptors in v-fps trnsformed Rt-2 cells, is result of the utocrine production of PDGF, nd constitutive stimultion nd down-regultion of the receptors. This hs een reported previously for cell trnsformed y vrious oncogenic viruses (Bowen-Pope et l., 1984). To test this hypothesis, we treted cells with surmin, compound known to lock the inding of PDGF to its receptor oth extr- nd intr-cellulrly (Grrett et l., 1984; Hwking, 1978; Hung nd Hung, 1988; Willims et l., 1984), nd determined the mount of PDGF receptor present in whole cell lystes using Western lot nlysis (Figure 3). Norml Rt-2 cells which re stimulted with PDGF for incresing times in the sence of surmin, show rpid reduction in PDGF receptor protein levels due to the degrdtion of receptor protein s prt of the down-regultion mechnism for the prolifertive signl (Figure 3; Heldin et l., 1982; Nilsson et l., 1983; Rosenfeld et l., 1984; Sorkin et l., 1991). When the sme experiment ws repeted in the presence of 1 mm surmin, no reduction in the level of PDGF receptors ws oserved, since the inding of PDGF to the receptors hd een prevented (Figure 3). If the down-regultion of the PDGF receptors in Rt-2 v-fps cells ws due to the constitutive production of PDGF BB nd utocrine stimultion of the endogenous receptors, we could mimic this e ect in Rt-2 cells y continully stimulting the cells with PDGF. If we then treted those cells with surmin, we would expect the inding of PDGF to the receptors to e locked, nd over time the receptor levels would return to norml. We stimulted Rt-2 cells with PDGF for 24 h, then dded surmin for vrious times nd determined the mount of PDGF receptor in cell lystes using Western lot nlysis (Figure 3c). Receptor levels incresed sustntilly within 2 h fter the ddition of surmin nd within 24 h were comprle to those prior to tretment with PDGF, suggesting tht surmin ws le to reverse the e ects of PDGF on PDGF receptor levels. Similr results were otined when PDGF ws simply removed from the medi (dt not shown). When surmin ws dded to Rt-2 v-fps cells, no increse in the low level of PDGF receptors ws oserved even fter 24 h (Figure 3d). This result strongly suggests tht the diminished numer of PDGF receptors in v-fps expressing cells is not result of the newly cquired ility of these cells to

3 2323 c d RNA stndrds yest RNA + RNse yest RNA RNse 1 µg 2 µg 4 µg 1 µg v-fps 2 µg 4 µg Figure 2 PDGF receptor mrna levels in v-fps expressing cells re somewht reduced s compred to cells lcking v-fps. ()A cdna encoding the indicted portion of the rt homologue of the PDGF receptor ws mpli ed y reverse trnscription polymerse chin rection (RT ± PCR), cloned into pgex2t nd sequenced. The region used to generte n RNA proe (see c) is lso indicted. () The sequence of the rt PDGF receptor prtil cdna is shown ligned to the corresponding sequences from the mouse nd humn PDGF receptor homologues. Identicl nucleotides re denoted y dots nd mismtches y lower cse letters. This region corresponds to the sequences encoding mino cids 549 ± 699 (mouse; (Yrden et l., 1986)) nd 55 ± 7 (humn: (Gronwld et l., 1988)) for the PDGF receptor. The PCR primers were derived from the sequences underlined. The loction of the HincII site used for sucloning is leled nd overlined. 1 Amino cid for mouse nd humn receptor is K. 2 Amino cid for humn receptor is M. (c) A frgment which contins the rt PDGF receptor prtil cdna in the reverse orienttion ws sucloned into pbluescript KS + (see Methods for detils). The resulting plsmid, pbsrevrtpdgfrc19, ws linerized with EcoRI nd used s templte for T7 RNA polymerse to generte rdioleled RNA proe (395 nt) (d) An RNse protection ssy ws performed, using the rdioleled RNA proe generted in (c). The proe ws hyridized to the indicted mount of totl RNA from Rt-2 cells (lnes 4 ± 6) or Rt-2 cells expressing v-fps (Rt-2 v-fps; lnes 7 ± 9). Smples were digested with RNse nd resolved on n crylmide-ure gel. Proe protected y the homologous mrna present in the RNA smples ws visulized y utordiogrphy (35 nt). Lne 1 contins RNA trnscript stndrds of the indicted sizes (nt). Control yest RNA ws lso hyridized with the PDGF proe RNA nd incuted in the presence (lne 2) nd sence of RNse (lne 3). nt=nucleotides produce PDGF BB. As control, the lot shown in Figure 3d ws stripped nd reproed with nti-gg ntiodies (Figure 3e). To con rm these results, we lso looked for the production of PDGF y the v-fps trnsformed cells oth in the cell culture medium, nd contined within the cells, using Western lot nlysis. Our lots re cple of detecting s little s 5 ng of PDGF BB, which corresponds to concentrtion of.5 ng/ml in the 1 ml of medi nlysed. In order to down-regulte the PDGF receptor, s shown in Figure 3, we require concentrtion of PDGF BB greter thn 2 ng/ml. Thus, our ssy would e sensitive enough to test for the presence of PDGF BB, t the concentrtions necessry to down-regulte the receptor. Anti-PDGF immunoprecipittes of the cell culture medium (1 ml) conditioned y the v-fps expressing cells, s well s whole cell lystes from v-fps trnsformed cells were resolved y SDS ± PAGE nd immunolotted with nti-pdgf ntiodies (dt not shown). No PDGF ws oserved in v-fps trnsformed cells, or in the medi conditioned y their growth.

4 2324 As well, quiescent Rt-2 rolsts were lso treted with the medi conditioned y v-fps trnsformed cells, to determine if the medi contined ny sustnces cple of ctivting the PDGF receptor. Anti-phosphotyrosine immunolots filed to detect the presence of ny tyrosine phosphorylted PDGF receptors (dt not shown). We hve oserved phosphotyrosine contining PDGF receptors when cells re exposed to s little s.1 ng/ ml PDGF BB. These experiments rgue tht v-fps trnsformed cells do not produce PDGF BB. Reltionship etween PDGF receptor protein expression nd the kinse ctivity of v-fps The fct tht the PDGF receptor protein expression ws very low in v-fps expressing cells suggested tht there my e link etween the kinse ctivity of v-fps nd the e ects on the PDGF receptor. To test this possiility, we used Rt-2 cell line (CNA7) expressing v-fps mutnt which is temperture sensitive for v-fps kinse ctivity c +PDGF surmin + surmin +PDGF, 24 hrs (tsax9m) (DeClue et l., 1987; Sdowski et l., 1986). At the non-permissive temperture (39.58C), the tyrosine kinse ctivity of this mutnt is very low s compred to wild type v-fps (Sdowski et l., 1986). Conversely, t the permissive temperture (348C), the mutnt v-fps tyrosine kinse is ctive, phosphorylting vriety of cellulr trgets. The level of mutnt v-fps protein remins constnt t the two tempertures (Figure 4c nd d). The level of PDGF receptor protein however, ecomes sustntilly reduced when the cells re shifted from 39.58C (inctive v-fps) to 348C (ctive v-fps) (Figure 4). This e ect is reversile, since shifting cells from 348C ck to 39.58C restores the receptor protein levels (Figure 4). This result supports the suggestion tht there is correltion etween reduced PDGF receptor protein levels nd the kinse ctivity of v-fps. Activtion of the v-fps tyrosine kinse ctivity induces rpid tyrosine phosphoryltion of the PDGF receptor If the decrese in PDGF receptor levels ws s result of ctivtion of the receptor, followed y the norml down-regultion process involving trgeting the receptor to the lysosomes nd nlly degrdtion, we would expect to see trnsient tyrosine phosphoryltion of the receptor. Therefore, we repeted the temperture.25 CNA C 34 C 34 C 39.5 C d v-fps c 39.5 C 34 C e v-fps Figure 3 Surmin ws not le to lock the down-regultion of the PDGF receptor protein in v-fps trnsformed cells. Rt-2 cells were treted with 5 ng/ml PDGF ( nd ) in the sence () or presence () of 1 mm surmin for the indicted times (hours). Whole cell lystes (15 mg) were resolved y SDS ± PAGE (7.5%) nd lotted onto nitrocellulose. Blots were proed with the indicted primry ntiody. (c) Rt-2 cells were stimulted with PDGF (5 ng/ml) for 24 h prior to the ddition of surmin (1 mm) for the indicted times (hours). Whole cell lystes (1 mg) were immunolotted with nti-pdgfr ntiodies. (d) Rt-2 v-fps expressing cells were treted with surmin for the indicted times nd detected s in (c). (e) The lot from (d) ws stripped nd reproed with nti-gg ntiodies. All ound ntiodies were detected using horserdish peroxidse conjugted secondry ntiody nd chemiluminescence d 34 C 39.5 C Figure 4 Correltion etween v-fps trnsformtion nd reduced PDGF receptor protein levels. Whole cell lystes (25 mg) of Rt-2 cells expressing v-fps mutnt which is temperture sensitive for trnsformtion (CNA7) hve een generted t tempertures which re either permissive (348C) or non-permissive (39.58C) for trnsformtion. () Cells growing t 39.58C were shifted to 348C for the indicted times (hours). Immunolots were proed with nti-pdgfr ntiodies. () Cells growing t 348C were shifted to 39.58C for the indicted times (hours) nd immunolotted with nti-pdgfr ntiodies. (c) The lot from () ws stripped nd reproed with nti-gg ntiodies. (d) The lot from () ws stripped nd reproed with nti-gg ntiodies. Bound ntiodies were visulized using chemiluminescence

5 shift experiment (39.58C to 348C) nd immunoprecipitted the receptor with nti-pdgf receptor ntiodies. Smples were resolved y SDS ± PAGE nd immunolotted with nti-p.tyr ntiodies (Figure 5). The PDGF receptor ws phosphorylted on tyrosine s erly s 5 min fter the temperture shift nd phosphoryltion peked fter out 2 h. The decresed receptor phosphotyrosine signl oserved t 4 nd 24 h re ects the concomitnt decrese in receptor protein levels lredy oserved (Figure 4), nd suggests tht the remining PDGF receptors re highly tyrosine phosphorylted. This result shows tht ctivtion of v-fps induces tyrosine phosphoryltion of the PDGF receptor, nd ultimtely leds to receptor down-regultion. Whether the receptor is phosphorylted y the ctivted v-fps tyrosine kinse, y utophosphoryltion, or y nother tyrosine kinse is not cler. The rpid kinetics of PDGF receptor phosphoryltion fter shifting to 348C would rgue in fvor of it eing direct sustrte. Cell lines expressing kinse defective mutnts of v-fps do not show mrked reduction in PDGF receptor mrna or protein levels In order to ssess the e ect of v-fps tyrosine kinse ctivity on the expression of the PDGF receptor, we otined two previously chrcterized cell lines which express di erent v-fps kinse mutnts (Figure 6). TO17 contins v-fps protein which hs point muttion chnging Tyr-173, the utophosphoryltion site of v-fps, to Glu. The mutnt protein hs only out 5% of wild type v-fps in vitro kinse ctivity ut is still le to trnsform cells; lthough signi cntly less well thn the wild type protein (Morn et l., 1988). TO29 contins v-fps mutnt with truncted kinse domin, generted y introducing stop codon in plce of the codon for Lys-118. This protein hs no kinse ctivity nd is unle to trnsform cells (Koch et l., 1989). The reltive expression of mutnt v-fps proteins in these cell lines is shown in Figure 6d, nd ws qunti ed using densitometry. When compred to the wild type, the reltive v-fps protein expression for the mutnts ws s follows: TO17 (.52) nd TO29 (.15). IP: nti-pdgfr Blot: nti-p.tyr CNA C 34 C 5 min 1 min 15 min 3 min 1 hr 2 hr 4 hr 24 hr PDGFR v-fps Figure 5 Activtion of the v-fps tyrosine kinse leds to tyrosine phosphoryltion of the PDGF receptor. CNA7 cells were shifted from the non-permissive (39.58C) to the permissive temperture (348C) for the indicted times, to ctivte the v-fps tyrosine kinse. () Anti-PDGF receptor immunoprecipittions of the cell lystes were resolved y SDS ± PAGE, immunolotted with nti-phosphotyrosine (nti-p.tyr) ntiodies nd detected y chemiluminescence. The loctions of the PDGF receptor nd v-fps proteins re indicted, s determined from susequent immunolotting (dt not shown) with nti-pdgfr nd nti-gg ntiodies nlogous to the lots in Figure 4 We mesured the levels of PDGF receptor mrna in these cell lines using n RNse protection ssy (Figure 6). Cells expressing either of the kinse defective v-fps proteins contined t lest s much mrna encoding the PDGF receptor s the Rt-2 cells devoid of v-fps expression. Further, they lso contined signi cnt quntities of receptor protein, s judged y e 2 µg 4 µg 4 µg 8 µg WT TO17 c d TO29 WT WT WT TO17 TO17 1 µg 2 µg TO17 TO29 TO TO29 1 µg 2 µg PDGF PDGFR v-fps RNse Protection Assy lystes lystes IP: PDGER Blot: P.Tyr Figure 6 Cells expressing kinse defective v-fps proteins contin stedy stte levels of PDGF receptor comprle to the prentl Rt-2 cells lcking ny v-fps proteins. () Wild type v-fps (WT) nd v-fps mutnt proteins: TO17 (Tyr-173 chnged to Glu) (Morn et l., 1988) nd TO29 (stop codon for mino cid 118, truncting the kinse domin) (Koch et l., 1989). Reltive kinse ctivities, s reported in Koch et l. (1989) nd Morn et l. (1988), re lso given. () An RNse protection ssy ws performed s descried for Figure 2 using the sme proe speci c for the rt PDGF receptor mrna nd the indicted mount of totl RNA prepred from the di erent cell lines. (c) Whole cell lystes (5 mg protein) from Rt-2 cells nd cells expressing wild type or mutnt v-fps proteins s descried in () were imunolotted with nti-pdgfr ntiodies. (d) Whole cell lystes (2 mg protein) were immunolotted with nti-gg ntiodies, (e) Anti-PDGF receptor immunoprecipittes of lystes from the indicted cell lines were immunolotted nd proed with nti-p.tyr ntiodies. The identities of the receptor nd v-fps proteins were determined y stripping this lot nd reproing with ntiodies speci c for the PDGF receptor nd v-fps in turn (dt not shown). Bound ntiodies were visulized using chemiluminescence 2325

6 2326 Western lot nlysis (Figure 6c). The extent of tyrosine phosphoryltion of these receptors ws nlysed in cells which hd een serum strved s well s strved nd stimulted for 5 min with PDGF. This ws ccomplished y recovering the PDGF receptor from cell lystes using nti-receptor ntiodies nd proing the Western lotted smples with nti-p.tyr ntiodies (Figure 6e). In the sence of PDGF stimultion, the PDGF receptors present in the TO17 nd TO29 cell lines did not contin pprecile levels of phosphotyrosine (Figure 6e). TO17 nd TO29 cells treted with PDGF did contin sustntil levels of phosphorylted receptor, nlogous to control Rt-2 cells. Admittedly the low level of mutnt v-fps expression in the TO29 cells my hve resulted in it hving little e ect on the cells, however the TO17 cell line expresses signi cnt mounts of mutnt v-fps nd still shows no e ect on the PDGF receptor mounts. These oservtions suggest tht v-fps proteins with impired kinse ctivity, re unle to induce downregultion of the PDGF-receptor either t the level of mrna or protein. These results lso suggest tht v-fps tyrosine kinse ctivity is required for induction of PDGF receptor tyrosine phosphoryltion in the sence of exogenously dded PDGF. The wek trnsforming ility of the TO17 mutnt is likely result of its low kinse ctivity which would only give rise to low level of PDGF receptor tyrosine phosphoryltion nd correspondingly low receptor turnover. The synthesis of new receptors ppers to e su ciently rpid so tht no detectle decrese in the stedy stte levels of receptor is oserved. These results re consistent with the tyrosine phosphorylted PDGF receptor plying n importnt role in e ecting v-fps trnsformtion. v-fps cn induce tyrosine phosphoryltion of kinse inctive PDGF receptor, ut expression of oth kinse ctive PDGF receptors nd v-fps re required for cells to form colonies in soft gr To ddress the question of whether or not the PDGF receptor ws required for v-fps trnsformtion, nd if so whether the kinse ctivity of the receptor ws lso required, we used series of dog epithelil cell lines (Kzlusks et l., 199). These cell lines lck endogenous expression of the PDGF receptor (), or hve een engineered to express wild type humn PDGF receptor (WT-R) or mutnt receptor in which Lys-635 hd een chnged to Arg, rendering the receptor kinse inctive (R5). Ech of these cell lines ws cotrnsfected with plsmid encoding v-fps together with plsmid conferring hygromycin resistnce. Hygromycin resistnt colonies were then screened for v-fps expression using Western lot nlysis. Prentl cell lines were lso mde hygromycin resistnt nd were used s controls. A Western lot nlysis of whole cell lystes of the prentl cell lines nd those lso expressing v-fps con rmed expression of the PDGF receptor nd v-fps proteins (Figure 7 nd ). Note tht expression of v-fps does not cuse decrese in PDGF receptor levels since the receptor is constitutively expressed t high levels under the direction of the retrovirl LTR. Of prticulr note, the kinse inctive PDGF receptor is highly tyrosine phosphorylted in cells which lso express v-fps (Figure c 7c). This result strongly indictes tht v-fps cuses tyrosine phosphoryltion of the PDGF receptor, either y direct phosphoryltion, or y ctivting endogenous tyrosine kinses other thn the receptor itself. We oserve much lower level of tyrosine phosphoryltion on wild type receptors in v-fps expressing cells, most likely s result of the rpid turnover of ctivted receptors. We further investigted the ility of these di erent cell lines to form colonies in soft gr s mesure of whether the cells were trnsformed (Figure 8). Only cells expressing oth v-fps nd kinse ctive PDGF receptor were le to demonstrte nchorge independent growth. These results rgue tht v-fps trnsformtion requires the PDGF receptor, since cells expressing v- Fps yet lcking the receptor were not le to grow in soft gr. Further, our results demonstrte tht v-fps trnsformtion requires PDGF receptor which possesses tyrosine kinse ctivity, since coexpression of kinse inctive receptor nd v-fps ws not su cient to trnsform cells. Discussion WT-R R5 v-fps WT-R In this report we demonstrte tht v-fps expression resulted in lrge decrese in the expression of the PDGF receptor. In prt this ws found to e due to reduction in the stedy stte levels of PDGF receptor mrna, ut this lone could not ccount for the more signi cnt reduction in the mount of receptor protein. Our ndings with v-fps trnsformed cells re in R5 lystes lystes IP: PDGFR Blot: P.Tyr Figure 7 Expression of v-fps cn induce tyrosine phosphoryltion of kinse inctive PDGF receptor. Dog epithelil cells devoid of PDGF receptor (), expressing wild type PDGF receptor (WT-R), or kinse inctive receptor (R5) were ech engineered to express v-fps s indicted. Whole cell lystes (25 mg) from ech cell line were resolved y SDS ± PAGE, trnsferred to nitrocellulose nd immunolotted with ntiodies speci c for the PDGF receptor () or gg () s descried in Figure 1. (c) Anti-PDGF receptor immunoprecipittions of the cell lystes (4 mg totl protein) were resolved y SDS ± PAGE nd immunolotted with nti-phosphotyrosine (nti-p.tyr) ntiodies s descried in Figure 5

7 no v-fps + v-fps 2327 no PDGFR WT-R ctive PDGFR R5 inctive PDGFR Figure 8 Expression of kinse ctive PDGF receptor nd v-fps re oth required for nchorge independent growth of cells. Dog epithelil cell lines with nd without PDGF receptor nd/or v-fps s indicted, were tested for their ility to form colonies in soft gr. The cell lines were seeded into soft gr t 1 5 cells per 6 cm plte nd fed on weekly sis. Photogrphs were tken 19 dys fter plting contrst to reports demonstrting tht cells expressing trnsforming forms of the rs oncogene were unresponsive to PDGF stimultion s result of some inhiitory fctor preventing the ctivtion of the norml levels of the PDGF receptor (Lin et l., 1988; Rke et l., 1991; Zullo nd Fller, 1988). This suggests tht di erent mechnisms re contriuting to the trnsformed phenotype mnifested y these two oncogenes. Low levels of PDGF receptors hve lso een reported in simin srcom virus-trnsformed nd sistrnsformed cells due to down-regultion of the receptor y n utocrine mechnism (Grrett et l., 1984; Hung nd Hung, 1988). In oth cses, receptor downregultion could e locked y ddition of surmin, n inhiitor which prevents the inding of PDGF or v-sis to the PDGF receptor. Cells trnsformed y severl other viruses hve lso een shown to use n utocrine mechnism to ctivte PDGF receptor signling pthwys which result in 5 ± 1% decrese in ville PDGF receptors (Bowen-Pope et l., 1984). Surmin hd no e ect however, on the level of receptors in v-fps trnsformed cells, indicting tht v- fps trnsformtion does not occur vi n utocrine growth stimultory pthwy in which cells lredy expressing PDGF receptors re induced to produce PDGF. As control for our experimentl system, we used surmin to return the level of PDGF receptors present in norml Rt-2 cells which hd een downregulted y continul exposure to PDGF, to the high levels oserved in unstimulted cells. The mount of PDGF receptor present in v-fps expressing cells ws found to e t lest 1-fold less thn tht of prentl Rt-2 cells devoid of v-fps. Some of this reduction could e explined y the 4 ± 8-fold lower mount of PDGF receptor encoding mrna in Rt-2 v-fps cells. Reductions in PDGF receptor mrna (5 ± 1-fold) hve een reported previously in src nd rs trnsformed cells with similr decreses in receptor protein levels. This oservtion hs led to the suggestion tht the expression of the PDGF receptor my e regulted in growth stte-dependent mnner in order to provide mechnism y which cells cn modulte their PDGF responsiveness (Vziri nd Fller, 1995). Our dt is consistent with this hypothesis since we lso oserve modest reduction (4 ± 8-fold) in PDGF receptor mrna in cells expressing n ctive v-fps tyrosine kinse. The e ect of v-fps expression on PDGF receptor messge level seems to e secondry nd in ddition to

8 2328 wht ppers to e more direct e ect of v-fps on the PDGF receptor itself. Nmely, cells expressing temperture sensitive mutnt of v-fps displyed trnsient increse in tyrosine phosphoryltion of the PDGF receptor in response to ctivtion of the v-fps tyrosine kinse. In ddition, our dt showing v-fps induced tyrosine phosphoryltion of kinse inctive PDGF receptor rgues tht v-fps either phosphoryltes the receptor directly or vi the ctivtion of nother tyrosine kinse distinct from v-fps nd the PDGF receptor. In ny cse, constitutive tyrosine phosphoryltion of the PDGF receptor my ctivte the receptor nd/or provide inding sites for vrious SH2-domin-contining proteins such s PI3 kinse, Nck, SHC, GRB2, rsgap, phospholipse Cg1 (PLCg1), Src nd SHPTP2 (reviewed in Clesson-Welsh, 1994; Cohen et l., 1995; Pwson, 1995; Schlessinger, 1994)). Recruitment of these cytosolic signling proteins to the tyrosine phosphorylted PDGF receptor my ctivte downstrem signling pthwys nd/or fcilitte their phosphoryltion y v-fps or the receptor. Some of these signling proteins hve een shown to e tyrosine phosphorylted in v-fps trnsformed cells including: SHC (McGlde et l., 1992), rsgap (Ellis et l., 199), nd PI3 kinse (Fukui et l., 1991). Fps/Fes hs een shown to ind to nd phosphorylte BCR on tyrosine, inducing the ssocition of GRB2/SOS with BCR nd providing mechnism y which to ctivte the Rs pthwy in 3Y1 Rt rolsts (Mru et l., 1995). The uthors lso suggest tht BCR, once phosphorylted y Fps/Fes, my ind other SH2 contining e ector proteins in ddition to GRB2. Thus BCR my function s docking protein, much like IRS-1 in the insulin receptor signling cscde (Sun et l., 1991). Mru et l. lso reported tht overexpression of the SHC SH2 domin filed to reverse morphologicl trnsformtion in v-fps trnsformed Rt 3Y1 cells nd suggested tht multiple signling pthwys contriute to Rs ctivtion in v-fps trnsformed cells (Mru et l., 1995). Thus, tyrosine phosphoryltion of the PDGF receptor y v-fps my represent prllel signling pthwy to tht involving BCR. Not only would the phosphorylted receptor provide SH2-inding sites, ut ctivtion of the endogenous tyrosine kinse ctivity of the receptor would provide n even stronger prolifertive signl y contriuting to Rs ctivtion. Although v-fps is cple of inducing tyrosine phosphoryltion of mutnt PDGF receptor which lcks intrinsic kinse ctivity, this ws not su cient to trnsform the cells, nd indictes tht the receptor is not simply cting s pssive docking protein. Our results demonstrte cler requirement for kinse ctive PDGF receptor in v-fps trnsformtion suggesting tht the ctlytic ctivity of the receptor is essentil for the ctivtion of mitogenic signling pthwys. Similr constitutive phosphoryltion nd ctivtion of the epiderml growth fctor receptor (Wsilenko et l., 1991) nd the insulin-like growth fctor I receptor (Kozm nd Weer, 199) hve een oserved upon trnsformtion of cells with the v-src oncogene. These results hve led to the suggestion tht the unregulted interction etween v-src nd these receptors my rogte the receptors requirement for lignd inding to produce ctivted receptors, leding to the constnt trnsmission of signls for growth (Kozm nd Weer, 199; Wsilenko et l., 1991). Our results support n nlogous model where v-fps my constitutively phosphorylte nd ctivte the PDGF receptor, cusing internliztion nd down-regultion of the receptor molecules. It hs een suggested tht ctivtion of the PDGF receptor y its ssocition with the ovine ppillomvirus E5 protein is le to generte sustined prolifertive signl, resulting in rolst trnsformtion (Nilson nd DiMio, 1993). A similr model for trnsformtion y v-fps is proposed here. Fps/Fes is normlly expressed in hemtopoietic cells, prticulrly in myeloid cells (Feldmn et l., 1985; Greer et l., 199; Lnfrncone et l., 1989; McDonld et l., 1985; Smrut et l., 1985; Smithgll et l., 1988). It hs een implicted to ply role in signl trnsduction from the grnulocytemcrophge colony-stimulting fctor (GM ± CSF) nd interleukin-3 (IL-3) receptors (Brizzi et l., 1996; Feldmn et l., 1985; Hnzono et l., 1993; Lnfrncone et l., 1989). In response to stimultion y GM ± CSF or IL-3, Fps/Fes ecomes tyrosine phosphorylted nd ctivted, nd it cn physiclly ssocite with the GM ± CSF receptor (Hnzono et l., 1993) nd JAK2 (Brizzi et l., 1996). More recently, fps/fes expression hs lso een descried in rpidly proliferting emryonic tissues (CreÁ et l., 1994) nd in vsculr endothelil cells (Greer et l., 1994). The vsculr hyperplsi oserved in trsgenic mice overexpressing n ctivted, memrne trgeted mutnt Fps/Fes protein, hs led to the suggestion tht Fps/Fes plys role in ngiogenesis. This muttion in Fps/Fes, which promotes its ssocition with the plsm memrne, could provide ccess to positive regultors or sustrtes nd promote hyperprolifertion in the sence of the norml signling mechnisms (Greer et l., 1994). It is interesting to note tht severl fctors which promote ngiogenesis re in fct lignds for receptor tyrosine kinses, some of which re structurlly relted to the PDGF receptor (DeVries et l., 1992; Klgsrun nd D'Amore, 1991; Milluer et l., 1993; Termn et l., 1992). PDGF itself hs een reported to stimulte the growth of primitive hemtopoietic cells y inducing IL- 1 production in suset of mcrophges, suggesting tht the PDGF receptor my e expressed in some of the sme cell types s Fps/Fes (Young nd Mrtin, 1984). Oservtions tht monocytes nd mcrophges exhiit ctivtion nd chemotctic responses to PDGF lend further support to this ide (Pntzis et l., 199; 1991; Sieghn et l., 199; Tzeng et l., 1985). There hs lso een report tht PDGF BB overexpression in hemtopoietic precursors induces myeloprolifertive syndrome (Yn et l., 1994). The uthors suggest tht this my provide new mechnism for the initition of myeloprolifertive disorders, prticulrly those lcking the cr ± l trnsloction. There hs lso een report showing PDGF receptor mrna expression in mcrophges (Yn et l., 1993) nd in di erentited cells of the myelomonocytic linege (Pntzis et l., 199; 1991). Moreover, sugroup of chronic myelomonocytic leukemi (CMML) ptients hve een reported to possess Tel-PDGF receptor fusion product resulting from

9 chromosoml trnsloction (Golu et l., 1994). The tel portion of the fusion encodes helix ± loop ± helix domin which my fcilitte dimeriztion of the PDGF receptor in the sence of PDGF, s well s providing n ctive promoter. Another lterntive put forwrd suggests tht overexpression of truncted nd constitutively ctive PDGF receptor in cells of the myeloid linege, my e su cient to drive cell prolifertion (Golu et l., 1994). Golu et l. lso suggest tht the errnt expression of tel-pdgf receptor erly in the development of CMML could predispose ptients to further trnsloctions nd more ggressive disese stte. Given tht Fps/Fes is normlly expressed in these cells, we re curious to know whether Fps/Fes plys role in this process. Mterils nd methods Antiser The nti-pdgf receptor ntiody used for immunoprecipittions ws rit polyclonl rised ginst GST fusion protein contining mino cids 89 ± 116 of the humn PDGF receptor generted in our lortory. For ll nti-pdgf receptor immunolots the rit polyclonl ntiserum, nti-pr4 ((Kypt et l., 199); gift from S Courtneidge), rised ginst the croxy-terminl 13 mino cids of the humn PDGF receptor, ws used. Both nti- PDGF receptor ntiodies were speci c for the PDGF receptor nd showed no cross rectivity towrds the PDGF receptor. Anti-gg mouse monoclonl ntiodies (R254E; Ingmn-Bker et l., 1984) hve een previously descried (Letwin et l., 1988). The nti-phosphotyrosine (nti-p.tyr) ntiodies were purchsed from Snt Cruz Biotechnology. Cell lines Rt-2 cells (Topp, 1981) were grown in Dulecco modi ed Egle medium contining 1 U penicillin G nd 1 mg of streptomycin per ml (DMEM) supplemented with 1% fetl ovine serum (FBS) in humidi ed incutor t 378C with 5% CO 2. Cell lines expressing wild type v-fps nd the vrious v-fps mutnts hve een descried previously (Koch et l., 1989; Morn et l., 1988; Sdowski et l., 1986; Weinmster et l., 1984). TO17 contins t point muttion in Tyr-173 chnging it to Glu. TO29 hs stop codon t position 118, truncting the protein within the kinse domin. CNA7 cells express v-fps mutnt which contins dipeptide insertion of Leu-Glu etween Glu-832 nd Leu-833 in the SH2 domin of v-fps, nd is temperture sensitive for trnsformtion (tsax9m). Dog epithelil cells () contining wild type (WT-R) or kinse inctive (R5) PDGF receptors hve een descried previously (Kzlusks et l., 199) nd were grown in DMEM supplemented with 1% FBS nd 4 mg/ml G418. Cell culture techniques For PDGF stimultions, pltes of cells which were out 8% con uent were serum strved in DMEM+.5% FBS for 2 dys. Cells were then stimulted with 5 ng/ml humn homodimer PDGF BB (Cedrlne) for 5 min or left unstimulted. They were then wshed nd lysed, s descried elow. To down regulte the level of PDGF receptors in Rt-2 cells, cells were stimulted with 5 ng/ml PDGF in 1% FBS for 24 h. To lock the inding of PDGF to the receptor, cells were treted with 1 mm surmin (Cliochem #574625). Conditioned medi ws prepred from cells which were left in medi for n dditionl two dys fter reching 1% con uence. The medi (1 mls) ws then lyophilized, resuspended in 1 ml PLC LB (see elow), preclered nd immunoprecipitted with 5 mg of nti-pdgf BB ntiody (Oncogene Science) s descried elow. To generte dog cell lines expressing v-fps, we cotrnsfected them with oth the v-fps plsmid (piv2.3; Morn et l., 1988, 1 mg) s well s hygromycin resistnce plsmid (psv 2 hph; Americn Type Culture Collection, 1 mg), since these cells hd previously een mde resistnt to G418 (Kzlusks et l., 199). Clones were selected in hygromycin B (Sigm, 4 mg/ml) nd tested for v-fps protein expression y sujecting whole cell lystes to Western lot nlysis using nti-gg ntiodies. After screening, cells were mintined in medi contining 8 mg/ml hygromycin. Prentl cell lines were lso trnsfected with psv 2 hph lone nd hygromycin resistnt clones were used s control cell lines in susequent experiments. The ility of cells to form colonies in soft gr ws ssyed y seeding cells (1 5 ) in suspension of top gr (2 ml of: DMEM, 1% FBS, 3.756MEM vitmins (Cndin Life Technologies),.36% gr) ove of lyer of ottom gr (5 ml of: DMEM, 1% FBS, 56MEM vitmins,.61% gr) in 6 cm pltes. Colonies were fed weekly y pplying n dditionl 1 ml of top gr to ech plte. The photogrphs shown re representtive of two independent experiments, ech done in duplicte. Preprtion of cell lystes, immunoprecipittions nd immunolot nlysis To prepre whole cell lystes, cells from ech 1 cm plte (when out 8% con uent) were wshed with PBS (137 mm NCl, 2.7 mm KCl, 4.3 mm N 2 HPO 4, 1.4 mm KH 2 PO 4 ) nd lysed with.3 ml of 18C SDS smple u er (5 mm sodium phosphte [ph 6.8], 2% sodium dodecylsulfte [SDS], 5% -mercptoethnol, 1% glycerol,.5% romophenol lue). Cells were scrped with Te on scrper nd forced through 27 guge needle severl times to reduce the viscosity of the lyste. Smples were incuted t 18C for 5 min nd n liquot ws tken for protein determintion using Lowry kit (Sigm #P5656). Smples were stored t 788C nd reheted to 18C for 5 min prior to loding onto n SDS-polycrylmide gel (SDS ± PAGE). To prepre cell lystes for immunoprecipittions, pltes of cells (out 8% con uent) were plced on ice nd wshed once with ice cold PBS. Ech 1 cm plte of cells ws scrped into 1 ml PLC LB (5 mm HEPES [ph 7.], 15 mm NCl, 1% glycerol, 1% Triton X-1, 1.5 mm MgCl 2, 1 mm EGTA, 1 mm NF, 1 mm NPP i,1mmn 3 VO 4,1mM phenylmethylsulfonyl uoride, 1 mg/ml protinin, 1 mg/ml protinin, 1 mg/ml leupeptin). Lystes were clri ed y centrifugtion t 14 g for 1 min. Lystes were stored t 788C nd thwed quickly t 158C for use. Anti-PDGF receptor immunoprecipittions were crried out using 3 ml of crude nti-pdgf receptor rit polyclonl nti-serum per ml of cell lyste nd 1 ml of protein A sephrose eds. After gentle mixing t 48C for 9 min the immune complexes were spun rie y nd wshed three times with 5 ml HNTG (2 mm HEPES [ph 7.], 15 mm NCl,.1% Triton X-1, 1% glycerol, 1 mm N 3 VO 4 ) nd resuspended in 4 ml SDS smple u er. Smples were heted t 18C for 5 min prior to loding onto n SDS ± PAGE. Immunolots of whole cell lystes or immunoprecipittes were prepred y rst resolving the proteins on 7.5% SDS ± PAGE, soking the gel in trnsfer u er (48 mm Tris, 39 mm glycine,.375% SDS, 2% methnol) for 1 ± 3 min nd trnsferring the proteins to nitrocellulose with semi-dry lotting pprtus t.8 mm cm 2 for 6 min. 2329

10 233 Immunolots were locked in milk locking (3% Crntion skim milk powder in TBST (2 mm Tris-HCl [ph 7.4], 15 mm NCl+.5% Tween-2), overnight t 48C or 1 h t room temperture. The lots were incuted in milk locking contining the speci c ntiody for 1 ± 2 h t room temperture, with gentle rocking (1 mg/ml nti-p.tyr; 1 : 5 dilution nti-pr4; 1 : 1 dilution nti-gg). Unound ntiody ws removed during three 5 min wshes in TBST. Immunolots were then incuted for 1 ± 2 h t room temperture in milk locking solution contining horserdish peroxidse conjugted secondry ntiody (1 : 2; Snt Cruz Biotechnology). Bound ntiodies were visulized using chemiluminescent detection regents (Dupont) ccording the the mnufcturers recommendtions. Figures shown re representtive of t lest two independent experiments. Qunti ction of reltive nd intensities ws performed y scnning densitometry (Biord Model 62 Video Densitometer nd 1D Anlyst 1.1 softwre (Biord)) using severl exposures to otin dt within the liner rnge of the lm. Blots were stripped y incuting them in stripping u er (62.5 mm Tris-HCl [ph 6.8], 2% SDS, 1 mm -mercptoethnol) for 3 min, wshing four times for 5 min ech in TBST nd relocking in milk locking solution prior to reproing with di erent ntiody. The e ectiveness of this stripping ws tested previously y incuting the lot in chemiluminescence regents prior to the relocking step, to detect ny horserdish peroxidse conjugted ntiodies remining ound to the lot. RT ± PCR of rt PDGF homologue nd RNse protection ssy A smple of mrna ws isolted from Rt-2 rolst cells using QuickPrep Micro mrna puri ction kit (Phrmci) ccording to the mnufcturers instructions. The corresponding cdnas were synthesized using Reverse Trnscription System kit (Promeg) which uses n oligo dt primer. The smple ws digested with RNse H nd the cdna used directly in polymerse chin rection (PCR). The oligonucleotide primers used to speci clly mplify portion of the cdna encoding the rt homologue of the PDGF receptor were: 5'-ATC- ATCCTCATCATGCTTTG-3' nd 5'-GAA GGT GTG TTT GTT GCG GT-3'. These primers correspond to regions with high degree of nucleotide sequence identity etween the known cdna sequences of the mouse nd humn PDGF receptor. The region of the PDGF receptor mpli ed y this PCR rection is shown in Figure 2 nd sequence lignment of the rt, mouse nd humn receptor is shown in Figure 2. In order to fcilitte the lunt end cloning of the PCR product, the PCR primers were phosphorylted prior to the PCR rection using T4 polynucleotide kinse. The PCR rection contined primers (.2 mm ech, phosphorylted), 25% of the cdna (i.e. 5 ml) generted y reverse trnscription, 5% dimethylsulphoxide, dntps (.2 mm ech) nd Pfu enzyme (Strtgene, 5 units) in the Pfu rection u er supplied with the enzyme. Minerl oil ws lyered on top nd the rection heted to 958C for 4 min. Thirty-six cycles of: 958C for1min,498cfor1minnd728c for 2 min llowed mpli ction of 453 se pir (p) product which ws ethnol precipitted nd gel puri ed from 1% low melting grose gel. This lunt end product ws ligted into pgex2t (Phrmci) which hd een digested with SmI nd treted with clf intestinl lkline phosphtse to minimize vector religtion in the sence of insert. Competent RR1 cells were trnsformed with the ligtion nd mpicillin resistnt colonies were screened for the presence of insert y digesting with BmHI nd EcoRI, two sites which nk the SmI site. Two independent clones contining inserts (pgexrtr) were sequenced using ech of the PCR primers s well s vector speci c primer (5'- TTT GCA GGG CTG GCA AGC-3') nd the T7 Sequencing kit (Phrmci; see Figure 2). In order to generte n RNA proe cple of inding to the rt PDGF receptor mrna isolted from di erent cell lines, we sucloned portion of the rt PDGF receptor cdna into vector which contins RNA polymerse inding sites. The 35 p HincII ± EcoRI frgment from pgexrtrc19 (which contins prtil cdna encoding the rt PDGF receptor cloned in reverse orienttion) ws sucloned into pbluescript II KS + (Strtgene) which hd een digested with SmI nd EcoRI. The resulting plsmid, pbsrevrtpdgfrc19 (Figure 2c), ws linerized with EcoRI nd treted with proteinse K to remove ny RNses present. An SP6/T7 MAXIscript kit (Amion) ws used to synthesize n RNA proe rdioleled with - 32 P-UTP (395 nt.), corresponding to the non-coding strnd of the rt PDGF receptor cdna using T7 RNA polymerse. The unincorported [- 32 P]UTP ws removed from the RNA proe using ProeQunt G-5 Micro Column (Phrmci). Totl RNA ws isolted from the di erent cell lines ccording to the method of Chomczynski nd Scchi (1987). The rdioleled RNA proe ws hyridized with the totl RNA smples (1 ± 8 mg s indicted) nd digested with RNse in n RNse protection ssy, crried out using Rionuclese Protection Assy kit (Amion) ccording to the mnufcturers instructions. RNA smples were resolved on 6% crylmide/8m ure gel, dried, nd visulized y utordiogrphy for severl dys with n intensifying screen. Results shown re representtive of t lest two experiments. RNA stndrds were synthesized using the sme pproch s ws used to mke our PDGF receptor proe using Century mrker templte preprtion (Amion, ct. 778). Control yest RNA, provided in the Amion kit, ws hyridized with the PDGF receptor RNA proe nd incuted with nd without RNse, s indicted. Acknowledgements We thnk B Trowell nd C Howells for technicl ssistnce. We re indeted to M Morn for TO17 nd TO29 cells; T Pwson for Cl-1 nd CNA7 cells; A Kzlusks for dog epithelil cell lines, WT-R, R5; nd S Courtneidge for nti-pr4 ntiodies. We thnk A Kzlusks nd R Wrrington for helpful discussions in ddition to M Morn nd P Greer for the criticl review of this mnuscript. This reserch is supported y the Ntionl Cncer Institute of Cnd with funds from the Cndin Cncer Society References Bowen-Pope DF, Vogel A nd Ross R. (1984). 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