125I-FGF-1 binding to recombinant receptors expressed on. 2.5 x 104 insect ... baculovirus bearing cDNAs coding for FGF-R cDNA vari- ants were prepared ...
Vol. 13, No. 7
MOLECULAR AND CELLULAR BIOLOGY, July 1993, p. 3907-3918 0270-7306/93/073907-12$02.00/0 Copyright © 1993, American Society for Microbiology
Control of Fibroblast Growth Factor Receptor Kinase Signal Transduction by Heterodimerization of Combinatorial Splice Variants ERGANG SHI,t MIKIO KAN, JIANMING XU, FEN WANG, JINZHAO HOU, AND WALLACE L. McKEEHAN* W. Alton Jones Cell Science Center, Inc., 10 Old Barn Road, Lake Placid, New York 12946 Received 4 January 1993/Returned for modification 10 February 1993/Accepted 9 April 1993 A differentiated liver cell (HepG2), which exhibits a dose-dependent growth-stimulatory and growthinhibitory response to heparin-binding fibroblast growth factor type 1 (FGF-1), displays high- and low-affinity receptor phenotypes and expresses specific combinatorial splice variants al, f1, and a2 of the FGF receptor (FGF-R) gene (flg). The extracellular domains of the a and 13 variants consist of three and two immunoglobulin loops, respectively, while the intracellular variants consist of a tyrosine kinase (type 1) isoform and a kinase-defective (type 2) isoform. The type 2 isoform is also devoid of the two major intracellular yrosine autophosphorylation sites (Tyr-653 and Tyr-766) in the type 1 kinase. An analysis of ligand affinity, dimerization, autophosphorylation, and interaction with src homology region 2 (SH2) substrates of the recombinant ael, 01, and a2 isoforms was carried out to determine whether dimerization of the combinatorial splice variants might explain the dose-dependent opposite mitogenic effects of FGF. Scatchard analysis indicated that the a and 13 isoforms exhibit low and high affinity for ligand, respectively. The three combinatorial splice variants dimerized in all combinations. FGF enhanced dimerization and kinase activity, as assessed by receptor autophosphorylation. Phosphopeptide analysis revealed that phosphorylation of Tyr-653 was reduced relative to phosphorylation of Tyr-766 in the type 1 kinase component of heterodimers of the type 1 and type 2 isoforms. The SH2 domain substrate, phospholipase C-yl (PLCOyl), associated with the phosphorylated type 1-type 2 heterodimers but was phosphorylated only in preparations containing the type 1 kinase homodimer. The results suggest that phosphorylation of Tyr-653 within the kinase catalytic domain, but not Tyr-766 in the COOH-terminal domain, may be stringently dependent on a trans intermolecular mechanism within FGF-R kinase homodimers. Although phosphotyrosine 766 is sufficient for interaction of PLC-yl and other SH2 substrates with the FGF-R kinase, phosphorylation and presumably activation of substrates require the kinase homodimer and phosphorylation of Tyr-653. We propose that complexes of phosphotmsine 766 kinase monomers and SH2 domain signal transducers may constitute unactivated presignal complexes whose active or inactive fate depends on homodimerization with a kinase or heterodimerization with a kinase-defective monomer, respectively. The results suggest a mechanism for control of signal transduction by different concentrations of ligand through heterodimerization of combinatorial splice variants from the same receptor gene.
taining src homology region 2 (SH2) domains (4, 15, 16, 28, 30, 43). Alternate splicing of an exon coding for an NH2terminal immunoglobulin-like disulfide loop results in isoforms in the extracellular domain of FGF-R composed of three (a isoform) and two (1 isoform) immunoglobulin loops (17). Although the two immunoglobulin loops that compose the 1 isoform are qualitatively sufficient for ligand binding, the NH2-terminal loop of the three-loop a isoform is interactive with the two-loop ligand-binding site (47). In liver cells which exhibit the inhibitory response to FGF-1, an alternate donor site splice results in a kinase-defective COOH-terminal isoform (type 2) that is devoid of the complete catalytic domain and the two major intracellular tyrosine autophosphorylation sites (Tyr-653 and Tyr-766) of the intact type 1 kinase (17, 18). Phosphotyrosine 766 is required for interaction of the FGF-R kinase with the SH2 domain signal transduction substrate, phospholipase C-yl (PLC-yl) (32, 33, 35). The role of phosphotyrosine 653 and phosphotyrosine 766 in receptor kinase activity and phosphorylation is unclear. The lack of kinase activity and phosphorylation sites in the type 2 intracellular domain linked to dimerization-competent variant extracellular domains predicts that the type 2 variant potentially mediates
Heparin-binding fibroblast growth factors (FGF) elicit dose-dependent growth-stimulatory and growth-inhibitory effects in cells which display high- and low-affinity receptor sites (23, 24). The FGF receptor (FGF-R) tyrosine kinase family consists of splice variants in the extracellular ligandbinding domains which combine with splice variants in the intracellular kinase, autophosphorylation, and substrate interaction domains (17). Transmembrane tyrosine kinase receptors are activated by a ligand-induced stabilization of dimers which occurs through the receptor extracellular domain (15, 44, 46, 50, 51). Kinase-defective mutants in the intracellular domain act as dominant negative suppressors of ligand-induced biological activities when coexpressed with wild-type receptors (1, 28, 42, 44, 45). This is thought to result from competition with the formation of kinase homodimers and prevention of the intermolecular trans phosphorylation of tyrosines within monomeric units of kinase homodimers which is obligatory for binding, phosphorylation, and activation of signal transduction substrates con*
Corresponding author.
t Present address: Department of Microbiology and Immunol-
ogy, Vanderbilt University, Nashville, TN 37232.
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the growth-inhibitory role of FGF through heterodimerization with the type 1 FGF-R kinase. The purpose of this study was to determine whether dimerization of monomers arising from regulated combinatorial splicing of coding sequences for the extracellular domain and the intracellular kinase domain from a single FGF-R gene might explain the concentration-dependent effects of a single FGF ligand on growth stimulation and growth inhibition in a single cell type. MATERIALS AND METHODS Materials. Constructs encoding human FGF-R variants and development and documentation of specificity of antibodies have been described in detail elsewhere (17, 47). Throughout this study, FGF-R refers to products of the human FGF-R gene, flg, which has been designated the FGF-R1 gene to distinguish it from the other three genes of the FGF-R family, FGF-R2 (bek), FGF-R3, and FGF-R4 (19). In this study, the a and 1B suffixes refer to the three- and two-immunoglobulin loop extracellular domain splice variants, while the numerical suffixes 1 and 2 refer to the kinase and kinase-defective intracellular domain splice variants. Native bovine heparin-binding FGF type 1 (FGF-1), previously called HBGF-1 or acidic FGF, was isolated as previously described (25). Grace's insect cell culture medium (modified) was from GIBCO (Grand Island, N.Y.). Fetal bovine serum and antibodies against PLCy1, phosphoinositol-3-kinase subunit (PI3Kp85), and ras-GTPase activating protein (ras-GAP) were from Upstate Biotechnology, Inc. (Lake Placid, N.Y.). [y-32P]adenosine 5'-triphosphate (3,000 Ci/mmol) was from DuPont (Wilmington, Del.). Triton X-100 was from Boehringer Mannheim (Germany). Protein A beads (Sepharose Cl-4B) were from Pharmacia/LKB Biotechnology (Uppsala, Sweden). Affi-gel 10 and 15 were from Bio-Rad Laboratories (Richmond, Calif.). Alkaline phosphatase-conjugated or horseradish peroxidase affinitypurified goat anti-rabbit immunoglobulin G and goat antimouse immunoglobulin G were from Organon Teknika Corp. (West Chester, Pa.) and Bio-Rad, respectively. Enhanced chemiluminescence Western blotting (immunoblotting) reagents were from Amersham International (Amersham, United Kingdom). Nitrocellulose nucleic acid and protein transfer membranes (pore size, 0.45 ,um) were from Schleicher & Schuell (Keene, N.H.). Dimethyl pimelimidate and disuccininidyl suberate were from Pierce (Rockford, Ill.). Molecular weight protein standards and prestained molecular weight protein standards were from Bio-Rad and Bethesda Research Laboratories (Gaithersburg, Md.), respectively. Sodium orthovanadate (Na3VO4), magnesium chloride (MgCl2), and manganese chloride (MnCl2) were from Sigma Chemical Co. (St. Louis, Mo.). Recombinant magnesium-dependent phosphotyrosine phosphatase (PTPase) (13) was obtained as a gift from M. Gu and P. W. Majerus (Washington University School of Medicine). Binding and cross-linking of 1251-FGF-1. The binding and covalent cross-linking of 1"I-FGF-1 to mammalian cells was performed and analyzed as previously described (23-25). Unless otherwise indicated, the heparin-to-FGF-1 ratio was 1,000 to 1 (wt/wt) in binding assays. 125I-FGF-1 binding to recombinant receptors expressed on 2.5 x 104 insect cells was determined 60 h after infection with recombinant baculovirus. Cells were collected and diluted in 1.7 ml of binding assay buffer in Eppendorf tubes. The binding was carried out in suspension at room temperature for 90 min by gently rolling the tubes. Cells from
MOL. CELL. BIOL.
binding assays were collected on a Millititer 96-well filtration plate (Millipore Corp., Bedford, Mass.) (5-,um pore size) previously blocked with bovine serum albumin (BSA) (10 mg/ml). After three washes with phosphate-buffered saline (PBS; pH 7.0), one wash with PBS containing heparin (250 ,ug/ml), and then two washes with PBS, the filters were air dried and counted. Similar levels of nonspecific binding resulted from noninfected Sf9 cells, from Sf9 cells infected with an unrelated recombinant virus, or by addition of a 100-fold excess of unlabeled FGF-1. Expression of recombinant FGF-R variants. Recombinant baculovirus bearing cDNAs coding for FGF-R cDNA variants were prepared, identified, and purified with Spodoptera frugiperda (Sf9) cell hosts (47). Each recombinant virus stock was titrated by end point dilution (41) and by level of expression of antigen determined by protein immunoblot with a common antibody against the FGF-R extracellular domain. FGF-R cDNAs in the P91023B vector were transiently transfected into monkey kidney cells (COS-1) by the DEAEdextran method as previously described (17). Antibodies and immunochemical analysis. COOH-terminal isoform-specific rabbit polyclonal antibodies Rl and R2 were prepared against synthetic polypeptides corresponding to residues E-799 to K-820 (E22K) of the type 1 full-length kinase and R-631 to R-650 (R20L) corresponding to the type 2 kinase-defective isoforms, respectively (47). Domain-specific mouse monoclonal and rabbit polyclonal antibodies were prepared against bacterium-derived recombinant extracellular domains of FGF-R (47). Unless otherwise indicated, the specificity and titer of antibodies, ratios of antibodies and protein A beads per cells extracted, and amounts of secondary antibodies used for immunoblot and immunoprecipitation analyses were those previously described (47). Primary rabbit or mouse antibodies on immunoblots were visualized with second antibodies conjugated to alkaline phosphatase or horseradish peroxidase (47). 125I-FGF-1-labeled transmembrane receptor species from HepG2 cells and transfected COS-1 cells were analyzed as follows: COOH terminus-specific rabbit polyclonal antibodies Rl and R2 were affinity purified on polypeptide antigen columns (14). Affinity-purified antibodies were then bound and covalently linked to protein A beads with dimethyl pimelimidate (38). 125I-FGF-1-labeled receptor species were extracted with PBS containing 1% Triton X-100 and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride and 1 ,ug [each] of pepstatin A and leupeptin per ml) (47). The detergent-soluble fraction was incubated with immobilized affinity-purified Rl antibody at 4°C for a minimum of 4 h. After a wash with PBS, the bound species was eluted with 5 ml of 0.1 M glycine-HCl (pH 2.5) plus 0.1% Triton X-100, and the eluates were immediately neutralized. The eluates were then concentrated and dialyzed against PBS containing 0.2% Triton X-100 and the protease inhibitors. The dialyzed sample was then divided into two portions. One portion was mixed with an equal volume of 2x concentrated sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiled for 3 min. The other portion of the sample was incubated with monoclonal antibody 2F12 and protein A beads at 4°C for 4 h. After a wash with PBS containing 0.35 M NaCl, the immunoprecipitates were extracted with the 2x SDS-PAGE sample buffer for 3 min. 1"I-FGF-1-labeled material from both the first- and second-step immunoprecipitations was analyzed by SDS7.5% PAGE and autoradiography. Preparation and analysis of FGF-R heterodimers. 1251
VOL. 13, 1993
FGF-1-labeled heterodimers in transiently transfected COS-1 cells were analyzed as described above. Recombinant heterodimers were prepared and isolated from Sf9 cells coinfected with recombinant virus coding for the 13 kinase- and a2 kinase-negative isoforms at ratios of a2 virus to 13 virus in PFU ranging from 0 to 25. For each coinfection, the input of ,11 or al virus was performed at 3 x 108 PFU/3 x 106 cells and the amount of a2 virus indicated in the text. After infection at 25°C for 1 h, the viruscontaining medium was replaced with medium containing 5% fetal bovine serum, and the infected cells were incubated for an additional 60 to 65 h at 27°C. After incubation with FGF-1 (10 ng/ml) and heparin (25 ,ug/ml), cells were then collected by centrifugation and lysed with 100 ,ul of lysis buffer (30 mM HEPES [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; pH 7.4], 0.15 M NaCl, 5% glycine, 2 mM EDTA, 5 mM MgCl2, 1% Triton X-100, protease inhibitors) per 3 x 106 cells for 10 min. The detergent-soluble fraction was transferred into Eppendorf tubes containing 0.5 ,1 of mouse ascites fluid containing monoclonal antibody 2F12 and 20 ,ug of protein A beads. After being shaken for 4 h at 4°C, the immune complexes were washed three times with lysis buffer and then extracted by boiling for 3 min with 30 pl of 2x SDS-PAGE sample buffer. The samples were separated by SDS-7.5% PAGE and electroblotted onto nitrocellulose paper. Duplicate blots were stained with COOH terminus antibody Rl to detect the coprecipitated ,13 kinase or monoclonal antibody 2F12 to detect the a2 antigen. al-a2 heterodimers were similarly prepared and analyzed by coinfection of Sf9 cells with the two respective recombinant viruses. COOH-terminal antibody R2 was used to coprecipitate the al isoform complexed to immobilized a2. Phosphorylation of immobilized FGF-R isoforms. Immobilized ,1l-a2 and al-a2 heterodimeric complexes were prepared as described above. Where indicated, single 131 and al isoforms were prepared from detergent extracts of Sf9 cells infected with a single virus and immobilized with the common FGF-R rabbit polyclonal antibody A40 or a-specific monoclonal antibody 2F12, respectively (47). The immobilized immune complexes were washed three times with extraction buffer and three times with phosphorylation buffer (30 mM HEPES [pH 7.4], 0.15 M NaCl, 5% glycerol, 0.2% Triton X-100, 100 ,uM sodium vanadate, 12 mM MgCl2, 2 mM MnCl2, protease inhibitors) prior to addition of phosphorylation buffer. After incubation with FGF-1-heparin as indicated in the text, phosphorylation reactions in a total volume of 0.25 p.l were started by the addition of [-y-32P]ATP (10 ,uCi) and continued for 5 min on ice. Reactions were stopped by the addition of 10 p.1 of 4x SDS-PAGE sample buffer, and the samples were immediately boiled for 3 min. The amount of kinase receptor antigen in each sample was calibrated on immunoblot with COOH-terminal antibody Rl. Phosphopeptide analysis. The phosphorylated receptor kinase isoform from immunoprecipitates incubated in the phosphorylation conditions described above was excised from the electroblot and subjected to reduction, alkylation, and digestion with trypsin according to described procedures (18, 40). The resultant tryptic peptides were separated on a narrow-bore C18 reverse-phase high-performance liquid chromatography (HPLC) column (2.1 by 250 mm) as described here and elsewhere (18). Association and phosphorylation of PLC-yl, PI3Kp85, and ras-GAP to immobilized FGF-R isoforms. Immunoprecipitates of the 11-a2 heterodimer and 13 kinase alone were prepared from Sf9 cells coinfected with optimum ratios of ,13 and a2 virus or 131 virus alone, respectively, as described
FGF-R
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above. Immunoprecipitates were washed three times with extraction buffer and three times with phosphorylation buffer and then incubated with FGF-1 (200 ng/ml) and heparin (25 ,ug/ml) at room temperature for 20 min. ATP was added to each sample to a concentration of 2 mM, and incubation continued at room temperature for 10 to 30 min. The samples were then washed three times with PBS and three times with the extraction buffer. HepG2 cell lysate was prepared by extraction of cell monolayers with the phosphorylation buffer. Freshly prepared lysate from 5 x 106 HepG2 cells was then added to each sample, and incubation was continued at 10°C for 1 h. The samples were then washed three times with extraction buffer and three times with phosphorylation buffer. Where indicated, the immunoprecipitates were treated with PTPase prior to substrate binding and phosphorylation. After three washes with the extraction buffer and one with phosphatase buffer (10 mM imidazoleHCI [pH 7.2], 0.1% BSA, 0.1% 13-mercaptoethanol), PTPase (100 ng) (13) was added into each sample of 50 p.l (total volume), and incubation was carried out at room temperature for 30 min. The samples were then washed three times with extraction buffer prior to continuation of the analysis. For autoradiography, a second phosphorylation reaction was initiated by adding [_y-32P]ATP (20 ,uCi) to each sample (total volume, 40 p.l), and incubation was continued on ice for 10 min. Reactions were terminated by extracting the samples with 2 x SDS-PAGE sample buffer and boiling them for 3 min prior to analysis by immunoblot and autoradiography. RESULTS Specific combinatorial splice variants of FGF-R are expressed in human hepatoblastoma HepG2 cells. Human hepatoblastoma HepG2 cells, which exhibit a dose-dependent growth-stimulatory and growth-inhibitory response to FGF-1 and both high- and low-affinity FGF-1 receptors (23), expressed mRNAs coding for the three-immunoglobulin loop a and two-loop 1 extracellular domains and kinase (type 1) and kinase-defective (type 2) intracellular domains of FGF-R (17). The expression of combinatorial variants of the a and 1 extracellular domain isoforms and the type 1 and type 2 intracellular domains was analyzed by using a monoclonal antibody, 2F12, against the unique NH2-terminal immunoglobulin loop of the at extracellular domain and polyclonal antibodies Rl and R2 against synthetic polypeptide sequences in the distinct COOH termini of the type 1 and the type 2 species, respectively (Fig. 1A). 125I-FGF-1 was bound and affinity cross-linked to and extracted from HepG2 cells, applied to and eluted from immobilized Rl and R2 antibodies, and then analyzed by SDS-PAGE and autoradiography. A single 135-kDa "1I-FGF-1-labeled species with apparent molecular mass corresponding to that of the three-immunoglobulin loop kinase-negative receptor isoform a2 bound to the immobilized R2 antibody (Fig. 1B, lane 1). A 115-kDa labeled species indicative of the presence of the 12 isoform could not be detected. In contrast, 165- and 135-kDa bands corresponding to apparent molecular masses of the three-loop kinase al and the two-loop kinase 11 (lane 2) were detected with the immobilized Rl antibody. Secondary immunoprecipitation with a-specific antibody 2F12 confirmed that the 135 (lane 3)- and 165 (lane 4)-kDa species from the immobilized R2 and Rl antibodies, respectively, were the three-loop a isoforms. The same analysis applied to a human hepatoma cell line, Hep3B, which exhibits only a positive growth response to FGF-1 (23), revealed the pres-
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FIG. 1. Analysis of combinatorial transmembrane FGF-R splice variants. (A) Schematic of FGF-R isoforms expressed in HepG2 cells. The hydrophobic signal sequence (SS), a characteristic sequence rich in acidic residues (the acidic box), the transmembrane domain (TM), and the intracellular tyrosine kinase domain split by a 14-amino-acid interkinase sequence are indicated. Immunoglobulinlike loops with putative disulfides and the two intracellular tyrosine autophosphorylation sites are indicated. Epitopes for antibodies described in the text are indicated. The epitope for a-specific monoclonal antibody 2F12 has been mapped to within a 19-residue sequence in the NH2 terminus of the unique a immunoglobulin loop (47). The estimated molecular weights of the mature transmembrane glycosylated isoforms from mammalian (M) and Sf9 insect cells (I) (47) are indicated. Differences are due to extent of glycosylation. (B) Immunochemical analysis of FGF-R isoforms expressed in HepG2 and Hep3B cells. Detergent-solubilized 125I-FGF-1-labeled receptor species from HepG2 cells were first concentrated with immobilized COOH-terminal specific antibody Rl (lanes 2, 4, 6, and 7) or R2 (lanes 1, 3, and 5). A portion of the eluate from each primary antibody was reacted with immobilized a-specific monoclonal antibody 2F12 (lanes 3, 4, and 7). Samples were analyzed by SDSPAGE and autoradiography. Lanes 1 and 2, primary eluates from immobilized R2 and Rl antibodies, respectively; lanes 3 and 4, secondary immunoprecipitates with antibody 2F12 of the samples in lanes 1 and 2, respectively; lanes 5, 6, and 7, the same analyses as lane 1, 2, and 4, respectively, of labeled receptor species from human hepatoma Hep3B cells. (C) Expression of a and a mRNA in different cell types. Expression was analyzed by nuclease protection (49) with a 169-base PstI fragment of human FGF-Ra cDNA. The 2P-labeled antisense probe contained 66 bases of the a immunoglobulin loop coding sequence and shared 103 bases of coding sequence shared by FGF-Ra and FGF-R1. The following RNAs were analyzed: lane 1, HepG2 cells (5 pug); lane 2, human foreskin fibroblasts (50 pg); lane 3, human umbilical vein endothelial cells (50 pg); lane 4, tRNA (50 kg). ence of only the 135-kDa 131 (lane 6) and 165-kDa al (lane 7) kinase species. Type 2 kinase-defective isoforms at 145 or 115 kDa (lane 5) could not be detected. The results demonstrate that alternate splicing of coding sequences for the NH2-terminal extracellular domain and the COOH-terminal intracellular kinase domains of FGF-R is coordinated and cell type specific. In HepG2 cells, both the a and extracellular domains combined with the type 1 kinase; only the a extracellular domain combined with the kinase-defective
type 2 domain, while the combination of the ,B extracellular domain with the type 2 kinase-defective isoform was excluded. Expression of the kinase-defective intracellular domain splice variant a2 in HepG2 cells correlates with the high dose-dependent inhibitory effect of FGF-1 on HepG2 cell growth which is not apparent in the Hep3B cells (23). The correlation of expression of the a extracellular domain with display of low-affinity receptor sites was confirmed at the mRNA level by nuclease protection analysis of coding sequences for the NH2 terminus of the extracellular domain of FGF-R (Fig. 1C). HepG2 cells (lane 1) exhibited a high ratio of expression of the a isoform to the 1 isoform, while human endothelial cells and fibroblasts which exhibit predominately high-affinity receptor sites (27) expressed predominately the 1 isoform of mRNA (lanes 2 and 3). Alternate splicing generates low-affinity three-immunoglobulin loop (a) and high-affinity two-immunoglobulin loop (1) isoforms of FGF-R. To determine whether the individual FGF-Rao and -1 isoforms exhibited differences in ligand affinity independent of endogenous host FGF-R isoforms, recombinant baculovirus bearing cDNAs for the FGF-Ra2, -al, and -13 isoforms were prepared and infected into Sf9 insect cells (41, 47). In contrast to mammalian cells in which an undetermined combination of over 100 different combinatorial splice variants of the four FGF-R genes may be expressed and with which a transfected isoform may interact, the Sf9 insect cell hosts exhibited negligible levels of endogenous FGF-R and hosted expression levels of individual isoforms which approached 0.5% of cellular proteins (47). Except for a reduction in extent of glycosylation, the insect cell-derived human receptor isoforms were accurately processed and transported to the cell membrane and exhibited ligand binding, kinase, and autophosphorylation activities predicted from their structure and immunochemical properties similar to those of the receptor isoforms expressed in mammalian cells (47). In addition, the same isoforms retained the same activities in vitro after extraction and purification to homogeneity by immunoaffinity chromatography (47). Viral titers and infection times (45 to 60 h) that resulted in optimal expression of each recombinant receptor isoform on the cell surface of infected Sf9 cells as indicated by FGF-1 binding and immunoassay was established. Scatchard analyses of 125I-FGF-1 binding to the infected Sf9 cells demonstrated that both the a2 and al isoforms exhibited similar affinities for FGF-1, with apparent Kd of 200 pM, while the 11 isoform exhibited an apparent Kd of about 25 pM (Fig. 2A). The difference in affinities between the a and extracellular domains was magnified in absence of exogenous heparin, which substitutes for native heparan sulfates (Fig. 2B). Heparin or native heparan sulfate proteoglycans are an obligatory and integral component of the FGF-R complex (26). In the Sf9 cell hosts, exogenous heparin was required for FGF-1 binding to the recombinant FGF-Ral isoform (lower panel), while the 131 isoform exhibited a reduced, but detectable, apparent Kd of 260 pM in the absence of heparin (upper panel). Dimerization and autophosphorylation of the FGF-Ral, -131, and -ca2 isoforms. cDNAs coding for FGF-Ral, -a2, and -131 were transiently transfected into monkey kidney COS-1 cells, and 1251I-FGF-1 binding and covalent affinity cross-linking were performed. Immunoprecipitation with domain-specific antibodies and subsequent SDS-PAGE and autoradiographic analysis revealed ligand-labeled species corresponding to the apparent molecular mass of homodimers of each transfected receptor isoform (Fig. 3,
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closed triangles) in addition to the monomeric species (open triangles). Heterodimers between the kinase-negative oa2 and the kinase isoforms, 13 and al, were produced in quantities for biochemical analysis by coinfection of Sf9 cells with separate recombinant viruses bearing cDNAs coding for the different monomers and subsequent coprecipitation of dimers with an immobilized domain-specific antibody against one of the monomer components. Individual components of the heterodimer were then analyzed by protein immunoblot analysis with monomer-specific antibodies. The ratio of the two recombinant viruses which gave rise to equal amounts of coprecipitated 13 and oal with a set level of antibody 2F12-protein A bead complexes was determined by
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FIG. 3. 251I-FGF-1-labeled recombinant homodimers of the FGF-Ral, -a2, and -13 isoforms in transfected monkey kidney (COS) cells. Detergent extracts of COS cells transiently transfected with cDNAs coding for the indicated receptor isoforms after '"IFGF-1 binding and covalent cross-linking were immunoprecipitated with the indicated domain-specific antibodies (bottom) and analyzed by SDS-PAGE and autoradiography. Homodimers and monomers are indicated by closed and open triangles, respectively. Antibody epitopes are indicated in the schematics at the top.
immunoblot with kinase-specific antibody Rl (Fig. 4A, upper panel). a2 antigen in the immunoprecipitates was assessed on the same blot with the a-specific 2F12 antibody that was used for primary immunoprecipitation of the heterodimer (Fig. 4A, lower panel). Production of a2-al heterodimers was similarly optimized by primary immunoprecipitation of coinfected Sf9 cell lysates with the R2 antibody against the kinase-negative type 2 COOH terminus and subsequent secondary analysis of the monomer components with the Rl and R2 antibodies. The effect of FGF-1 on dimer formation in Sf9 cells coinfected with viruses bearing cDNAs for a2 and ,B1 isoforms was examined. At 60 h after infection at the optimum ratio of the two recombinant viruses (Fig. 4A), FGF-1 was incubated with the coinfected Sf9 cells for 2 h, and extraction and immunoprecipitation were then performed with a-specific antibody 2F12. Increasing FGF-1 significantly increased the amount of ,13 monomer (Fig. 4B, upper panel) which coprecipitated with the constant levels of the a2 isoform immobilized on the 2F12 antibody beads (Fig. 4B, lower panel). Kinase activity measured by autophosphorylation of FGFRal, -11, and -a2 isoforms was first examined by immunoprecipitation of the indicated isoforms (Fig. 5) from extracts
SHI ET AL.
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FIG. 4. Preparation of heterodimers of FGF-Ra2 and -01 from coinfected Sf9 insect cells. (A) Optimization of coinfections. Recombinant virus encoding FGF-R13l was coinfected with the indicated amounts of virus (PFU, 108) encoding FGF-Ra2 (bottom) into 3 x 106 cells. After incubation with FGF-1-heparin, the detergentsoluble fraction from the infected cells was immunoprecipitated with at-specific monoclonal antibody 2F12. The immune complexes were extracted with SDS-PAGE sample buffer and subjected to immunoblot analysis with antibody Rl (upper panel) and 2F12 (lower panel). (B) Effects of FGF-1 on dimerization. The FGF-Ra2 and -13 isoforms were coexpressed in Sf9 cells by infection with 3 x 108 PFU each of recombinant baculovirus. Coinfected Sf9 cells were incubated in the presence of heparin (25 ,ug/ml) and the indicated amounts of FGF-1 prior to extraction and immunoprecipitation with monoclonal antibody 2F12. The immunoprecipitates were extracted with SDS-PAGE sample buffer and subjected to immunoblot analysis with antibody Rl (upper panel) or 2F12 (lower panel).
of virus-infected Sf9 cells with a polyclonal antibody, A40, which reacts with both the a. and extracellular domains (47) (Fig. 1A). Kinase activity assessed by phosphorylation of the atl and 1 isoforms in vitro was significantly enhanced by FGF-1 (Fig. 5A). As predicted from its structure (17, 47), the a2 isoform was not autophosphorylated. Phosphorylation of the kinase monomer occurs within heterodimers of type 1 kinase and the kinase-defective type 2 isoform. To test whether phosphorylation of the type 1 kinase monomer was impaired when it was complexed with the type 2 isoform, we compared phosphorylation of the al and ,13 monomers complexed with the a.2 isoform in immunoprecipitates from coinfected Sf9 cells to the phosphorylation of al and 13 isoforms in immunoprecipitates from cells infected with single oli and 1 viruses (Fig. 6). Phosphorylation of either al (lane 1) or 131 (lane 3) in the heterodimeric complex with a2 was reduced to only 60 to 80% of the phosphorylation level of an equivalent amount of single cxl (lane 2) or 13 (lane 4). This result suggests that maximal phosphorylation of all tyrosine sites within the type 1 kinase monomer may not be stringently dependent on trans inter-
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FIG. 5. Phosphorylation of FGF-Ral, -11, and -a2 isoforms. (A) FGF-R isoforms were expressed in Sf9 cells and then extracted and immobilized with polyclonal antibody A40 which recognizes all three isoforms (47). After incubation with heparin (25 ,ug/ml) with (+) or without (-) FGF-1 (200 ng/ml) in phosphorylation buffer for 20 min, the samples were incubated with [-y-32P]ATP for 5 min. The samples were then extracted and subjected to autoradiography (upper panel) and immunoblot analysis (lower panel) with monoclonal antibody 17D10 against a common epitope in the extracellular domain of all three FGF-R1 variants (47). (B) Portions of FGF-R(xl immobilized with a-specific antibody 2F12 were incubated with the indicated concentrations of FGF-1 and heparin (25 p,g/ml) and then incubated in the phosphorylation reaction mixture as described in for panel A. The samples were then subjected to autoradiographic analysis (upper panel) and immunoblot analysis with antibody Rl (lower panel).
molecular phosphorylation of the type 1 kinase within homodimers. Comparative phosphopeptide analysis of the kinase monomer in homo- and heterodimers. To determine whether phosphorylation of the two major intracellular sites, Tyr-653 and Tyr-766, was similar in type 1 kinase homodimers and the type 1-type 2 heterodimers, the 32P-labeled kinase component from immunoprecipitates of the 1 kinase and the ,B1-c2 heterodimer were separated by SDS-PAGE and electroblotted onto nitrocellulose paper. The excised labeledkinase band was then subjected to digestion with trypsin, and resultant phosphopeptides were separated by HPLC. Relative to the digest from an equivalent amount of the phosphorylated 1 kinase alone (Fig. 7A), the digest of the kinase from the ,B1-a2 heterodimer exhibited a significant decrease in the 32P-labeled doublet peaks which exhibit retention times characteristic of the two tryptic peptides, DIHHIDY(653)YKK and DIHHIDY(653)YK (18) (Fig. 7B).
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FIG. 6. Phosphorylation of FGF-Roal and -131 alone and complexed to FGF-Ra2. The indicated immobilized isoforms were prepared from single or coinfected Sf9 cells and then incubated with FGF-1-heparin and immobilized with the antibodies indicated below as described in the legend to Figure 4 and Materials and Methods. Immunocomplexes were incubated in phosphorylation reaction mixtures (Fig. 5) containing [-y-32P]ATP on ice for 5 min. The samples were then extracted, separated by SDS-PAGE, and electroblotted, and duplicate blots were analyzed by autoradiography (A) and immunoblot analysis with antibodies Rl (B) and R2 (C). Lane 1, a2-al heterodimers immobilized with Rl antibody; lane 2, al immobilized with a-specific 2F12; lane 3, a2-11 heterodimers immobilized with 2F12; lane 4, 13 immobilized on common polyclonal antibody A40. Open and closed circles in receptor schematics indicate putative unphosphorylated and phosphorylated tyrosine sites, respectively (see Fig. 10).
These results show that phosphorylation of Tyr-653 was selectively reduced relative to phosphorylation of Tyr-766 in the FGF-R kinase when it was heterodimerized with the kinase-negative type 2 isoform. Phosphorylation of Tyr-653 appears to occur within kinase homodimers, while phosphorylation of Tyr-766 is significant independent of whether the kinase monomer is within a homo- or heterodimer. PLC-yl associates with, but is not phosphorylated, by FGF-R type 1 kinase-type 2 kinase-defective heterodimers. Since the kinase monomer within the kinase-kinase-defective heterodimers appears to be phosphorylated on Tyr-766, we tested whether PLC-y1 as well as SH2 substrates PI3Kp85 and ras-GAP associated with the type 1-type 2 heterodimers (Fig. 8). 11-a2 heterodimers from lysates of Sf9 cells coinfected with 131 and a2 viruses were immobilized with at-specific antibody 2F12. The immobilized heterodimer was first incubated with ATP to phosphorylate the kinase monomer and then incubated with a lysate of HepG2 cells which was used as the source of the substrates. Associated substrates were then analyzed by extraction of the complex and immunoblot with antibodies against human PLCy1, PI3Kp85, and ras-GAP. Figure 8A and B show that PLC-y1 and PI3Kp85 antigens associated with immunoprecipitates of the 13 isoform alone (lane 2) and immobilized a2-1l heterodimers containing similar amounts of the 1 antigen (lane 4). Neither PLCY1 nor PI3Kp85 antigens could be detected in association with the immobilized a2 isoform
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FIG. 7. Analysis of phosphopeptides from FGF-R,1l from phosphorylated immunoprecipitates of ,13 and the a2-,1l heterodimer. Phosphorylated 131 and the a2-,1l heterodimer were prepared from singly infected and coinfected Sf9 cells, respectively, as described in the legend to Fig. 6 (lanes 3 and 4). The 12P-labeled 13 band was excised from electroblots, subjected to tryptic digestion, and analyzed by C18 narrow-bore reverse-phase HPLC at a flow rate of 150 p1/min with an acetonitrile gradient in 0.1% aqueous trifluoroacetic acid. The following gradient was used: 0 to 10 min, 0% CH3CN; 10 to 11 min, linear gradient to 5% CH3CN; 11 to 60 min, linear gradient of 5 to 40% CH3CN (dashed lines); 60 to 70 min, linear gradient of 40 to 70% CH3CN; 70 to 72 min, linear gradient of 70 to 100% CH3CN; 72 to 80 min, 100% CH3CN. Fractions of 0.2 ml were collected and counted by liquid scintillation. (A) Digest of 131 from phosphorylated immunoprecipitates of 131 alone; (B) 131 component of immobilized a2-1l heterodimers. Peaks with retention times correlating to tryptic peptides containing phosphotyrosine 653 and phosphotyrosine 766 (18) are indicated. The chromatograms are a representative pair of eight independent analyses from separate preparations.
alone (lane 5). ras-GAP antigen could not be detected in immunoprecipitates of any of the phosphorylated FGF-R isoforms despite the presence of the antigen in the HepG2 cell lysates (Fig. 8C). Treatment of the immunoprecipitates of phosphorylated a2-1l heterodimer or the 13 isoform alone with recombinant PTPase prior to incubation with the HepG2 cell lysates confirmed that the association of PLC-yl and PI3Kp85 with the dimeric FGF-R isoforms was dependent on the state of tyrosine phosphorylation of the ,13
3914
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