Cloning and expression of human platelet-derived growth factor α and β receptors

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[7]

conjugated with [125I]NGF. These cross-linking results indicate that the primary 75,000-85,000 dalton receptor species appears in cells with both high and low affinity forms of the receptor and supports the conclusion that the cloned NGF receptor gene is capable of giving rise to both high and low affinity receptors. 5 The heightened interest in the role of NGF in the central nervous system and in neurodegenerative diseases such as Alzheimer's dementia requires molecular and biochemical techniques to characterize the interaction of NGF and its receptor. The detection of NGF receptors by in situ rosetting, immunofluorescence, and affinity cross-linking provides the opportunity to use in vitro mutagenesis of NGF and its receptor to map the binding site and to delineate which structural features of the receptor are responsible for the functional responses of NGF.

[7] C l o n i n g a n d E x p r e s s i o n o f H u m a n P l a t e l e t - D e r i v e d G r o w t h F a c t o r a a n d fl R e c e p t o r s By LENA CLAESSON-WELSH, ANDERS ERIKSSON, BENGT WESTERMARK,

and CARL-HENgIK HELDIN Platelet-derived growth factor (PDGF), a major mitogen for connective tissue cells, is composed of disulfide-bonded A and B polypeptide chains. The three isoforms of PDGF have been found to bind to two distinct receptor types with different affinities (reviewed in Refs. 1 and 2). The PDGF/3 receptor (also denoted PDGF B-type receptor) binds PDGF-BB with high affinity, PDGF-AB with somewhat lower affinity, but appears not to bind PDGF-AA. The PDGF a receptor (also denoted PDGF Atype receptor) binds all PDGF isoforms with high affinity. Both receptors possess an intrinsic kinase activity, which becomes activated after ligand binding. Cloning and Expression of Human Platelet-Derived Growth Factor fl Receptor Partial amino acid sequence information of tryptic fragments from the purified murine PDGF/3 receptor was used to clone the corresponding C.-H. Heldin and B. Westermark, Trends Genet. 5, 108 (1989). z R. Ross, E. W. Raines, and D. F. Bowen-Pope,Cell (Cambridge, Mass.) 46, 155 (1986). METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cDNA from murine fibroblast and placenta libraries) Using synthetic oligomers based on the murine receptor cDNA sequence as probes, we isolated a number of cDNA clones from a human foreskin fibroblast hgtl0 cDNA library. 4 The RNA was isolated from human AG 1523 foreskin fibroblasts using the LiC1 m e t h o d : and mRNA was collected through oligo(dT) affinity chromatography according to standard procedures. cDNA was synthesized and cloned using commercially available kits (Amersham International Ltd., Buckinghamshire, UK). The resulting cDNA, cloned into hgtl0, was divided into 11 pools which were amplified independently. About 300,000 plaque-forming units (pfu) from each pool were seeded on 23 × 23 cm agar plates and transferred in duplicate to nitrocellulose filters. 6 The probes used for hybridization were constructed by annealing two partially overlapping synthetic oligomers, each 40 base pairs long. After a fill-in reaction using Klenow polymerase and [a-32p]dCTP, a 3zp-labeled 70-mer was obtained. Two different probes, both located in regions of the cDNA corresponding to conserved areas in the intracellular tyrosine kinase domain, were used for the two replica filters from each agar plate. Using a high stringency protocol for hybridization [50% formamide, 5 x SSC (1 x SSC is 15 mM sodium citrate, 150 mM NaCl, pH 7.0), 5 x Denhardt's solution, 7 0.1% sodium dodecyl sulfate (SDS), and 0.1 mg/ml salmon sperm DNA, at 37°] and mild washing conditions (2 x SSC, 0.1% SDS at 37° for 30 min), we identified four unique positive plaques in the first round of screening. A third 70-mer corresponding to a region in the extracellular domain was used to identify one of the isolated clones as covering most of the open reading frame (hhPDGFR-2A3). This clone was used in a second round of screening of a new seeding of agar plates with aliquots from the 11 cDNA library pools. One new unique clone, denoted hhPDGFR-8, was identified, which based on the size of the insert, was close to fulMength. The nucleotide sequence of both strands of hhPDGFR-8 was determined using M13 dideoxy sequencing. 8 After restriction mapping of the cDNA subcloned into p U C I 9 , 9 conveniently sized fragments were cloned into M13. A sche3 y . Yarden, J. A. Escobedo, W.-J. Kuang, T. L. Yang-Feng, T. O. Daniel, P. M. Tremble, E. Y. Chen, M. E. Ando, R. N. Harkins, U. Francke, V. A. Fried, A. Ullrich, and L. T. Williams, Nature (London) 32,3, 226 (1986). 4 L. Claesson-Welsh, A. Eriksson, A. Mor6n, L. Severinsson, B. Ek, A. Ostman, C. Betsholtz, and C.-H. Heldin, Mol. Cell. Biol. 8, 3476 (1988). 5 C. Auffrey and F. Rougeon, Eur. J. Biochem. 107, 303 (1980). 6 T. Maniatis, E. F. Fritsch, and J. Sambrook, in "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 7 D. T. Denhardt, Biochem. Biophys. Res. Cornmun. 23, 641 (1966). 8 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 9 C. Yannisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985).

74

IGF, NGF, AND PDGF SS

ED1

ED2

ED3

PDGFR- c¢ ~ ) - O ~ O - C ' ~ C ' ~ O

%IDENTITY

PDGFR-B

ED4

ED5 TMJM TK1 ~

30

~

[7]

i

48 83

~

KI I

87

I

TK2 I

35

I

CT I

74

I

27

I

FIG. 1. Schematic representation of the two PDGF receptor types. The receptors are drawn to indicate the presence of 10 evenly distributed cysteine residues in the extracellular parts and the split tyrosine kinase domains in the intracellular parts of the receptors. The level of amino acid identity between the various domains of the molecules are indicated. SS, Signal sequence; ED, extracellular domain; TM, transmembrane domain; JM, juxtamembrahe domain; TK, tyrosine kinase domain; CT, carboxy-terminal tail.

matic representation of the deduced structure of the PDGF/3 receptor is shown in Fig. 1. In order to express the/3 receptor stably, the full-length cDNA was subcloned into an SV40-based expression vector. 10Chinese hamster ovary (CHO) cells were transfected with the cDNA, mixed with a plasmid containing the neomycin resistance gene to allow selection for neomycinresistant cell colonies coexpressing the PDGF/3 receptor. Calcium phosphate-mediated transfection H was performed in order to introduce the fl-receptor cDNA into the CHO cells. To screen for cells expressing the PDGF 13 receptor, 20 colonies were picked 3 weeks after transfection, during which time the cells were cultivated in the presence of 0.5 mg/ml of Geneticin 418 (neomycin). The colonies were expanded in duplicate 12-well dishes, and, when confluency was reached, the cells were analyzed for their ability to bind lzSI-labeled PDGF-BB. Clones which expressed 105 cell surface receptors per cell, that is, a number similar to that of human fibroblasts, were obtained and analyzed with regard to their ability to bind and respond to different isoforms of PDGF. 4

~0M. A. Truett, R. Blacher, R. L. Burke, D. Caput, C. Chu, D. Dina, K. Hartog, C. H. Kuo, F. R. Masiarz, J. P. Merryweather, R. Najarian, C. Pachl, S. J. Potter, J. Puma, M. Quiroga, L. B. Rail, A. Randolph, M. S. Urdea, P. Vaienzuela, H. H. Dahl, J. Favalaro, J. Hansen, O. Nordfang, and M. Ezban, DNA 4, 333 (1985). H M. Wigler, R. Sweet, G. K. Sim, B. Wold, A. Pellicier, E. Lacy, T. Maniatis, S. Silverstein, and R. Axel, Cell (Cambridge, Mass.) 16, 777 (1979).

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Cloning and Expression of Platelet-Derived Growth Factor a Receptor In order to structurally characterize the PDGF a receptor, a panel of cell lines were analyzed for binding of the PDGF isoforms, with the aim of obtaining one that expressed the a receptor, but not the/3 receptor. One glioma cell line, U-343 MGa 31L, bound 125I-labeled PDGF-AA as well as ~25I-labeled PDGF-BB. Unlike the situation in human fibroblasts, however, all the binding of ~25I-labeled PDGF-BB could be competed for by PDGF-AA. This indicated that these cells expressed the PDGF a receptor only. A rabbit antiserum raised against purified porcine PDGF/3receptor preparations ~2was found to react with a 170-kDa component from the U-343 MGa 31L cells. The 170-kDa component was downregulated when the cells were exposed to PDGF-AA, indicating that it was the mature PDGF a receptor. The cross-reactivity of the antiserum, and the fact that the a and/3 receptors yielded similar peptide maps, indicated that the two receptors were structurally similar.13 We therefore examined whether a /3-receptor cDNA probe would react with any potential areceptor transcript in the U-343 MGa 31L cell line. Whereas no crossreactivity could be seen under high stringency conditions, a 6.5-kilobase (kb) glioma cell transcript was detected when the intracellular part, but not the extracellular part, of the/3 receptor was used as a probe in Northern blotting, under lower stringency conditions [hybridization performed in 40% (v/v) formamide, 5 × SSC, 10 × Denhardt's solution, 50 mM phosphate buffer, pH 6.5, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA; washing in 2× SSC, 0.1% (w/v) SDS, 2 times, 20 min each, at 45°]. In order to clone the a receptor, a cDNA library was constructed from mRNA purified from the glioma cells, again using cDNA cloning and synthesis kits from Amersham. From 1 × 10 6 plaques, seeded without prior amplification of the cDNA library and screened using the tyrosine kinase part of the/3-receptor cDNA as a probe and the lower stringency conditions described above, we isolated one positive clone (phPDGFRA 1). M13 dideoxy sequencing of the 4.5-kilobase pair (kbp) insert revealed that the most 5' segment contained an open reading frame with a sequence which was up to 87% similar to that of the tyrosine kinase domain of the /3 receptor. The cDNA library made from the U-343 MGa 31L cells was then screened with the most 5' part of the phPDGFRA1 clone as a probe; 12 L. R6nnstrand, M. P. Beckmann, B. Faulders, A. 0stman, B. Ek, and C.-H. Heldin, J. Biol. Chem. 262, 2929 (1987). 13 L. Claesson-Welsh, A. Hammacher, B. Westermark, C.-H. Heldin, and M. Nist6r, J. Biol. Chem. 264, 1742 (1989).

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however, since we failed to find extended clones after having screened about 5 x 106 pfu, we constructed a new cDNA library. The previous libraries were made using synthetic linkers with internal EcoRI sites to tail the cDNA. Instead, we now employed synthetic, double-stranded adaptors, with one nonphosphorylated EcoRI overhang and one blunt end. In this way we avoided having to cleave the cDNA at internal EcoRI sites. We purified mRNA from AG 1518 human foreskin fibroblasts; the cDNA kit from Pharmacia-LKB (Uppsala, Sweden) was used for the first and second cDNA strand synthesis. The adaptors were ligated to the cDNA using T4 DNA ligase from New England Biolabs (Beverly, MA); gel chromatography using Sephadex G-50 equilibrated in 1 mM Tris-HC1, pH 7.5, 1 mM EDTA was performed to remove free adaptors and short cDNA molecules. After ethanol precipitation, the cDNA ends were phosphorylated using components in the Pharmacia-LKB kit. After a second ethanol precipitation, the cDNA was ligated to dephosphorylated Xgtl0 arms purchased from Promega Corp. (Madison, WI), at a ratio of 100 ng cDNA to 2 ~g hgtl0 arms, in a final volume of 20/xl, using T4 DNA ligase from New England Biolabs. The ligation mixture was packaged using components from Amersham; 3.4 x 10 6 pfu/100 ng cDNA were thus obtained. One million plaque-forming units from the adaptor-tailed library were seeded without prior amplification. The most 5' segment from the phPDGFRA1 clone was labeled using [a-3Zp]dCTP to a specific activity of about 108 counts/min (cpm)//zg using the multiprime labeling kit from Amersham; 1 x 10 6 cpm of the probe was added per milliliter of hybridization solution. The nitrocellulose filters were hybridized and washed under high stringency conditions. Phage DNA was prepared from 16 positive clones. From two of these clones four fragments were released on EcoRI cleavage. The combined size of these fragments was 6.5 kbp for the clone denoted phPDGFRA15. The individual fragments were subcloned and hybridized one by one to U-343 MGa 31L mRNA, to ensure that they originated from the same 6.5-kb transcript. M13 dideoxy sequencing showed that this new clone covered and extended the first clone. A schematic representation of the structure of the molecule encoded by the cDNA, based on the deduced amino acid sequence, is shown in Fig. 1. As described below, expression of the cDNA and PDGF-binding analyses showed that it encoded the PDGF ot receptor.~4 The degree of identity between the two PDGF receptors ranges from 27% identity in the Cterminal tails to 87% in the first part of the tyrosine kinase domains, as indicated in Fig. 1. 14 L. Claesson-Welsh, A. Eriksson, B. Westermark, and C.-H. Heldin, Proc. Natl. Acad. Sci. U . S . A . 86, 4917 (1989).

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For expression of the a receptor, a 3.5-kbp subclone covering the entire open reading frame of the a receptor was cloned into the same SV40-based expression vector used for B-receptor expression, to yield a clone denoted pSV7dl5.1 + 5. Calcium phosphate-mediated transfection was then performed to achieve transient expression in COS-1 cells. The transient expression was reproducible enough to allow Scatchard analyses of binding of the PDGF isoforms; all three PDGF isoforms bound to COS-1 cells transfected with pSV7dl5.1 + 5 with high affinities. Immunoprecipitation of metabolically labeled, transfected COS-1 cells also yielded results supporting the conclusion that the 6.5-kbp cDNA clone encoded the PDGF a receptor. To achieve stable expression of the PDGF a receptor, we subcloned the parts of the clone containing the open reading frame, as well as 1I00 bp of the 3' untranslated region. The 3' part was included since it facilitated cloning of the cDNA into a retroviral expression vector, containing the murine leukemia virus long terminal repeats and the neomycin resistance gene. Electroporation ~5rather than calcium phosphate-mediated transfection was used to introduce the DNA into CHO cells. We picked 20 neomycin-resistant colonies and screened for binding of ~25I-labeled PDGF-AA. A cell line expressing about 0.2 × 105 a-receptor molecules on the cell surface was obtained. Unlike the case of/3-receptor-expressing CHO cells, we found it to be crucial to grow the a-receptor-expressing CHO cells in the absence of serum overnight before performing the binding assay. If this was not done, the amount of binding of 125I-labeled PDGF-AA was low, probably owing to downregulation of the a receptor by constituents of the fetal calf serum.

Conclusions To facilitate cloning of long cDNA molecules, it is necessary to ensure a high quality of mRNA preparations. We have limited the processing of the mRNA to as few steps as possible and do not enrich for long transcripts, to avoid exposure to degrading enzymes. Instead, a larger number of recombinants have to be screened, preferentially using a nonamplified library. The availability of high quality modifying and restriction enzymes and the use of adaptors rather than linkers for the cDNA tailing reaction make it possible to construct successfully a high quality cDNA library without much previous experience.

r5 D. Rabussay, L. Uher, G. Bates, and W. Plastuch, Focus 9, 1 (1987).