©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Demonstration of Functionally Different Interactions between Phospholipase C- and the Two Types of Platelet-derived Growth Factor Receptors (*)

(Received for publication, September 8, 1994; and in revised form, January 18, 1995)

Anders Eriksson (§) Eewa Nånberg (1) Lars Rönnstrand Ulla Engström Ulf Hellman Eva Rupp Graham Carpenter (2) Carl-Henrik Heldin Lena Claesson-Welsh (¶)

From the  (1)Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden, the Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden, and the (2)Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-0146

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C- (PLC-). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC- are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC- SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC- and the PDGF alpha-receptor is more stable than that of PLC- and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC- and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-.


INTRODUCTION

Platelet-derived growth factor (PDGF) (^1)is a polypeptide mitogen that acts on a broad spectrum of cells, including fibroblasts and glial cells (for reviews, see (1) and (2) ), and that consists of disulfide-linked dimers of A and B polypeptide chains, forming three possible isoforms, PDGF-AA, -AB, and -BB. PDGF mediates its biological effects by interacting with two structurally related receptor types that differ in their interactions with the PDGF isoforms (for a review, see (3) ). Thus, the alpha-receptor binds PDGF A- and B-chains, whereas the beta-receptor binds only the B-chain. Binding of ligand induces receptor dimerization, activation of the receptor tyrosine kinase, and autophosphorylation on multiple tyrosine residues in the intracellular region of the receptors (for a review, see (4) ). The phosphorylated tyrosine residues of the receptor form specific binding sites for downstream signaling components, containing one or more 100-amino acid residue motifs denoted Src homology 2 (SH2) domains (reviewed in (5) ). The specificity of the association is defined both by the amino acid residues surrounding the phosphotyrosine and the structure of the SH2 domain. The signal transduction molecules either have endogenous catalytic activities or serve as adaptor molecules, which lack enzymatic activity but interact with catalytic components. The SH2 domain-containing proteins that associate with PDGF beta-receptors and that have been identified include phospholipase C- (PLC-), the regulatory subunit (p85) of phosphatidylinositol 3` kinase, members of the Src family of protein tyrosine kinases, the Ras GTPase activating protein, and most recently the phosphatase SH-PTP 1D/SH-PTP 2/Syp and the adaptor proteins Grb2, Nck, Shb, and Shc (reviewed in (3) ). In addition, the two PDGF receptors interact with other unidentified substrates(6) .

Both alpha- and beta-receptors mediate mitogenic signals; however, signals leading to chemotaxis and membrane edge ruffling are mediated through the beta-receptor, while the alpha-receptor, in certain cell types, conveys a negative signal that inhibits chemotaxis. Eight autophosphorylation sites have been mapped in the PDGF beta-receptor: Tyr-579 and Tyr-581 in the juxtamembrane region(7) , Tyr-740, Tyr-751, and Tyr-771 in the kinase insert(8, 9) , Tyr-857 in the kinase domain (10) , and Tyr-1009 and Tyr-1021 in the carboxyl-terminal tail(11, 12, 13, 14) . Seven of these residues are conserved in the PDGF alpha-receptor at positions that are homologous with those in the beta-receptor. We report the mapping of two autophosphorylation sites, Tyr-988 and Tyr-1018, in the carboxyl-terminal tail of the human PDGF alpha-receptor and show that these sites are required for the interaction of the alpha-receptor with PLC-. Furthermore, we show that the human PDGF alpha- and beta-receptors differ quantitatively in their abilities to associate with and phosphorylate PLC- and to stimulate inositol phosphate production.


MATERIALS AND METHODS

Cell Culture and Transfection

Porcine aortic endothelial (PAE) cells(15) , which lack endogenous PDGF receptors (16) were cultured in Ham's F12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin. The mutated alpha-receptors were expressed in PAE cells after insertion of the cDNA into the expression vector pcDNA I/neo (Invitrogen), and transfection followed by selection as described earlier(16) . By use of Scatchard analysis, performed as previously described in detail(6) , the number of receptors were estimated to 25,000 for the Y988F cell line and 30,000 for the Y1018F cell line. The wild-type human PDGF alpha-receptor-expressing cell line (ZalphaR15; 40,000 receptors per cell) as well as the wild-type human beta-receptor-expressing cell line (beta-SM; 32,000 receptors per cell) have been described before(6, 17) . The PAE cell line co-expressing both PDGF receptors, denoted Palphabeta:1, has 18,000 alpha-receptors and 10,000 beta-receptors(18) . Human foreskin fibroblasts line AG 1523 (30,000 alpha-receptors and 100,000 beta-receptors) (19) was bought from the Human Genetics Mutant Cell Repository in Camden, NJ and was kept in Eagle's minimal essential medium, 10% FCS, and penicillin/streptomycin.

Site-directed Mutagenesis

A 3.4-kilobase pair BamHI fragment of the human PDGF alpha-receptor (20) was subcloned into the pALTER vector and subjected to site-directed mutagenesis using the Altered Sites system (Promega). Point mutations changing tyrosine residues Tyr-988 and Tyr-1018 to phenylalanine residues were introduced using the oligonucleotides 5`-GACAATGCATTCATTGGTGTC-3` (Y988F) and 5`-GACAGTGGCTTCATCATTCCTC-3` (Y1018F). Introduction of the expected mutations were confirmed by nucleotide sequencing.

Antibodies

The rabbit peptide antiserum PDGFR-7, specifically recognizing the PDGF alpha-receptor, has been described earlier(6) . The rabbit peptide antiserum AbPalpha1 was raised against a synthetic peptide corresponding to amino acids 993-1009 of the human PDGF alpha-receptor (20) and was kindly provided by Dr. S. Bishayee(21) . The mouse phosphotyrosine monoclonal antibody (PY20) was from ICN Biomedicals, Inc. The rabbit antibody against phosphotyrosine (22) was purified using affinity chromatography(23) . Rabbit PLC- antiserum has been described earlier(24) . Peroxidase-conjugated swine anti-rabbit immunoglobulins were from Dakopatts (Copenhagen, Denmark). Peroxidase-conjugated sheep anti-mouse immunoglobulins were from Amersham Corp.

[H]Thymidine Incorporation Assay

The ability of PDGF-BB to stimulate DNA synthesis, measured by the incorporation of [^3H]thymidine, in the different transfected PAE cell lines was performed as previously described(6) .

Identification of Carboxyl-terminal Phosphorylation Sites

[P]Orthophosphate labeling of cells and immunoprecipitation with PY20 was performed as described before (13) . Samples were separated on a 7.5% polyacrylamide gel, transferred to nitrocellulose membranes (0.2 µm; Schleicher and Schuell), followed by autoradiography. The band corresponding to the PDGF alpha-receptor was cut from the nitrocellulose, washed with water, and incubated for 12 h at 25 °C with cyanogen bromide (CNBr) (10 mg/ml; Sigma) in 70% formic acid essentially as described earlier(25) . The supernatant was dried under nitrogen, dissolved in water, dried in a rotor evaporator, and redissolved in 50 mM NH(4)HCO(3). Protein A-Sepharose CL-4B beads cross-linked to the peptide antiserum AbPalpha1 as described (26) was then added. After incubation at 4 °C for 2 h, immune complexes were washed three times with phosphate-buffered saline containing 0.05% Triton X-100, followed by two washes with 50 mM NH(4)HCO(3). The immunoprecipitated material was then eluted from the beads with 70% formic acid. A fraction of the purified peptide was coupled to Sequelon-AA membrane (Milligen/Bioresearch, Burlington, MA). Edman degradation was performed in a protein sequencer (Applied Biosystems, model 477A), adopted for solid phase sequencing, using a modified program where the ATZ amino acids were extracted with 100% trifluoroacetic acid and collected without high pressure liquid chromatography analysis. The remaining purified peptide was dried in a rotor evaporator and dissolved in 8 M urea, which was then diluted to 4 M by addition of 0.1 M Tris-HCl, pH 8.8. The sample was further digested with 0.5 µg of a lysylendopeptidase, Achromobacter lyticus protease I (Wako Chemicals, Inc.), for 12 h at 30 °C and desalted on a Fast Desalter in a SMART system (Pharmacia Biotech Inc.) equilibrated in 20% acetonitrile and 0.065% trifluoroacetic acid. The P-containing fraction was subjected to Edman degradation as described above. The fractions from each Edman cycle were dried in a rotor evaporator, dissolved in trifluoroacetic acid, spotted on cellulose thin layer chromatography plates, and quantitated using a PhosphorImager (Molecular Dynamics).

In vitro labeling of wild-type and tyrosine-mutated PDGF alpha-receptors was performed essentially as follows. PDGF-BB-stimulated and control cells were solubilized as described above and immunoprecipitated with PDGFR-7. The protein A-Sepharose beads were washed four times with lysis buffer and once with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. In vitroP labeling was performed by incubating in a solution containing 20 mM HEPES, pH 7.5, 10 mM MnCl(2), 1 mM dithiothreitol, and 50 µCi [-P]ATP (Amersham; specific activity, 3000 Ci/mmol) for 10 min at 25 °C. The samples were then digested, immunoprecipitated, and washed as described above, except that the final wash was with 20 mM Tris-HCl, pH 7.5. The peptide was eluted from the beads with 8 M urea in 0.1 M Tris-HCl, pH 8.8. Digestion with A. lyticus protease I, desalting, and Edman degradation was performed as described above.

Ligand Stimulation of Cells, Immunoblotting, and Immunocomplex Kinase Assay

To the medium of transfected cell lines was added 20 mM HEPES, pH 7.5, and 50 µM Na(3)VO(4) with or without 100 ng/ml PDGF-AA or PDGF-BB. After incubation on ice for 1 h, the cells were washed with an ice-cold solution containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 50 µM Na(3)VO(4) and solubilized in a CHAPS lysis buffer (20 mM HEPES, pH 7.5, 10% glycerol, 1% CHAPS, 150 mM NaCl, 10 mM EDTA, 30 mM Na(4)P(2)O(7), 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol, 250 µM Na(3)VO(4)). The lysates were centrifuged to remove unsolubilized material and incubated for 2 h with antiserum, followed by adsorption to protein A-Sepharose CL-4B. In certain experiments, the cell lysates were incubated with wheat germ lectin Sepharose 6 MB (Pharmacia Biotech Inc.). The Sepharose beads were washed four times with lysis buffer, and the samples were subjected to SDS-gel electrophoresis in 7.5% polyacrylamide gels before electroblotting to Hybond-C extra membranes (Amersham). Immunoblotting was further performed essentially as described elsewhere (27) using the ECL Western blotting detection system (Amersham). In some cases, the filters were reprobed after stripping, as described(27) .

Immunocomplex kinase assays were performed as previously described(6) . Essentially, P labeling of the immunoprecipitates, generated as described above, was performed in a 40-µl reaction volume containing 20 mM HEPES, pH 7.5, 10 mM MnCl(2), 1 mM dithiothreitol, and 5 µCi [-P]ATP (Amersham; specific activity, 3000 Ci/mmol) that was incubated for 10 min at 25 °C. The kinase reactions were terminated by addition of 40 µl of twice-concentrated SDS sample buffer and incubated at 95 °C for 4 min. Samples were analyzed by SDS-PAGE followed by cross-linking with glutaraldehyde and treatment with 1 M KOH at 55 °C for 1 h to preferentially decrease serine phosphate levels. The gels were then dried and exposed to Fuji RX film with intensifying screen. Quantitation of precipitated PDGF alpha-receptor was performed using a PhosphorImager (Molecular Dynamics).

Synthesis and Purification of Phosphorylated Peptides

Peptides were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and phosphorylated by use of the global phosphorylation method(28, 29) using di-t-butyl-N,N-diisopropylphosphoramidite, as described earlier(7) . All peptides had free carboxyl termini. The peptides were purified by reversed phase chromatography. The collected fractions were analyzed by plasma desorption mass spectrometry using a Bio Ion 20 instrument (Applied Biosystems). The sequences of the peptides were as follows: peptide Tyr-988, MRVDSDNAYIGVTYKNE; peptide Tyr-988(P), MRVDSDNAY(P)IGVTYKNE; peptide Y1018, LDEQRLSADSGYIIPLPDID; peptide Tyr-1018(P), LDEQRLSADSGY(P)IIPLPDID; (P) indicates a phosphorylated tyrosine residue.

Peptide Competition Experiment

Stock solutions of the peptides were made in 20 mM HEPES, pH 7.4. All solutions were adjusted to neutral pH. Dilutions of peptides were made in a solution containing 1% Triton X-100, 10% glycerol, 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 250 µM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol, 1 mM dithiothreitol (lysis buffer). In a total volume of 100 µl, peptide was incubated with P-labeled PDGF alpha-receptor, generated by in vitro complex kinase assay as described above, for 30 min at 4 °C. 50 µl of a 1:1 slurry of glutathione-Sepharose (loaded with 2 µg of GST-PLC- N/C SH2 fusion protein) was added to each tube. After 1 h incubation end over end at 4 °C, samples were washed three times in lysis buffer. 50 µl of SDS sample buffer was added to each sample. After boiling for 5 min, samples were separated on a 7% SDS-polyacrylamide gel. The amount of PDGF alpha-receptor precipitated in each lane was quantitated using a PhosphorImager (Molecular Dynamics).

Measurement of Inositol Phosphate Production

The production of inositol phosphates was measured using 60-mm dishes of PDGF alpha- (ZalphaR15) or beta-receptor (beta-SM) expressing PAE cells and in PAE cells co-expressing the alpha- and beta-receptors (Palphabeta:1), which were labeled for 48 h in Ham's F12 supplemented with 0.1% bovine serum albumin and 1 µCi/ml myo-[^3H]inositol. The cultures were washed and incubated for 15 min with Ham's F12 containing 0.1% bovine serum albumin, 15 mM HEPES, pH 7.5, and 20 mM LiCl. The medium was then aspirated, fresh medium with or without 50 ng/ml PDGF-AA or PDGF-BB or with 10% FCS was added, and the incubation at 37 °C continued for 30 min. Human foreskin fibroblasts were treated similarly, except that MCDB 104, containing 0.5 mM CaCl(2), was used instead of Ham's F12. The culture dishes were placed on ice, and the medium was removed before the addition of 1.5 ml of ice-cold methanol/HCl (100:1)(30) . The quenched samples were collected, and the dishes rinsed with an additional 1.5 ml of methanol/HCl (100:1). To each sample, 1.5 ml of H(2)O and 3 ml of CHCl(3) was added, the tubes were mixed by vortexing and left on ice for 30 min. The water-soluble phase was collected, diluted with 2 volumes of H(2)O, and 1 ml of AG1-X8 (formate; Bio-Rad) resin was added and incubated end over end for 2 h(31) . The samples were extensively washed with H(2)O and then with a solution of 5 mM disodium tetraborate and 60 mM sodium formate. Finally, the total inositol mono-, bis-, tris-, and tetrakisphosphates were eluted from the resin with a solution of 0.1 M formic acid and 1.0 M ammonium formate and subjected to scintillation counting. The CHCl(3)-phase samples containing the phospholipids were dried, redissolved in methanol, and subjected to scintillation counting. The number of cells was determined in parallel culture dishes for each experiment.


RESULTS

Identification of Autophosphorylation Sites at the Carboxyl Terminus of the PDGF alpha-Receptor

The carboxyl-terminal tail of the PDGF alpha-receptor contains three tyrosine residues, Tyr-988, Tyr-993, and Tyr-1018. Tyr-1018 is conserved in the PDGF beta-receptor as Tyr-1021, which recently was shown to be an autophosphorylation site (11, 13, 14) . To characterize the potential role of the PDGF alpha-receptor carboxyl-terminal tyrosine residues in receptor signaling, we analyzed whether they serve as autophosphorylation sites. PAE cells expressing the wild-type human PDGF alpha-receptor were labeled with [P]orthophosphate and stimulated with PDGF-BB. The PDGF alpha-receptor binds both PDGF-AA and PDGF-BB with high affinity; the same extent of kinase activation and indistinguishable biological activity is seen independent of which PDGF isoform is used for stimulation of the receptor(6) . Cell lysates were subjected to immunoprecipitation with PY20, a phosphotyrosine monoclonal antibody, followed by SDS-PAGE and transfer to nitrocellulose membranes. The band containing the phosphorylated PDGF alpha-receptor was excised from the nitrocellulose sheet and fragmented with CNBr, which is expected to produce a large carboxyl-terminal fragment encompassing residues 981-1072. Precipitation of the digest with AbPalpha1, an antiserum raised against a synthetic peptide corresponding to amino acid residues 993-1009 in the carboxyl-terminal tail of the alpha-receptor, identified a major P-containing band of approximately 10,000, as demonstrated by SDS-PAGE (data not shown). The immunoprecipitated peptide was subjected to Edman degradation, and the P radioactivity in each cycle was measured. The three tyrosine residues, Tyr-988, Tyr-993, and Tyr-1018, are expected to appear at cycles 8, 13, and 34, respectively. As shown in Fig. 1A, increased radioactivity was collected at cycle 8 and not at cycle 13, indicating that Tyr-988, but not Tyr-993, is an autophosphorylation site in the human PDGF alpha-receptor. The decrease in yield with successive cycles during the Edman degradation precluded a conclusion as to whether Tyr-1018 is an autophosphorylation site. Therefore, the isolated carboxyl-terminal peptide was digested with a lysylendopeptidase, A. lyticus protease I, followed by Edman degradation and measurement of P radioactivity in each cycle. After A. lyticus protease I degradation of the carboxyl-terminal CNBr fragment, without further fractionation, radioactivity from Tyr-988 is expected to still appear at cycle 8 and potential radioactivity from Tyr-1018 at cycle 17. As shown in Fig. 1B, radioactive peaks appeared at cycles 5, 8, and 17. Radioactive material in cycle 9 most likely represents material trailing from cycle 8 (compare Fig. 1A and Fig. 2). These data suggest that Tyr-1018 is also an autophosphorylation site in the human PDGF alpha-receptor. Examination of the amino acid sequence of the carboxyl-terminal CNBr fragment indicates that Ser-1041 or Thr-1066 could account for the radioactivity in cycle 5. Thr-1066 appears indeed to be a phosphorylation site in vivo; (^2)no data are available concerning Ser-1041.


Figure 1: Edman degradation of in vivo labeled carboxyl-terminal PDGF alpha-receptor fragments and quantification of P radioactivity in each cycle. A, PAE cells expressing the wild-type alpha-receptor were labeled with [P]orthophosphate, stimulated with PDGF-BB, lysed, and immunoprecipitated with anti-phosphotyrosine monoclonal antibodies. The immunoprecipitates were separated by SDS-PAGE and transferred onto a nitrocellulose filter, and the band corresponding to the phosphorylated receptor was cut out and treated with CNBr. The fragments were immunoprecipitated with AbPalpha1, an antiserum raised against a synthetic peptide located in the carboxyl terminus of the alpha-receptor. B, an aliquot of the purified CNBr peptide was further degraded with A. lyticus protease I, which cleaves carboxyl-terminal of lysine residues. The resulting digest was subjected to Edman degradation followed by measurement of P radioactivity in each cycle. The amino acid sequences of the carboxyl-terminal peptides are presented along with the fraction numbers. Tyr-988 is shown in boldlettertype in A, and Tyr-988, Tyr-1018, Ser-1041, and Thr-1066 are shown in boldletters in B. Quantification of the radioactivity in each cycle was performed by use of a PhosphorImager instrument, as described under ``Materials and Methods.''




Figure 2: Edman degradation of carboxyl-terminal fragments derived from in vitro labeled wild-type and tyrosine residue-mutated PDGF alpha-receptors. PAE cells expressing the wild-type alpha-receptor, or Y988F or Y1018F mutant receptors, were stimulated with PDGF-BB, lysed, and immunoprecipitated with PDGFR-7, an alpha-receptor-specific antiserum. The immunoprecipitates were labeled in vitro using [-P]ATP, separated by SDS-PAGE, and transferred to a nitrocellulose filter; the band corresponding to the phosphorylated receptor was cut out. The samples were treated with CNBr, immunoprecipitated with AbPalpha1 (reactive with the carboxyl-terminal PDGF alpha-receptor tail), and further digested with A. lyticus protease I. The digests were subjected to Edman degradation followed by measurement of P radioactivity in each cycle. The amino acid sequences of the carboxyl-terminal peptides are presented along with the fraction numbers; Tyr-988 and Tyr-1018 are shown in boldletters.



To confirm that Tyr-988 and Tyr-1018 are autophosphorylation sites, we used site-directed mutagenesis to change the residues individually to phenylalanine residues. The mutated receptors were stably expressed in PAE cells (see below), which were stimulated with PDGF-BB, immunoprecipitated with an alpha-receptor-specific antiserum (PDGFR-7), and phosphorylated in vitro. The receptors were purified and fragmented with CNBr, followed by isolation of the carboxyl-terminal peptide, which was further digested with A. lyticus protease I, as described above. Edman degradation was performed on the peptides followed by analysis of P radioactivity in the different fractions. As shown in Fig. 2, radioactivity from the wild-type receptor was detected at cycles 8 and 17. In the Y988F mutant receptor, radioactivity was found at cycle 17 but not at cycle 8, whereas in the Y1018F mutant receptor, radioactivity was detected at cycle 8 but not at cycle 17, as expected. The results demonstrate that Tyr-988 and Tyr-1018 are autophosphorylation sites both in vivo and in vitro.

Characterization of PAE Cells Expressing PDGF alpha-Receptor Mutants Y988F and Y1018F

The mutated PDGF alpha-receptors Y988F and Y1018F, expressed in PAE cells, were identified by binding of I-labeled PDGF-AA and analyzed by metabolic labeling followed by immunoprecipitation with PDGFR-7. Analysis by SDS-PAGE and fluorography revealed 140- and 170-kDa components, which correspond to precursor and mature forms of the PDGF alpha-receptor, respectively (data not shown). Through Scatchard analysis of the specific binding of I-labeled PDGF-AA at 0 °C, the number of binding sites on the Y988F and Y1018F cell lines were estimated to 25,000 and 30,000 per cell, respectively. The number of binding sites on the cell line expressing wild-type alpha-receptors has previously been estimated to 40,000 per cell(6) .

The ability of the wild-type and mutant PDGF alpha-receptor to transduce mitogenic signals was investigated by a [^3H]thymidine incorporation assay. We have previously shown that the wild-type alpha-receptor responds mitogenically to PDGF-AA and PDGF-BB stimulation (6) . As seen in Fig. 3, both wild-type and mutant receptor-expressing cell lines were found to respond, in a similar dose-dependent manner, to PDGF-BB stimulation by increased incorporation of [^3H]thymidine. Non-transfected PAE cells were, as expected, negative. The slight deviation by the mutant receptor cell lines, in response to lower concentrations of PDGF, compared with that of wild-type receptorexpressing cells, was not due to a lower affinity of PDGF (data not shown); it is possible that the differences are related to the slightly lower receptor number on the mutant receptorexpressing cells. Thus, the tyrosine-mutated receptors are able to induce DNA synthesis.


Figure 3: Stimulation of [^3H]thymidine incorporation by PDGF-BB in cells expressing wild-type and tyrosine residue-mutated PDGF alpha-receptors. Serum-starved PAE cells expressing the wild-type alpha-receptor (closedcircles), Y988F (opensquares), Y1018F (closedsquares) mutated receptors, and non-transfected control cells (opencircles) were incubated with [^3H]thymidine at the indicated concentrations of PDGF-BB for 24 h. Trichloroacetic acid-precipitable radioactivity was determined as described under ``Materials and Methods'' and expressed as percent of the incorporation in control cultures.



Tyr-988 and Tyr-1018 Are Involved in the Interaction with PLC-

Through phosphorylation of tyrosine residues outside the kinase domains in the PDGF alpha- and beta-receptors, binding sites for proteins participating in downstream signaling are created (reviewed in (3) ). Autophosphorylation sites in the carboxyl-terminal tails of the PDGF beta-receptor(11, 13, 14) , the epidermal growth factor (EGF) receptor(32, 33, 34) , as well as the fibroblast growth factor (FGF) receptor-1(35) , have been shown to be involved in the association and phosphorylation of PLC-. We explored the possibility that binding and phosphorylation of PLC- in vivo is dependent on autophosphorylation of Tyr-988 and/or Tyr-1018 in the PDGF alpha-receptor. Cells expressing the wild-type or the mutant receptors were exposed to PDGF-AA or PDGF-BB, lysed and immunoprecipitated with PDGFR-7 (Fig. 4A), or an antiserum recognizing PLC- (Fig. 4B). The immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose filters; the blots were probed with the phosphotyrosine monoclonal antibody PY20. As shown in Fig. 4A, PDGF-AA and PDGF-BB induced tyrosine phosphorylation of the wild-type and the two mutant receptors. The extent of tyrosine phosphorylation of the wild-type and mutant receptors was similar, irrespective of which PDGF isoform was used, taking into account the differences in expression levels. Therefore, the mutant receptors are functional kinases in vivo.


Figure 4: Tyrosine phosphorylation and association of PLC- with ligand-activated wild-type and tyrosine residue-mutated PDGF alpha-receptors. PAE cells expressing the wild-type alpha-receptor or Y988F or Y1018F mutated receptors were incubated with or without 100 ng/ml PDGF-AA or PDGF-BB for 1 h at 4 °C. The cells were solubilized, and the lysates were subjected to immunoprecipitation using the receptor antiserum PDGFR-7 (A) or the PLC- antiserum (B). The immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with the phosphotyrosine monoclonal antibody PY20, and the reactions were visualized using the ECL Western blotting detection system. The filter shown in panelB was stripped and reprobed with PLC- antiserum (C) and, after a second stripping, reprobed with PDGF alpha-receptor antiserum (D). Maybe due to the low sensitivity of the PDGF receptor antiserum in immunoblotting, several components appeared to be immunoreactive in the Y1018F sample (rightside of panelD) in a ligand-independent manner. None of these components had a migration rate corresponding to the alpha-receptor and could be regarded as unspecific. The migration positions of the PDGF alpha-receptor and PLC- are indicated, as well as the migration positions of molecular weight standards (myosin, 200,000; phosphorylase b, 97,000), run in parallel. ip, immunoprecipitation.



Stimulation with PDGF-AA and PDGF-BB also induced a low level of tyrosine phosphorylation of PLC- in the wild-type receptor cells and in the Y988F mutant receptor-expressing cells (Fig. 4B). In addition, a 170-kDa component most likely corresponding to the autophosphorylated alpha-receptor was co-immunoprecipitated with PLC- in the wild-type and Y988F mutant receptor-expressing cells. In contrast, the Y1018F mutant receptor failed to associate with or phosphorylate PLC-. Reprobing the filter in Fig. 4B with PLC- antiserum showed similar levels of PLC- in all lanes (Fig. 4C), demonstrating that the difference in tyrosine phosphorylation of PLC- was not due to differences in the amount of PLC- immunoprecipitated from the various cell lines. To show that the component coprecipitating with PLC- indeed was the alpha-receptor, the filter used in Fig. 4, B and C, was stripped again and reprobed with PDGF alpha-receptor antiserum (Fig. 4D). These results indicate that in vivo, the Y988F mutant had reduced capacity to associate with PLC-, whereas the Y1018F mutant had almost completely lost its capacity to associate with PLC-, irrespective of whether PDGF-AA or PDGF-BB were used for stimulation.

To quantify the fraction of phosphorylated wild-type, Y988F, and Y1018F receptors that associate with PLC-, in vitro kinase assays were performed. The different cell lines were stimulated with PDGF-BB, lysed, and immunoprecipitated with PY20 or PLC- antiserum. The immune complexes were then incubated with [-P]ATP and analyzed by SDS-PAGE, followed by autoradiography. As seen in Fig. 5, stimulation with PDGF-BB increased autophosphorylation of the wild-type receptor, as well as receptor-mediated phosphorylation of PLC-. PDGF-BB stimulation of the Y988F and Y1018F mutant receptor-expressing cells induced the kinase activity of the mutant receptors, but the extent of complex formation with PLC- was changed. The total pool of phosphorylated receptors, as well as the pool of phosphorylated receptors co-immunoprecipitated with PLC-, was quantified for each cell line using a PhosphorImager instrument. About 55% of the phosphorylated wild-type receptors were co-immunoprecipitated with PLC- (compare lanes2 and 4). Changing Tyr-988 to Phe reduced the pool of phosphorylated receptors associated with PLC- to 35% of the total phosphorylated receptor pool (compare lanes6 and 8). The association with PLC- to the Y1018F receptor was drastically reduced, since only 3% of these phosphorylated receptors were coprecipitated with PLC- (compare lanes10 and 12). Repeated experiments gave similar figures for the reduction in complex formation between PLC- and the mutant receptors. These data indicate that Tyr-1018 is the major binding site for PLC- and that Tyr-988 could serve as a minor binding site.


Figure 5: Quantification of the association of PLC- to ligand-activated wild-type and tyrosine residue-mutated PDGF alpha-receptors. PAE cells expressing the wild-type alpha-receptor or Y988F or Y1018F mutant receptors were incubated with or without 100 ng/ml PDGF-BB for 1 h at 4 °C. The cells were solubilized, and the lysates were subjected to immunoprecipitation using the phosphotyrosine monoclonal antibody PY20 or the PLC- antiserum. Kinase assays were performed on the immune complexes, and the samples were analyzed by SDS-PAGE. The gel was treated with 1 M KOH at 55 °C for 1 h prior to autoradiography. The 170-kDa alpha-receptor band was quantitated using a PhosphorImager instrument. ippt, immunoprecipitation.



To confirm the involvement of Tyr-988 and Tyr-1018 in PLC- binding, competition experiments were performed. The ability of unphosphorylated and phosphorylated synthetic peptides to compete with receptor binding to the PLC- SH2 domains was tested as follows. PDGF alpha-receptors were labeled with P through stimulation of the PDGF alpha-receptor-expressing cells with PDGF-BB, followed by lysis and immunoprecipitation using PY20. In vitro kinase assay was performed on the immobilized immunoprecipitate in the presence of [-P]ATP. Phosphorylated receptors were eluted with phenylphosphate, and, after desalting, aliquots were incubated with Sepharose-coupled fusion protein composed of glutathione S-transferase linked to a stretch covering the two PLC- SH2 domains. Incubations were performed in the presence and absence of different concentrations of unphosphorylated and phosphorylated synthetic peptides corresponding to residues 980-996 and 1007-1026. After washing the beads, the extent of association between the receptor and the PLC- SH2 fusion protein was estimated by SDS-gel electrophoresis, autoradiography, and quantification using a PhosphorImager (Fig. 6). Fig. 6shows that both types of phosphorylated peptides were able to compete with the intact alpha-receptor for binding to the PLC- SH2 domain fusion protein. The 1018(P) peptide displayed about 10 times higher affinity for the fusion protein compared with the 988(P) peptide. The unphosphorylated peptides were not able to compete to any significant extent.


Figure 6: Competition between synthetic peptides and P-labeled alpha-receptor for binding to PLC- SH2 domains. PAE cells expressing the PDGF alpha-receptor were stimulated with PDGF-BB and lysed, and receptors were immunoprecipitated with phosphotyrosine antibodies, followed by in vitro kinase assay. P-Labeled receptors were eluted with phenylphosphate, and the abilities of the receptors to interact with a bacterial fusion protein containing both PLC- SH2 domains were analyzed in the presence and absence of synthetic unphosphorylated and phosphorylated peptides at the indicated concentrations. The extent of binding of P-labeled alpha-receptor was analyzed by SDS gel electrophoresis and quantitated using a PhosphorImager.



Tyrosine Phosphorylation of PLC- by PDGF alpha- and betaReceptors in Vivo

During earlier characterizations of signal transduction and substrate phosphorylation by PDGF alpha- and beta-receptors, we observed that the degree of tyrosine phosphorylation of a protein, possibly corresponding to PLC-, was different in cells expressing PDGF alpha- and beta-receptors(6) . Therefore, we compared the abilities of the PDGF alpha- and beta-receptors to phosphorylate and interact with PLC-. PAE cells expressing similar levels of either the wild-type alpha-receptor or the wild-type beta-receptor were stimulated with PDGF-BB and lysed. The samples were enriched for glycosylated proteins using wheat germ lectin Sepharose or immunoprecipitated with the PLC- antiserum and then subjected to SDS-PAGE and transfer to nitrocellulose membranes. Probing the filters with PY20 revealed similar levels of PDGF-BB-induced phosphorylation of both the alpha- and beta-receptors (Fig. 7A). As seen in Fig. 7B, the level of tyrosine phosphorylation of PLC- was low in the alpha-receptor-expressing cells and considerably higher in the beta-receptor cells. The components coprecipitating with PLC- in Fig. 7B corresponded to the alpha- and beta-receptor, respectively, as shown previously (see Fig. 4D and (13) ). A greater proportion of phosphorylated alpha-receptor was found to be associated with PLC-, as compared with phosphorylated beta-receptor (Fig. 7B). Reprobing the filters in Fig. 7B with the PLC- antiserum revealed similar levels of PLC- in all lanes (Fig. 7C). These data indicate that although the major binding site for PLC- is conserved between the PDGF alpha- and beta-receptors, there is a difference between the two receptors in their ability to phosphorylate PLC-.


Figure 7: Abilities of the wild-type PDGF alpha- and beta-receptors interact with and phosphorylate PLC-. Cultures of PAE cells expressing either alpha- or beta-receptors were incubated with or without 100 ng/ml PDGF-BB for 1 h at 4 °C. The cells were solubilized, and a glycoprotein fraction was collected using wheat germ agglutinin Sepharose 6B (A) or the lysates were immunoprecipitated by the anti-PLC- antiserum (B). The samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with the phosphotyrosine monoclonal antibody PY20, and the reactions were visualized using the ECL Western blotting detection system. The filter shown in panelB was stripped and reprobed with PLC- antiserum (C) as a control for equal loading. The migration positions of the PDGF receptors and PLC- are indicated, as well as the migration positions of molecular weight standards (myosin, 200,000; phosphorylase b, 97,000) run in parallel. The molecular nature of the band around 130 kDa, seen in panelA for samples derived from the PDGF alpha-receptor cells, is unknown.



Inositol Phosphate Production by PDGF alpha- and beta-Receptors

Phosphorylation of PLC- has been reported to enhance its catalytic activity, seen as production of increased levels of inositol phosphates(36) . To analyze how the differences in extents of PLC- phosphorylation by the alpha- or beta-receptors correlated with activation of PLC-, we measured PDGF-induced formation of inositol phosphates in the transfected cell lines. PDGF alpha- or beta-receptor-expressing PAE cells were labeled with myo-[^3H]inositol, stimulated with PDGF-AA, PDGF-BB, or 10% FCS for 30 min at 37 °C, and quenched in acidified methanol. The samples were extracted to separate phospholipids from the inositol phosphate pool, as described under ``Materials and Methods,'' and the inositol phosphates were eluted using AG 1-X8 formate resin. PDGF-AA and PDGF-BB induced elevation of total inositol phosphates in the alpha-receptor-expressing cells 1.08 (±0.02)- and 1.16 (±0.03)-fold, respectively, while the increase in the PDGF beta-receptor cell line in response to PDGF-BB was 4.45 (±0.16)-fold. Thus, the level of production of inositol phosphate in the alpha- and beta-receptor-expressing cells (Fig. 8) correlated with the different abilities of the receptors to tyrosine-phosphorylate PLC- (Fig. 7B).


Figure 8: Formation of inositol phosphates (InsP) in PAE cells transfected with both alpha- and beta-receptors (Palphabeta:1) or human fibroblasts (AG 1523). Cultures pre-labeled with 1 µCi/ml myo-[^3H]inositol for 48 h under serum-free conditions were incubated in the presence of vehicle (openbars), 50 ng/ml PDGF-AA (stripedbars), 50 ng/ml PDGF-BB (solidbars), or 10% FCS (shadedbars) for 30 min at 37 °C. The samples were quenched by addition of acidified methanol, and the water-soluble and lipid fractions separated. The inositol phosphates were separated by anion-exchange chromatography, and the total amount of cpm eluted as inositol phosphate (InsP), inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were related to the amount of radioactivity in the corresponding lipid fraction in 10^6 cells. -Fold stimulation under the different conditions are shown, with the basal level in unstimulated cells for each cell line set to 1. The values represent means ± S.E. from two experiments with triplicate samples.



To investigate whether the alpha-receptor requires the presence of the beta-receptor to mediate a release of inositol trisphosphates, we used PAE cells co-expressing both receptor types (Palphabeta:1, (18) ), as well as human fibroblasts, AG 1523, in which both receptor types are expressed. In both cell types, a consistent response was seen, with a limited accumulation of inositol phosphate after stimulation with PDGF-AA, which activates the alpha-receptor, as compared with the levels seen after stimulation with PDGF-BB, which activates both the alpha- and the beta-receptor (Fig. 8). The magnitude of the response seen after stimulation with PDGF-BB was lower in Palphabeta:1 cells than in AG 1523, probably due to the fact that the Palphabeta:1 cells express about 10,000 beta-receptors per cell, whereas AG 1523 express about 100,000 beta-receptors per cell.


DISCUSSION

We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, near the carboxyl terminus of the human PDGF alpha-receptor, which are phosphorylated both in vivo and in vitro. These residues serve as binding sites for PLC-, as shown by the reduced levels (Y988F mutant receptor), or close to complete loss (Y1018F mutant receptor) of complex formation and tyrosine phosphorylation of PLC- in the PDGF-stimulated mutant receptor cell lines in vivo. The mutant receptors are functional kinases in vivo (see Fig. 4A) and are as efficient as the wild-type alpha-receptor in their interactions with other SH2 domain-containing proteins. (^3)Synthetic peptides encompassing Tyr-988(P) or Tyr-1018(P) were able to compete with the intact receptor for binding to the PLC- SH2 domains; the Tyr-988(P) peptide competed with considerably lower efficiency. Crystallographic analysis of the carboxyl-terminal SH2 domain of PLC- in complex with a peptide from the PLC- binding site in the PDGF beta-receptor (containing Tyr-1021), showed that the phosphotyrosine and the following six residues make specific contact with the SH2 domain(37) . The amino acid sequences following Tyr-1018 in the PDGF alpha-receptor and Tyr-1021 in the beta-receptor are identical (Table 1). PLC- binding sites in other receptor tyrosine kinases, such as the FGF receptor-1(38, 39) , the hepatocyte growth factor receptor(40) , the nerve growth factor receptor(41) , and the low affinity binding sites in the PDGF alpha- and beta-receptors (Tyr-988 and Tyr-1009, respectively; see Table 1), are also related and consist mostly of hydrophobic residues, and proline residues are often found in position +5 or +6. The EGF receptor carboxyl-terminal tail has been shown to contain both high affinity binding (Tyr-992) and low affinity binding (Tyr-1068) binding sites(33) . The association of PLC-(1) with the EGF receptor, however, is complex as mutagenesis of individual autophosphorylation sites suggests that other autophosphorylation sites may compensate for the loss of primary sites (42) . Interestingly, the low affinity binding sites in the PDGF receptors appear to be structurally related to the binding sites in the hepatocyte growth factor and nerve growth factor receptors, since all have hydrophobic residues (T, I, V, or L) in position +1 and V in position +3 carboxyl-terminal of the phosphorylated tyrosine. Since the contribution of the alpha-receptor Tyr-988 autophosphorylation site in PLC- binding is relatively small, the role of this site in PLC- activation is unclear. It is possible that also other SH2 domain-containing proteins bind to this site. However, the tyrosine phosphatase SH-PTP ID/SH-PTP 2/Syp, which binds to Tyr-1009 in the PDGF beta-receptor(14) , does not appear to bind with high efficiency to Tyr-988 in the alpha-receptor (not shown). (^4)



PLC- catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate, two second messengers involved in the activation of protein kinase C and in the release of Ca from internal cellular stores, respectively (reviewed in (43) ). PLC- was recently implicated in mitogenic signal transduction, since restoration of its binding site in a PDGF beta-receptor mutated at multiple tyrosine residues was accompanied by a restoration of the mitogenic signaling capacity of the mutant receptor(44) . However, we show that mutant PDGF alpha-receptors, in which either Tyr-988 or Tyr-1018 were replaced with phenylalanine residues, mediated an increased DNA synthesis in response to PDGF-AA, comparable with that for the wild-type receptor. This is in agreement with a report by Hill et al.(45) who observed increased DNA synthesis in the absence of PLC- activation after treatment of PDGF-stimulated murine fibroblasts with genistein. Intact mitogenic signaling was also reported for mutants of PDGF beta-receptors (13) and FGF receptors(38, 39) , which were unable to mediate PLC- binding and phosphorylation. The recently characterized signal transduction pathway involving the SH2 domain-containing protein Grb2, which couples receptor tyrosine kinases to Ras, is believed to represent a major mitogenic pathway(46, 47) . It is possible that multiple parallel mitogenic pathways exist in the cell and that PLC- is part of one such pathway.

PLC- becomes phosphorylated on tyrosine residues in response to PDGF or EGF stimulation(48, 49) . Tyrosine phosphorylation has been shown to increase the enzymatic activity of PLC- (36) and to reduce its binding to the activated epidermal growth factor receptor(50) . Our results indicate that the interaction of PLC- with the PDGF alpha- and beta-receptors are qualitatively different. A stable complex between PLC- and the autophosphorylated alpha-receptor could readily be detected, but the degree of PLC- phosphorylation was low. In contrast to this, and in agreement with results from previous studies (11, 13) , only a small fraction of the activated beta-receptor pool occurred in complex with PLC- at each single moment. However, efficient phosphorylation of PLC- on tyrosine residues was produced by the beta-receptor. Most likely, the phosphorylated and activated PLC- was then released from the receptor complex. As a consequence, there was a higher production of inositol phosphates in the beta-receptor-expressing cells than in the alpha-receptor-expressing cells. Higher production of inositol phosphates in response to PDGF-BB (which binds to both alpha- and beta-receptors) than to PDGF-AA (which binds only to alpha-receptors) was also recorded for PAE cells co-expressing both receptor types after transfection and for human foreskin fibroblasts, AG 1523, which endogenously express the two PDGF receptor types. When 32D hematopoietic cells expressing alpha- or beta-receptors were compared, the receptors induced similar extents of phosphorylation of PLC-(51) , and the stimulation of inositol phosphate production was equally efficient for the two receptors(52) . Swiss 3T3 cells endogenously expressing both PDGF receptor types also respond equally well to PDGF-AA and -BB in the formation of inositol phosphates(53) . On the other hand, vascular smooth muscle cells, which express PDGF alpha- and beta-receptors, accumulate inositol phosphate in response to PDGF-BB but not PDGF-AA(54) . In agreement, different populations of vascular smooth muscle cells examined for PDGF-induced Ca fluxes were either unresponsive or only partially responsive to treatment with PDGF-AA, as compared with PDGF-BB(55) . In this work, we provide an explanation for the molecular mechanisms underlying these observations, since the efficiency with which PDGF alpha-receptor mediates tyrosine phosphorylation of PLC-, and consequently, inositol phosphate production, is considerably lower than that of the PDGF beta-receptor.


FOOTNOTES

*
This work was supported by the Swedish Natural Science Research Council, Magn. Bergvall Fund, and by National Institutes of Health Grant CA43720. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Immunex Research and Development Corp., 51 University St., Seattle, WA 98101.

To whom correspondence should be addressed. Tel.: 46-18-551688; Fax: 46-18-506867.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; CNBr, cyanogen bromide; EGF, epidermal growth factor; FGF, fibroblast growth factor; PAE cells, porcine aortic endothelial cells; PLC-, phospholipase C-; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

(^2)
L. Rönnstrand, unpublished data.

(^3)
K. Yokote, C.-H. Heldin, and L. Claesson-Welsh, manuscript in preparation.

(^4)
A. Eriksson, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Joseph Schlessinger and Dr. Tony Pawson for generous gifts of PLC- SH2 domain fusion proteins, Christer Wernstedt for skillfully performing the amino acid sequence analysis, and Ingegärd Schiller for valuable assistance in preparation of the manuscript.


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