MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2237-2246 0270-7306/90/052237-10$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 5

Isolation and Characterization of the ot Platelet-Derived Growth Factor Receptor from Rat Olfactory Epithelium KYU-HO LEE,' DANIEL F. BOWEN-POPE,2 AND RANDALL R. REED'* Laboratory of Genetics, Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,1 and Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 981052 Received 14 August 1989/Accepted 3 February 1990

We have cloned and characterized a new member of the receptor tyrosine kinase family. The cDNA clone, isolated from a rat olfactory cDNA library, has considerable homology to the family of receptors that includes the colony-stimulating factor 1 receptor, the c-kit proto-oncogene, and the platelet-derived growth factor (PDGF) receptors. Analysis of DNA sequence homology, ligand-binding, and ligand-stimulated phosphorylation data suggests that this clone encodes the rat PDGF-A/B or a-receptor. Comparison of its sequence to those of other receptors allows us to postulate a mechanism for receptor dimerization and activation. The expression of the rat a-PDGF receptor in nonneuronal cells of the olfactory epithelium and in the olfactory bulb is consistent with a role for PDGF in glial cell generation.

The receptor tyrosine kinases (RTKs) mediate growth, differentiation, and mitogenic activities in a variety of systems. These integral membrane receptors bind growth factors or other small molecules at the cell surface and catalyze the phosphorylation of tyrosine residues on target substrates. These substrates in turn induce various intracellular responses (48). These receptors share several general structural features: (i) an external ligand-binding domain, (ii) a hydrophobic transmembrane domain, and (iii) a highly conserved intracellular catalytic domain responsible for tyrosine kinase activity. The receptors can be classified into several families on the basis of their subunit structures and conserved sequence elements. One such family, designated class III (48), includes the colony-stimulating factor 1 receptor of monocyte and macrophage cells (33, 36), the c-kit receptor (31, 47), whose ligand and function are unknown, and the platelet-derived growth factor (PDGF) receptor (25, 46). The members of this class share a conserved pattern of 10 cysteine residues in the external ligand-binding domains and a characteristic kinase-insert (KI) region (9, 48) within their catalytic domains. PDGF exists as a disulfide-bonded homo- or heterodimer of A- and/or B-chain polypeptides, encoded by homologous but distinct genes (19). There are thought to be two receptor molecules capable of transducing the PDGF signal. These receptors, a and ,1, have differing affinities for the different PDGF isoforms. The ,8-receptor protein was the first to be cloned and characterized (46) and binds specifically the PDGF-B-chain (7, 13). The presence of PDGF-A-chainbinding sites on certain cell types (13, 16) indicated the existence of a second PDGF receptor, the a-receptor protein. Current evidence indicates that the a- and ,B-subunit proteins function as subunits in the active dimeric form of the PDGF receptor (18, 35). Since the 13-subunit binds only the B-chain with high affinity and the a-subunit can bind either the A-chain or the B-chain, the three different combinations of receptor subunits create high-affinity receptors with differing isoform specificities. For example, the aa form of the receptor can bind PDGF-AA, PDGF-AB, and PDGFBB isoforms, while the form of the receptor can bind only *

PDGF-BB. It seems likely that the isoform specificity of the receptor proteins allows different cell types to be targeted by different isoforms of PDGF. This possibility is supported by the observation that different cell types express different levels of the two receptor proteins. For example, adult human fibroblasts largely express p-subunit and are very responsive to PDGF-BB but are poorly responsive to PDGFAA (35). By contrast, the a-subunit is expressed on cells of central nervous system glial origin and PDGF-AA seems to play an important role in regulating astrocyte differentiation in the central nervous system (26, 32). We isolated a cDNA clone encoding an apparently novel protein from a rat olfactory cDNA library during a screen for olfactory tissue-specific gene products. Its sequence, as well as that of a partial murine clone obtained by high-stringency cross-hybridization, contains the conserved sequence elements of the class III RTKs, including a conserved pattern of cysteine residues and the existence of a unique KI domain. The clone is highly homologous to the recently reported human a-PDGF receptor (25). Expression of our cDNA clone in COS cells has allowed us to determine that our rat olfactory clone encodes the rat a-PDGF receptor. Sequence and RNA analyses have allowed us to examine possible mechanisms of receptor function and to postulate the role of the receptor in the olfactory system. MATERUILS AND METHODS Cloning and sequencing. cDNA clones were obtained from libraries made from the polyadenylated RNA fraction of adult rat (Sprague-Dawley) olfactory epithelium. Doublestranded cDNA molecules were synthesized by using the DNA polymerase I-RNAse H method of Gubler and Hoffman (14) and inserted into the EcoRI restriction site of the lambda phage cloning vector Xgt-10. The initial clone, 8023, was isolated fortuitously with a 32P-end-labeled oligonucleotide probe, 5'-AC(G/A/T/C)GC(G/A/T/C)GCCAT(A/G)TA CCA-3', during a screen for adenylyl cyclase in the olfactory library. Filters were washed at 42°C in 3 M tetramethylammonium chloride (45). In order to generate a full-length cDNA clone, clone 8028, containing the 3' sequences of 8028/3, was obtained upon rescreening the olfactory library with the central 1.3-kilobase (kb) BamHI fragment of clone

Corresponding author. 2237

2238

LEE ET AL.

8023. The mouse clone 4A4-1 was similarly isolated from a non-size-selected mouse placental library (Daniel Linzer, Northwestern University). Clones were excised from phage DNA and subcloned into Bluescript (Stratagene) plasmid vectors for sequencing, restriction mapping, and subcloning. Sequence was determined via oligonucleotide-primed synthesis on double-stranded plasmid DNA templates by the Sanger dideoxynucleotide method (34). Expression in COS cells. COS-1 cells (10) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, 100 U of penicillin G per ml, 100 ,ug of streptomycin per ml, and 10% fetal bovine serum (DMEM/10) and passaged 1:4 every 2 days. The 8028/3 expression vector pM802E was constructed by ligating together in Bluescript the overlapping cDNA clones 8023 and 8028 at a common SpeI site. An EcoRI fragment containing the entire cloned construct was then transferred to the EcoRI site of expression vector pMT2 (Genetics Institute) (44). Tissue culture plates (10 cm) of COS-1 cells were transfected with 5 jig of pM802E or pMT2 by using calcium phosphate precipitation (40) and maintained in DMEM/10 for 3 days before harvesting. Binding of PDGF isoforms. Binding of iodinated PDGF isoforms to monolayer cultures of transfected cells was determined essentially as described by Bowen-Pope and Ross (3). Briefly, 72 h after transfection, cells were subcultured in 24-well plates for an additional 24 h in DMEM/10. Medium was aspirated, and cell monolayers were incubated at 4°C with increasing concentrations of the various PDGF isoforms. Specific activities for the iodinated isoforms were as follows: [125I]PDGF-AA, 937 cpm/fmol; [125I]PDGF-AB, 1,186 cpm/fmol; and [125I]PDGF-BB, 697 cpmlfmol. Nonspecific binding was determined by preincubation with 200 ng of unlabeled ligand per ml. Parallel wells were incubated in medium without label to determine cell number for calculation of the number of binding sites per cell. Competition for ['2,I]PDGF-BB binding. The indicated control cell types were plated in 24-well dishes and then incubated overnight in DMEM-1% FBS (DMEM/1) prior to assay. Transfected COS cells were assayed 72 h following transfection. All cultures were incubated for 3 h at 4°C with either increasing concentrations (1 to 200 ng/ml) of unlabeled PDGF-AA or 100 ng of PDGF-BB per ml to determine nonspecific binding. Cultures were then washed and incubated for an additional 2 h with 1 ng of [1251]PDGF-BB per ml. The level of [125I]PDGF-BB binding which is not inhibited by the highest concentration of PDGF-AA is expressed as the percent of binding to cultures not exposed to PDGF (35). In vivo phosphorylation. At 72 h after transfection, cells were washed with phosphate-buffered saline and incubated for 6 h at 37°C with serum-free DMEM. Medium was then aspirated, and cells were incubated for 1 h at 4°C in test medium. Medium was again removed, and cells were harvested in 3 ml of phosphate-buffered saline, pelleted, and

MOL. CELL. BIOL.

then suspended in 100 ,ul of TE (10 mM Tris, 1 mM EDTA; pH 7.6). Following estimation of total protein (4), 100-,ug samples were incubated in sodium dodecyl sulfate sample buffer (23) at 65°C for 3 min and then separated electrophoretically on a sodium dodecyl sulfate-6% polyacrylamide gel and transferred to nitrocellulose filters (Schleicher & Schuell). Following fixation in 10% acetic acid-25% MeOH for 20 min, filters were rinsed with distilled water and blocked in 1% gelatin in TBS (15 mM Tris, pH 7.5, 150 mM NaCl)-0.1% bovine serum albumin-0.05% Tween 20. Blots were reacted with an affinity-purified rabbit polyclonal antibody made against phosphotyrosine-bovine serum albumin (R. Huganir, Johns Hopkins School of Medicine) followed by [251I]protein A (2 to 10 pRCi/4g, Dupont, NEN Research Products, Inc.). Blots were autoradiographed on Kodak XAR film. Anti-PDGF receptor immunoblot. Protein samples were prepared, resolved, and transferred to nitrocellulose as described above. Filters were blocked for 1 h in TBS-5% nonfat dry milk (Blotto) and reacted with a 1:200 dilution in Blotto of an anti-human a-PDGF receptor antibody (a kind gift from S. Aaronson, National Cancer Institute [25]) followed by [1251]protein A. Blots were autoradiographed for 24 h on Kodak XAR film. Northern (RNA) blot analysis. Total RNA samples were extracted from freshly dissected rat tissues in the presence of guanidinium thiocyanate by the method of Chirgwin et al. (5). Samples (10 ,ug) were treated with glyoxal and separated on a 1% agarose gel in 10 mM phosphate buffer, pH 6.8 (39). RNA from mouse tissues and cell lines was separated in the presence of 0.2 M morpholinepropanesulfonic acid (MOPS), pH 7.0, 50 mM sodium acetate, 1 mM EDTA, and 2.2 M formaldehyde (24). The RNA was transferred to filters in 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and hybridized to a random-primed 32P-labeled DNA probe containing the central 1.3-kb BamHI fragment of clone 8023 (39). Following overnight hybridization at 42°C, the filter was washed twice, for 20 min each time, at 65°C in 0.1 x SSC-0.1% sodium dodecyl sulfate, air dried, and autoradiographed for 5 days. Molecular sizes were determined from 1-kb DNA ladder fragments (Bethesda Research Laboratories, Inc.) run in adjacent lanes. Quantity and quality of RNA present in each lane were determined by hybridization of the blot to a labeled DNA probe specific for rRNA. Time course experiments. Subconfluent BALB/c 3T3 cells were grown on 15-cm tissue culture plates in DMEMI10. When cells were confluent, medium was replaced with DMEM-0.1% FBS (DMEM/0.1) and incubated for 3 days. Cells were then incubated for varying times with DMEM/10 in the presence or absence of 10 p.g of cycloheximide per ml, as indicated in the legend to Fig. 5. Medium was then aspirated, and RNA was extracted with a single phenolchloroform extraction step (6). Samples (20 ,ug) were separated and transferred in the presence of glyoxal, as described above. Blots were hybridized with a random-primed 32p-

FIG. 1. Restriction map and sequence alignment. (a) Restriction map. Restriction maps of rat olfactory library cDNA clones 8028 and 8023. Open reading frame, including transmembrane and tyrosine kinase regions, is indicated, as is the probable oligonucleotide-binding site. The oligonucleotide recognized the sequence 5'-ATGGTATAATGGCCGCTGT-3' on the noncoding strand of clone 8023. Size on scale below is in kilobases. B, BamHI; H II, HincII; H III, HindIII; P, PstI; Rl, EcoRI; RV, EcoRV; Sc, Sacl; Sl, Sall; Sp, SpeI; Symbols: =, coding region; 6, transmembrane domain; E, tyrosine kinase homology; 0, oligonucleotide-binding site. (b) Sequence alignment. The amino acid sequences of rat clone 8028/3 and mouse clone 4A4-1 are shown aligned with those of the other members of the class III RTKs. Alignment is based on pairwise alignments via the DFASTP protein homology program and adjusted by eye for overall comparison. Amino acid identities are shaded. Conserved N-terminal domain cysteines are boxed, as are the putative transmembrane domains. The C-terminal KI region is boxed in dotted lines. Asterisks indicate positions in the ligand-binding external domains with identity for at least 4 of 5 or 5 of 6 sequences or positions where all sequences contain highly compatible residues. Potential N-linked glycosylation sites are overlined.

VOL. 10, 1990

3li

lii I -a

>

C;

(ZZ; C)....I n

V)Z:Z::x (4C}

Or"m GA :4 alt1tv

04-er:)4

(4ZU

I

il

-C
Z -4(V4:4.

>1 Inn

l --V) -a > OK-4-a P1

K>44.4-4;4

1'r G

OD

rr

Z~'rTr

>-I

0000. K-IKK

-4-t -*jhr-I -C-Cl~~~~~~~~KKK ~ IlK> > T'IOZUO 11=1 K 0Z14*

>

(40(4V

=zzZ:

0 ;-4.(-< OKK K::OC

o-a~I

0(444')

1044

U~- * r-r--C -CI r>-14C ZK4=0 0> V(A) rm>o ,444K

C)i

==Or-

Zl =rvi ::;1)-»0>= -< ;rr

0K(A401m P1

-a0P1

4-4V-C



I

0*.'. :4QP1P1>:X _-4 : K

I

wr-00cK0 4~--4CRKP1

=

-4-4(4

1I) I> P1 { -a

1-"1

o "1 1 I

P1 I( -I r-I -

>

40

Zr-c

rq K4'1

01

11-0 ( C 4"1 ZP1 -C-C 1IZ >-II0-4-44-0

I

-CI KI

0

-a-, 0I

>

OK -Clr-Kq 0I

-

0(P-I'

I 0(4-i£

0:0

-

--6 r~"-'k0 *0 1) T d4v-4 0100 0 P1 o (4ZKP1KO »~~~~~~~~~~~~~~.O -4>>*

0)K(4,-M

:0004 0)

Is.t

Iu r;

0

>1n I:4-

-Cr-
w

o

-

MKI

'T

(4

»4flf

0 I

I.a

rs>

0

014 0-Cr--I -44 ZZKMMO -C-