Cell, Vol. 66, 775-765,

February

21, 1992, Copyright

0 1992 by Cell Press

Expression Cloning of the TGF-P Type II Receptor, a Functional Transmembrane Serine/Threonine Kinase Herbert Y. Lin,“t Xiao-Fan Wang,‘* Elinor Ng-Eaton,’ Robert A. Weinberg,‘t and Harvey F. Lodish’t ‘Whitehead Institute for Biomedical Research Cambridge, Massachusetts 02142 fDepartment of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Summary A cDNA encoding the TGF-P type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an ~80 kd protein that specifically binds radioiodinated TGF-bl. Excess TGF-Pl competes for binding of radioiodinated TGF-f31 in a dose-dependent manner and is more effective than TGF-B2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/ threonine phosphorylation as an important mechanism of TGF-P receptor-mediated signaling. Introduction The transforming growth factor-6 (TGF-6) family of peptide growth factors includes five members, termed TGF-61 through TGF-65, all of which form homodimers of m25 kd (reviewed in Roberts and Sporn, 1990; Massague, 1990). Sequence analysis of the five TGF-6 genes shows that their structures are highly conserved over great evolutionary distances. The mature processed hormones formed from individual members of this gene family show almost 100% amino acid identity between species, and the five peptides as a group exhibit m60%-80% identity. This TGF-6 family belongs to a larger, extended superfamily of peptide signaling molecules that includes the Miillerian inhibiting substance (Cate et al., 1986), decapentaplegic (Padgett et al., 1967) bone morphogenic factors (Wozney et al., 1988; Wang et al., 1988) vgl (Weeks and Melton, 1987) activins (Vale et al., 1986; Ling et al., 1966) and inhibins (Mason et al., 1965; Forage et al., 1986). These factors are similar to the TGF-6s in overall structure, but share only -25% amino acid identity with the TGF-6 proteins and with each other. All of these molecules are thought to play important roles in modulating growth, development, and differentiation. *Present Medical

address: Department Center, Durham. North

of Pharmacology, Carolina 27705.

Duke

University

TGF-6 was originally described as a factor that induced normal rat kidney fibroblasts to proliferate in soft agar in the presence of epidermal growth factor (Roberts et al., 1981). TGF-6 has subsequently been shown to exert a number of different effects in a variety of cells (reviewed in Roberts and Sporn, 1990; Massague, 1990). For example, TGF-6 can inhibit the differentiation of certain cells of mesodermal origin (Florini et al., 1986; Massague et al., 1986; Olson et al., 1986) induce the differentiation of others (Seyedin et al., 1985; lgnotz and Massague, 1985; Centrella et al., 1988) and potently inhibit proliferation of various types of epithelial cells (Tucker et al., 1984b; Moses et al., 1985; Masui et al., 1986; Shipleyet al., 1986; FraterSchroder et al., 1986; Jetten et al., 1986; Kurokawa et al., 1987; Boyd and Massague, 1969). This last activity has led to speculation that one important physiologic role for TGF-6 is to maintain the repressed growth state of many types of cells. Accordingly, cells that lose the ability to respond to TGF-6 are more likely to exhibit uncontrolled growth and to become tumorigenic. Indeed, the cells of certain tumors such as retinoblastomas lack detectable TGF-6 receptors at their cell surface and fail to respond to TGF-6, while their normal counterparts express cell surface receptors and their growth is potently inhibited by TGF-6 (Kimchi et al., 1988). Besides carcinogenesis, disregulation of TGF-6 function is also implicated in the pathological processes of diseases such as arthritis, ulcerative diseases, atherosclerosis, and glomerulonephritis (reviewed in Roberts and Sporn, 1990; Massague, 1990). An elucidation of the mechanism of TGF-6 action would be beneficial to an understanding of these diseases and may lead to improved therapeutics. The cellular mechanisms of action of TGF-6s are unknown, despite tremendous study over the past decade. None of the known pathways for intracellular signaling, including phosphatidylinositol turnover, modulation of intracellular CAMP levels, and tyrosine phosphorylation, have been directly linked to TGF-6 action (reviewed in Roberts and Sporn, 1990; Massague, 1990). The likely candidates for mediating signal transduction by TGF-6 are three cell surface proteins that have been identified through their ability to bind and be chemically crosslinked to radioiodinated TGF-6 (Frolik et al., 1984; Tucker et al., 1984a; Cheifetz et al., 1986, 1987, 1988; Fanger et al., 1986; Segarini and Seyedin, 1986; Segarini et al., 1969). They have been termed type I, II, and Ill receptors and have apparent molecular weights of 55, 80, and 280 kd, respectively. Several lines of evidence support the role of these cell surface proteins in the transduction of TGF-p-initiated signals First, the biological effects of TGF-6 can be achieved by addition of concentrations of hormone corresponding to the known binding constants for the type I and type II receptors (Roberts and Sporn, 1990; Massague, 1990). Second, several tumorigenic cell lines, such as pheochromocytoma cells, neuroblastoma cells, retinoblastoma cells (Kimchi et al., 1988) and breast carcinoma cells (Ar-

Cell 776

Figure

1. isolation

of Type

II TGF-B

Receptor

cDNA

by Expression

Cloning

(A) Photomicrograph of a positive cell on a glass slide of COS cells transfected with pool #97 of an LLC-PK, cDNA library representing *7,500 clones, as detected by dark-field illumination. COS cells transfected with plasmid pools were allowed to bind [1z51]TGF-~1 and were then washed, fixed, and dipped in photographic emulsion. After slides were developed, a positive cell was identified by the accumulation of srlver grams over it, and the corresponding cDNA pool was subdivided for rescreening. Bar: 100 pm. See Experimental Procedures for detarls (8) A combination dark-field and phase-contrast view of the same high-power field as (A). (C and D) Dark-field view of COS cells transfected with pure cDNA clone P2-3F. Binding of [‘251]TGF-61 was performed In the absence (C) or presence (D) of IOO-fold excess (5 nM) unlabeled TGF-61. Bar: 100 nm

teaga et al., 1988; Zugmaier et al., 1989) lack one or more of thesereceptorsandshownogrowth inhibition byTGF-6. Furthermore, several groups have shown that loss of type I or type II receptor expression corresponds to loss of responsiveness to TGF-6 (Laiho et al., 1990, 1991; Geiser et al., 1992). Cloning of the TGF-6 type III receptor gene reveals its product to be a membrane-anchored proteoglycan (LopezCasillas et al., 1991; Wang et al., 1991), as was first predicted by Segarini and Seyedin (1988). It has a short cytoplasmic region having noobvioussignaling motif. We have shown that the ability of the type II receptor in L6 myoblasts to bind TGF-61 is up-regulated by forcing the expression of the otherwise missing type Ill receptor (Wang et al., 1991) but it remains unclear whether the type Ill receptor protein can directly mediate a TGF+initiated signal across the plasma membrane. These results focused our attention on the type I and II receptors. We report here the cloning of the TGF-6 type II receptor, and show that it is related to the activin receptor (Mathews and Vale, 1991) and the daf-1 receptor of C. elegans (Georgi et al., 1990).

Results Expression Cloning of the Receptor cDNA In previous work, we isolated the TGF-6 type Ill receptor cDNA by expression cloning from a library made from cells which expressed all three TGF-6 receptor genes. We expected that isolation of the type Ill receptor cDNA would have been greatly favored, because the type Ill receptor is expressed in lo- to 50-fold higher amounts than the type I and type II receptors. To avoid recloning the type Ill receptor, we constructed a mammalian cDNA expression library from mRNA of a cell type that does not display type Ill receptors but does express the type I and type II receptors, LLC-PK, cells (Goldring et al., 1978). This porcine renal epithelial cell line lacks detectable type Ill receptor as determined by crosslinking of radioiodinated TGF-61 to cell-surface proteins (data not shown), a result confirmed by the lack of type Ill receptor mRNA in these cells (Wang et al., 1991). In brief, we constructed a size-selected cDNA library from LLC-PK, cells, and after linking adaptors to the ends, the cDNAs were inserted into the mammalian expression

Cloning 777

of the TGF-b

Type

II Receptor

A -241 -121 -1 ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACRCGACATGATAGTCACTGAC MetGlyArqGlyLeuLeuArqGlyLeuTrpProLeuHisIleValLeuT~pThrArgIleAl~S~rTh~ll~P~~P~~Hi~V~lGl"Ly~S~rValA~"A~"A~pM~tll~v~lTh~A~p T AACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCRACTGCAGCATCACCTCCATCTGTGAGAAGCCA As"AsnGlyAlaValLysPheProGl"LeuCysLysPheCysAspValArgPheSerThrCysA~pAsnGlnLysSerCy~M~tSerAsnCysSe~ll~ThrSerIleCy~Gl~Ly~P~~ + + + + + # + CAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACAT~CACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCA~G GlnGluValCysValAlaValTrpArgLysAsnAspGluAsnIleThrLeuGluThrValCysHisAspP~~LysLeuProTyrHisAspPheIl~LeuGluAspAlaAl~S~rProLys + + # TGCATTATGAAGGAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATAT~CACCAGCAATCCTGACTTG CysIleMetLysGluLysLysLysPKOGlyGluTh~PhePheMetCySSe~CySSe~Se~AspGluCy~A~nA~pAs"IleIl~PheSerGluGl"TyrAsnThrSerA~nP~~A~p~ + + + t # TTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTT~CCGGCAGCAG~GCTGAGTTCAACC +ArgValAsnArgGlnGlnLysLeuSerSerThr

120 40 240 80 360 120 480 160 600 200

TGGGAAACCGGCAAGACGCGGAAGCTCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGATGACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATCAACCACRACRCACAGAG 720 TrpGluThrGlyLysThrArgLysLeuMetGluPheSerGluRisCysAlaIleIleLeuGluAspAspArgSerAspIleS~rSerThrCysAlaAsnA~nIleAsnHisAsnThrGlu 240 s s CTGCTGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTTGCTGAGGTCTATAAGGCCAAGCTGAAGCAGAACACTTCAGAGCAGTTTGAGACAGTGGCAGTCAAGATCTTTCCC 840 LeuLeuProIleGluLeuAspThrLeUValGlyLysGlyAKgPheAlaGlUValTyrLysAlaLysLe"Ly~Gl"A~nThrS~~Gl"Gl"PheGl"Th~ValAlaValLy~Il~Ph~P~o 280 -3 TATGAGGAGTATGCCTCTTGGAAGGACAGGAAGGACATCTTCTCAGACATCAATCTGAAGCATGAGAACATACTCCAGTTCCTGACGGCTGAGGAGCGG~GACGGAGTTGGGGAAACAA 960 TyrGluGluTyrAlaSerTrpLysAspArgLysAspIlePheSerAspIleAsnLeuLysHisGluAs"IleLeuGlnPheL~~ThrAlaGluGl~ArgLysThrGluLeuGlyLysGln 320 s TACTGGCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACGCGGCATGTCATCAGCTGGGAGGACCTGCGCAACGTGGGCAGCTCCCTCGCCCG~GGATTGTCTCAC 1080 TyrTrpLeuIleThrAlaPheHisAlaLysGlyAs"LeuGlnGluTyrLeuThrArqHisValIleSe~T~pGluAspLeuA~gAsnValGlySerSerLeuAl~ArgGlyLeuSe~His 360 1200 400 1320 440

TCCCTGCGTCTTGGACCCTACTCTTCTGTGGATGACCTGGCTAACAGTGGGCAGGTGGGAACTGCAAGATACATGGCTCCAGAAGTCCTAGAATCCAGGATGAATTTGGAGAATGCTGAG SerLeuArqLeuGlyProTyrSerSerValAspAspLeuAlaAs"SerGlyGl"ValGlyThrAlaArgTyrMetAlaProGluValLeuGluSerArqMetAs"LeuGluAsnAlaGlu s s TCCTTCAAGCAGACCGATGTCTACTCCATGGCTCTGGTGCTCTGGGAAATGACATCTCGCTGTAATGCAGTGGGAGAAGTA~AGATTATGAGCCTCCATTTGGTTCCAAGGTGCGGGAC SerPheLysGlnThrAspValTyrSerMetAlaLeuValLeuTrpGluMetThrSerArgCysAsnAl~V~lGlyGluValLysAspTyrGluProP~oPheGlySe~LysValArgAsp $ $ CCTGTGGTCGAAAGCATGAAGGACAACGTGTTGAGAGATCGAGGCACCAGAATTCCAGCTTCTGGCTCAACCACCAGGGCATCCAGATGGTGTGTGAGACGTTGACTGAGTGCTGGGAC ProValValGluSerMetLysAspAsnV~lLeuArgAspArgGlyThrArqAsnSe~SerPh~TrpLe~A~"Hi~GlnGlyIl~GlnMetValCy~Gl~ThrLeuThrGl~Cy~T~pAsp s CACGACCCAGAGGCCCGTCTCACAGCCCAGTGTGTGGCAGAACGCTTCAGTGAGCTGGAGCATCTGGACAGGCTCTCGGGGAGGAGCTGCTCGGAGGAGAAGATTCCTGAAGACGGCTCC XisAspProGluAlaArqLeuThrAlaGlnCysValAlaGluArgPheSeKGluLeuGluHi~LeuAspArgLeuSerGlyArqSerCysSerGluGluLysIleProGluAspGlySer t s CTAAACACTACCAAATAGCTCTTATGGGGCAGGCTGGGCATGTCCAAAGAGGCTGCCCCTCTCACCA~ LeuAsnThrThrLys*** S

1440 480 1560 520 1680 560 1749 565

B human

(1)

Pig

(1)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVA~RKNDENITLETVCHDPKLPYHD MGRGLLGGLWPLHVVLWTRIASTIPPHVPKSVNSDMMVTDSNGAVKLPQLCKFCDVRSSTCDNQKSCLSNCSITAIC~KPQ~VCVA~RKND~NITI~TVCDDPKIAYHG l

human

(1111

pig

(111)

human pig

Figure

(2211 (221)

2. Sequence

I

****

*

*

*

*

.

l

FILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRK~EFSEHCAIIL FVLDDAASSKCIMKERKGSGETFFMCSCSSDECNDHIrFSEFSEHLAIIL f. * l l * * + I t *I l l fi ** l

EDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKDRKDIFSDI EDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKKLRQNTSEQFETVAVKIFPYEEYASWKDRKDIFSDL 1

of the TGF-8

f

i*.

l

(297) (297)

Type II Receptor

(A) DNA and translated amino acid sequences of clone H2-3FF. The open reading frame and flanking nucleotide sequences of clone H2-3FF (full insert size is -4.7 kb) are shown, The upstream inframe TGA stop codon (-21 to -19) is indicated by the wavy line. The potential signal peptide cleavage site after Thr-23, predicted by von Heijne’s algorithm (von Heijne, 1986), is indicated by the vertical arrow. Consensus N-linked glycosylation sites are indicated by # and extracellular cysteines by +. The single transmembrane domain is underlined. Consensus protein kinase C phosphorylation sites are indicated by $. Horizontal arrows mark the extent of the kinase domain. (B) Comparison of the amino acid sequence of the porcine and human receptors. The porcine receptor is encoded by clone P2-3F and the human receptor by clone H2-3FF. Asterisks denote differences in amino acids between the two receptors. Amino acid 297 of the porcine sequence is encoded by vector sequences, A line over the sequences represents the transmembrane domain.

Cell

778

I32 !nM) fll type

:ype type

(nM) III

-

r-----7-

5

-

initiation codon is favorable for translational initiation (Kozak, 1987). These data alone, however, did not allow us to identify the product of this cDNA as the type I or type II receptor. - - - .25.5

- .25 5 -

kd -200

-92.5

iT AL -

-

69

-

46

I-

12345678 Figure 3. Crosslinking of [‘251]TGF-b1 Mock-Transfected CO.? Cells

to Hep G2 and Transfected

and

Hep G2 cells (lane l), COS cells mock-transfected with the endothelin receptor cDNA (Lin et al., 1991a) (lanes 2 and 3) and COS cells transfected with the human TGF-6 type II receptor clone H2-3FF in pcDNA-1 (lanes4-8)were incubated with 50pM [‘ZSI]TGF-61 and crosslinked with disuccinimidyl suberate before analysis on SDS-PAGE as described in Experimental Procedures. Lanes 3 and 7 contain 1 OO-fold (5 nM) excess TGF-31; lane 5, 5-fold (0.25 nM) excess TGF-61; lane 6, 1O-fold (0.5 nM) excess TGF-61; lane 8, 5fold (0.25 nM) excess TGF-52. The different TGF-6 receptor subtypes are indicated, as are molecular weight markers.

vector pcDNA-1 (InVitrogen). Aftertransfection into E. coli, pools of rulO,OOO independent clones were separated and grown on LB agar plates. Plasmidswith cDNA inserts were harvested from each pool. COS cells grown on glass slide flaskettes were transfected with cDNA pools, allowed to bind radioiodinated TGF-81, fixed with glutaraldehyde, and overlaid with photographic emulsion for autoradiography. We screened for positive cells using dark-field microscopy. One pool (#97) produced a COS cell that could clearly bind radioiodinated TGF-81 (Figures 1A and 1 B). The subsequently isolated single pure clone, P2-3F, produced ligand binding in ~10% of transfected COS cells that was specifically inhibited by excess unlabeled TGF-81 (Figures 1 C and 1 D) (see Experimental Procedures). Isolation of a Full-Length Human Receptor cDNA The P2-3F cDNA isolated by expression screening contained an open reading frame of 297 amino acid residues, of which residue Leu-297 was encoded by vector sequences, clearly indicating that clone P2-3F was an incomplete cDNA. This porcine cDNA clone was used as a probe to isolate the full-length coding region of the corresponding human receptor by screening a h-ZAP II Hep G2 human hepatoma cell cDNA library (see Experimental Procedures). The resulting 4.7 kb human clone, termed H2-3FF, encoded a protein of 565 amino acids (Figure 2A); its amino acid sequence was colinear with that of clone P2-3F, with 88% amino acid identity (Figure 28). Clone H2-3FF encoded the complete receptor protein, since there is an in-frame stop codon upstream of the initiator ATG and the nucleotide context surrounding this

Characterization of the Expressed Human Receptor in COS Cells In order to characterize the product of the clone H2-3FF, we subcloned it into the EcoRl site of the plasmid pcDNA-1 and introduced the construct by transfection into COS cells. Transfected COS cells were treated with radioiodinated TGF-61 and incubated with the chemical crosslinker disuccinimidyl suberate, and any resulting labeled receptors were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 3, lane 2, shows that mocktransfected COS cells express mainly the type I and type Ill TGF-8 receptors, and lane 3 shows that competition with lOO-fold unlabeled ligand inhibits crosslinking of radioiodinated TGF-61 to all three surface receptors. Transfecting the H2-3FF cDNA into COS cells caused a several-fold increase in expression of a protein comigrating with the TGF-8 type II receptor (lane 4). Crosslinking of radioiodinated TGF-61 to this protein was reduced by incubation with unlabeled TGF-61 in a concentrationdependent manner (lanes 5-7). The abilities of TGF-81 and TGF-82 to compete with [1251]TGF-f31 for binding to endogenously expressed type Ill receptor were roughly equivalent, but binding of TGF-81 to the overexpressed type II receptor was competed far more effectively by TGF-61 than TGF-62 (compare lanes 5 and 8) a characteristic of the type II receptor (Roberts and Sporn, 1990; Massag&, 1990). We conclude that clone H2-3FF encodes a human protein with the properties of the TGF-6 type II receptor. Expression of Type II Receptor mRNA Figure 4 shows expression of the type II mRNA in several different tissue culture cell lines described by Wang et al. (1991). In most cells a single m5.5 kb message exists; a second species of ~3 kb is also present in normal AD1 2 retinoblasts. This species may represent an alternatively spliced message. The type II receptor mRNA is absent in retinoblastoma cells (Y79, Weri-1, Weri-24, and Weri-27 cells), which lack cell-surface expression of the receptor and lack responsiveness to TGF-6 (Kimchi et al., 1988). Breast carcinoma cells that lack responsiveness toTGF-8, MCF-7(-), express fewer cell surface type II receptors (Arteaga et al., 1988; Zugmaier et al., 1989) and correspondingly less mRNA than cells that do express type II receptor protein and are growth inhibited by TGF-6 (MCF-7(+)). Thus, expression of the TGF-8 type II mRNA corresponds with the expression of type II receptors and with responsiveness to ligand. Note that the L6 and LLC-PK, cells which do not express detectable type Ill receptor mRNA (Wang et al., 1991) express abundant type II receptor mRNA, confirming that expression of the mRNAs for the type II and type Ill receptors is independently regulated. Sequence Analysis The H2-3FF cDNA clone (Figure 2A) encodes a protein of 565 amino acid residues with a calculated mature molecu-

Cloning 779

of the TGF-6

Type II Receptor

Figure 4. RNA Blot Analysis Receptor mRNA

of TGF-I)

Type

II

Polyadenylated mRNA (2 ug) was prepared from various cell lines, resolved on a 1% agarose gel containing formaldehyde, blotted onto I I I I I I I I II I I I I I III1 a nylon membrane (Biotrans, ICN), and probed with clone P2-3F. AlO, rat embryonic thoracic i. .:.! smooth muscle; PC12, rat pheochromocy‘I 28Stoma; GH3, rat pituitary; L6, rat myoblast; N2A, mouse neuroblastoma; MEL, mouse erythro18Sleukemia; ATT20, mouse pituitary; LLC-PK,, pig kidney epithelial; Hep G2. human hepatoma; MCF-7 (+) and MCF-7 (-), human breast carcinoma; AD12, human retinoblasts; Y79, tubulin Weri-1, Weri-24, and Weri-27, human retinoblastoma; HT-29, human colonic epithelial; YH16, mouse B cell; 3-4, mouse BAF3 clone. A parallel filter probed with rat a-tubulin cDNA (Lemischka et al., 1961) probed filter is shown as control for mRNA loading. Note: This blot represents an exact duplicate of the one utilized in Wang et al. (1991). This tubulin control figure is identical to that shown previously in Wang et al. (1991) and is included for completeness

lar weight of m60 kd. This compares well with the ~65 kd mass estimated for the deglycosylated TGF-8 type II receptor and unfavorably with the ~48 kd mass estimated for the type I receptor (Roberts and Sporn, 1990; Massag&, 1990). The porcine (clone P2-3F) and human (clone H2-3FF) amino acid sequences align without gaps, and they share 88% amino acid identity in the sequenced portion (Figure 28). There is a predicted hydrophobic signal sequence (von Heijne, 1986) followed by a cysteinerich, hydrophilic, presumed extracellular domain containing three consensus N-linked glycosylation sites. Further towards the C-terminus is found a 30 residue region of strong hydrophobicity (residues Leu-160 to Tyr189) which represents the single putative transmembrane domain (Kyte and Doolittle, 1982). These features are schematically illustrated in the scaled drawing in Figure 5A. Noteworthy is the lackof homology between the extracellular domains of the TGF-8 type II and type Ill receptors (Wang et al., 1991).

The cysteine-rich extracellular region of the type II receptor does show a small region of relatively high amino acid identity, underlined in Figure 58, with two other putative transmembrane serine/threonine kinases, daf-1 (Georgi et al., 1990) and the activin receptor (Mathews and Vale, 1991). The cysteines within this region align quite well, as do several polar residues. Other parts of the cysteine-rich domains also appear to share homology with these other receptor proteins (Figure 58) although the sequence homology and alignment of the cysteines becomes less pronounced towards the N-terminus. The type II receptor extracellular region shares no regionsof homologywith other receptors, such as the epidermal growth factor (Ullrich et al., 1984) and the platelet-derived growth factor receptor (Yarden et al., 1986). Comparison of the cytoplasmic domain of the TGF-8 type II receptor with sequences in databases reveals that the most similar proteins are the activin receptor and daf-1 gene product, both of which have been predicted to be

Figure 5. Domain II Receptor

mACTRI1

;zGFf;PRII mACTRI1

~$LPY$FILE

gSVTpFmTEFH . . wLnp&

ii..

(143) (152) (110)

Structure

of the TGF-6 Type

(A) A scaled schematic representation of the human receptor (hTGF+RII) encoded by clone H2-3FF, indicating the extracellular cysteinerich domain in relationship to the transmembrane domain and the serine/threonine kinase domain. (6) Comparison of the cysteine-rich extracellular domains of receptor serine kinases. Regions were aligned initially by the UWGCG program GAP (Devereaux et al., 1964) and then the cysteines were manually spaced to produce maximal alignment. The underlined region represents the region of highest homology between the receptors. hTGF-pRII, human TGF-6 type II receptor; mACTRII, mouse activin receptor (Mathews and Vale, 1991); daf-1, daf-1 receptor (Georgi et al., 1990). Reversed script is used to highlight all the cysteine residues, while shaded print is used to highlight those residues found in two or all three of the receptors.

Cdl 780

A I

receptor

serine

kinase

subfamily 1

DAFl

(33%)

tyrosine kinase subfamlly

cdc2/casein kinasc subfamily

calmodulin

kinase/ ‘c-MOS

wee1

ZmPKl

(20%)

(23%)

(26%)

B Conserved hTGF+RII mACTRI1 daf-1 Subdomains

aa

Conserved hTGF+RII mACTRI1 daf-1 Subdomains

aa

Conserved hTGF+RII mACTRII daf-1 SubdomaIns

aa

Conserved h' TGF-PRII mACTRI1 daf-1 Subdomains

aa

Figure

6. Simrlanty

AK KQNTSEQ LN..... RG..... I

II

V

TRHVISWEDLRNVGSS KAN@~NELcHI~T LENTVNIETYYNLM& VI-A

E YDHY~WKDRKD~FjSD IQDKQSWQNEYEVYSL ALOEPAFHKETEIFET III

ELGKQ WDVD GFVTE IV

DLK

RL...GPYSSVDDLANSG KF...EAGKSAGD..THG SKPEDAASDIIAN..ENY VII

CF

VI-B

D WS G

APE

G

N

RJTP . CG WGLKD QIGGSKE

n? YM RE

ENAEXFK QR.hFL TVFESYQ IX

VIII

R E'PFG....SKVRDPWESMKD LPFE....EEIGQEiPSLEDMQ AATVIPYIEWTDRDPQDA@lJ X

of the TGF-f3 Type

II Receptor

to Other

(542)

(466) (592) XI Protein

Kinases

(A) Relationship of the TGF-b receptor kinase domain to other protein kinases. The kinase domains of representative sequences were aligned and their evolutionary relatedness calculated as described (Hanks and Quinn, 1991). Scale bar represents a relative score of 25. ActRII, mouse activin receptor (Mathews and Vale, 1991); DAF-1. daf-1 receptor (Georgi et al., 1990); ZmPKl, ZmPKl receptor (Walker and Zhang, 1990). References for the other sequences are found in Hanks and Quinn (1991). The amino acid identity in the kinase domain between the TGF-6 type II receptor (hTGF-bRII) and the other kinases are noted as a percentage. We thank A. M. Quinn and L. S. Mathews for preparing this figure. (6) Comparison of the TGF-8 type II receptor kinase domain to the activin receptor and the daf-1 receptor kinase domains. Residues found rn all three receptors are in reverse print those common in two of the three are in shaded print. “Conserved aa” refers to those residues found to be invariant or highly conserved in the serine/threonine kinase subfamily, as described in Hanks and Quinn (1991). “Subdomains” refers to the subdomains of the kmases (see Hanks and Quinn, 1991).

Cloning 761

of the TGF-b

Type II Receptor

B

A

Figure 7. Autophosphorylation of the Cytoplasmic Domain of the TGF-f3 Type II Receptor

Phosphoamino Acid Analysis of GST-type II Receptor Fusion Protein

123

kd - 200 - 118 - 97 - 68

free

PO4 -

ser-PO4 thr-PO4

-

tyr-PO4

-

origin

-

- 40

serinelthreonine kinases on the basis of their similarly structured primary sequences. Indeed, comparison of the cytoplasmic segment of the type II receptor with other known kinases, both serinelthreonine and tyrosine kinases, reveals a ~20% overall amino acid identity (Figure 6A). The cytoplasmic domain of the type II receptor can be parsed into subdomains based on regions of invariant and highly conserved residues (Figure 66) as has been described for other kinases (Hanks et al., 1988; Hanks and Quinn, 1991). For example, in subdomains I and II of kinases is the highly conserved motif GXGXXGXVXU,+K, which is thought to form the ATP binding site. The type II receptor in the corresponding region has the sequence GKGRFAEVX(,,K, which aligns well with this consensus (Figure 6A). The substitution of alanine for the third conserved glycine has also been observed in several other kinases (Hanks and Quinn, 1991). Noted in Figure 66 are 22 invariant or highly conserved amino acids found in the serine/threonine kinase subfamily of protein kinases (Hanks and Quinn, 1991). The TGF-6 type II receptor exhibits 18 of 22 of these residues in its putative kinase domain. Closer examination of subdomains VI-B and VIII, which are thought to distinguish tyrosine kinases from serinelthreonine kinases and have been used to classify predicted kinases as one or the other, reveals that the type II receptor has the signatures of the serinelthreonine family (Figure 6B) (Hanks et al., 1988). Thus, the relevant type II receptor sequences in subdomain VI-B (DLKSSN) are more like that of the serinelthreonine kinase consensus (DLKPEN) than the tyrosine kinase consensus (DLAARN). Comparison of the relevant residues in domain VIII yields the same conclusion: the type II receptor has the sequence GTARYM, which corresponds well to GTISXXYIPX for serine/threonine kinases and poorly to XPINKIRWTIM for tyrosine kinases (Hanks et

(A) Kinase assay of GST fusion proteins. Purified proteins bound to glutathione-agarose beads were incubated with 200-300 mCi of y[“P]ATP for 30 min at room temperature before analysis on 7% SDS-PAGE (see Experimental Procedures). Lane 1, GST; lane 2, GST-type II TGF-5 receptor cytoplasmic domain fusion: lane 3, GST-type III TGF-5 receptor fusion (Wang et al., 1991). (B) Phosphoamino acid analysis. HCI-hydrolyzed material derived from Y[~P]ATPlabeled fusion protein was spotted on a thin layer cellulose paper and resolved by electrophoresis at pH 3.5. Phosphoamino acid spots were visualized by spraying with a ninhydrin solution, then the paper was exposed to an X-ray film. The positions of all phosphoamino acids and the origin are marked. We thank J. Kyriakis and J. Avruch for their help in preparing this figure.

al., 1988). Based on these analyses, we predict that the type II receptor is a kinase having specificity for serine and threonine residues. Kinase Activity of the Cytoplasmic Domain In order to test the prediction that the cytoplasmic domain is indeed a kinase, we fused nearly the entirety of the reading frame encoding the cytoplasmic domain (Asn-192 to Lys-565) to a glutathione S-transferase (GST) gene (see Experimental Procedures). The resultant fusion protein was expressed in E. coli and purified using glutathioneagarose beads in a one-step procedure as described by Smith and Johnson (1988). Purified fusion protein coupled to glutathione-agarose beads was incubated with $*P]ATP for 30 min at room temperature before the reaction was stopped and the samples were analyzed by SDS-PAGE analysis. Figure 7A, lane 2, shows that the m66 kd GST type II receptor kinase chimeric protein can autophosphorylate itself, while GST itself (lane 1) and GST fused to the TGF-8 type Ill receptor (lane 3) show no evidence of phosphorylation, even after prolonged exposure of the autoradiogram. This provides the first direct evidence that these receptor proteins indeed contain functional kinase domains. Whether the individual kinase molecules can autophosphorylate themselves or only transphosphorylate one another will require further study. The identity of the phosphoamino acid was examined by analysis of an acid hydrolysate by thin layer chromatography, and showed that the fusion protein is autophosphorylated only on serine and threonine residues (Figure 78). Since there are numerous serines and threonines in the intracellular domain of the receptor protein, and many of these are consensus targets for other kinases such as protein kinase C (Figure 2A), the location of these phosphorylation sites will require detailed analysis.

Cell

782

Discussion A number of receptors for ligands such as GM-CSF (Gearing et al., 1989), endothelin-1 (Lin et al., 1991a), calcitonin (Lin et al., 1991 b), parathyroid hormone (Juppner et al., 1991), leukemia inhibitory factor (Gearing et al., 1991) and activin (Mathews and Vale, 1991) have been isolated by utilizing an expression cloning strategy, in which pools of cDNAsare transfected into COS cells followed byvisualization of positive clones using single cell autoradiography. Most recently, we have employed this strategy to isolate the TGF-6 type Ill receptor (Wang et al., 1991). This method of expression cloning is advantageous for several reasons. First, the proceduredoes not require prior information regarding the structure of the receptor, thus eliminating the need for often difficult, expensive, and time-consuming purification of the receptor protein. Second, direct visualization of positive cells (see Figure 1) permits an unparalleled signal-to-noise ratio, allowing one to distinguish easily true positives from false positives, which may be caused by nonspecific aggregation of radioligand. This resolution was crucial in the cloning of the TGF-6 receptor genes because untransfected COS-7 cells express a high number of TGF-6 binding sites (estimated at >200,000 per cell) (Wang et al., 1991). However, a single, positively transfected COS cell can express several million cell-surface receptors per cell (Gearing et al., 1989; our unpublished data), creating enough binding sites to allow one to distinguish it easily from nontransfected neighboring cells. We were surprised that the porcine and human TGF-6 type II receptors shared only88% amino acid identity(85% in the extracellular domain) (Figure 2l3), because the mature forms of human and porcine TGF-8 ligand are 100% identical. The differences in the residues between the porcine and human receptors may reflect those residues that are not exposed to the environment or are not essential for the structure of or ligand binding to the mature receptor protein. The absolute conservation of the TGF-8 ligand structure across species may be reflective of the multiple interactions that these molecules must participate in to function properly. Since activin and TGF-6 are members of the same superfamily of ligands and share the same overall structural features, it is not surprising that the extracellular regions of their receptors share asegment of relatively high amino acid sequence identity (Figure 58). An alignment of this highly conserved region shows that the cysteines in it align quite well. Since cysteines are thought to form disulphide linkages, their conservation may indicate the presence of a biologically important structural framework. Another putative receptor serine kinase, the C. elegans daf-1 (Figure 56) a protein involved in larval development (Georgi et al., 1990) also shares this conserved motif. We would speculate that the still unknown ligand for daf-1 will be found to be a member of the TGF-8 superfamily of ligands. Other members of the TGF-6 superfamily of ligands besides activin and TGF-8 may have receptors that are serine/threonine kinases and that contain this conserved extracellular receptor serine kinase (RSK) motif. Indeed,

while the gene product described here binds TGF-61, we do not address the possibility that this receptor binds other members of the TGF-8 family of ligands. In the kinase region, the type II receptor shares 45% and 32% amino acid identity with the activin receptor and daf-1 gene product, respectively (Figure 6B). A phylogenetic analysis of the kinase domains of these receptors indicates that these receptors define a newly recognized and emerging subfamily of receptor serine kinases (Figure 6A) (Hanks and Quinn, 1991). The other known receptor serine kinase, ZmPKl (Walker and Zhang, 1990), appears to be much more distantly related to the other receptors in both its extracellular and intracelluar domains (Figure 6A). Although kinase activity has not yet been demonstrated in these receptors, the ability of the TGF-8 type II receptor to autophosphorylate on serine and threonine residues makes it likely that other proteins also function as serinelthreonine kinases. Recall that there are several cell surface receptors that bind TGF-6. In particular, we showed that expression in L6 myoblasts of the type Ill receptor increases the ability of the type II receptor to bind radioiodinated TGF-61 (Wang et al., 1991). While L6 cells do not require type Ill receptors for functional responsiveness toTGF-6 (Florini et al., 1986; Massague et al., 1986), the type III receptor presumably increases the sensitivity of the type II and possibly the type I receptors to ligand. How the receptors for TGF-6 interact with each other to transduce the TGF-6 signal is not known, but our results implicate serinelthreonine phosphorylation as an important mechanism in TGF-6 signaling. Polypeptide growth factors for transmembrane receptors with intrinsic tyrosine kinases have been extensively studied (reviewed in Ullrich and Schlessinger, 1990) and much detail has emerged regarding their function. In general, these receptors act to transduce mitogenic or trophic signals into the cell, and we might speculate that in certain cell types, the growth-stimulating signals released by their associated tyrosine kinase domains may be countered by antimitogenic signals emitted by the serinelthreonine kinase of the TGF-f3 receptor. In the case of these tyrosine kinase receptors, a group of receptor-associated effecters has already been identified, including phospholipase C-r, phosphatidylinositol 3-kinase, and GAP (GTPase-activating protein). Comparable effecters mediating TGF-8 signaling are still obscure. Their identification will be essential to understanding further steps in the signaling pathway triggered by TGF-8. Experimental

Procedures

Materials Recombinant humanTGF$l wasgenerouslyprovided by Ark Derynck of Genentech, and TGF-62 was purchased from R+D Systems (Minneapolis). COS-M6 ceils were provided by Brian Seed of Massachusetts General Hospital and Alejandro Aruffo of Bristol-Myers Squibb. LLCPK, cells were a gift of Dennis Ausiello of Massachusetts General Hospital. Expression Cloning Mammalian Expression Library Construction Polyadenylated mRNA (10 ug) was prepared from (ATCC CCI 101) using a proteinase-Wsodium dodecyl

LLC-PK1 ceils sulfate (SDS)

Cloning 783

of the TGF-P

Type

II Receptor

digestion method (Gonda et al., 1982). After synthesis of doublestranded cDNA and addition of nonpalindromic BstXl adaptors (Seed and Aruffo, 1987), cDNA was layered onto a 5%-20% potassium acetate gradient and size fractionated. Inserts greater than 2 kb in size were ligated to the plasmid vector pcDNA-1; the ligation mixture was subsequently electroporated into the E. coli strain MC10611P3. This procedure yielded a predicted library of >lO’ recombinants. Pools of 5 x lo3 to 104 recombinant bacteria were grown on Luria broth agar petri dishes containing ampicillin (12.5 pglml) and tetracycline (7.5 wglml). Bacterial colonies were scraped from the dishes and aliquots saved in 15% glycerol stock at -70°C. Plasmid DNA was purified from the remainder of the sample using the alkaline lysis mini-prep method (Sambrook et al., 1989). Transfection of COS Cells and Generation of Plasmid Subpools COS-M6 cells, a subclone of COS-7 cells, were grown on glass slide flaskettes (Nunc) and transfected with an aliquot from each pool of plasmid cDNA using a DEAE dextranlchloroquine procedure (Aruffo’ and Seed, 1987). Forty to sixty hours later, 50 pM [‘251]TGF-~1 (loo200 Cilmmol) was added to the transfected cells and allowed to bind at 4“C for 4 hr before the cells were washed four times in BSA (bovine serum albumin)-free binding buffer and fixed with a 1% glutaraldehyde solution as described (Wang et al., 1991). Following fixation, the plastic chamber and rubber gasket were removed from the glass slide. The glass slides were then subjected to autoradiographic analysis essentially as described (Gearing et al., 1989). Positive COS cells covered with silver grains were identified using dark-field microscopy. After screening nearly 1 million clones, we identified one pool (LLCPK, #97) representing -7500 independent recombinant cDNAs as being positive. cDNA from this pool was transfected into MC1061/P3 cells, and subpools of cDNA were prepared and retransfected into COS cells. Several rounds of iterative subpooling and rescreening eventually yielded a single positive clone (P2-3F) containing a 0.9 kb insert that encoded a 297 amino acid protein (Figure 28). Cloning of H2-3FF A cDNA library was generated in h ZAP II (Stratagene) from sizeselected cDNA made from the human hepatoma cell line, Hep G2 (ATCC HB 8065). This library was screened at high stringency with the clone P2-3F and yielded several clones of -4.7 kb size. One such clone was selected for further study and termed H2-3FF. It encoded an open reading frame of 565 amino acids, of which the first 296 were colinear with clone P2-3F (Figure 28). DNA Sequencing and Sequence Analysis The dideoxy chain termination method was used to sequence doublestranded DNA utilizing reagents from Sequenase (United States Biochemicals). Comparisons of the sequences to the data bases was performed using BLAST (Altschul et al., 1990). A comparison of the human TGFj3 type II receptor kinase domain to other kinases in the database was performed as described by A. M. Quinn (Hanks and Quinn, 1991). L. S. Mathews constructed the phylogenetic tree in Figure 6A based on information generated by the comparison. lodination of TGF-PI TGF-Pl was iodinated (Cheifetz et al., 1988).

using

the chloramine

T method

as described

Chemical Crosslinking of [‘z51JTGF-~1 to Cells Binding and crosslinking of [‘251]TGF-P1 to Hep G2 cells or transfected COS cells grown on six-well trays (Costar) was as described by Wang et al. (1991). In brief, cells were incubated with 50 pM [‘ZSl]TGF-~l for 4 hr at 4OC in KRH buffer (Krebs-Ringer; 20 mM HEPES [pH 7.51, 5 mM MgSO$with0.5% BSA.Afterwashingcellsfourtimeswith ice-cold KRH buffer, cells were incubated for an additional 15 min in KRH with 60 pglml of disuccinimidyl suberate under constant rotation. The crosslinking reaction was stopped by addition of 7% sucrose in KRH, and cells were scraped, collected, and pelleted by centrifugation before lysis in buffer containing IO mM Tris (pH 7.4), 1 mM EDTA (pH 8.0), 1% Triton X-100, 10 mglml pepstatin, 10 mglml leupeptin, 10 mglml antipain, 100 mglml benzamidine hydrochloride, 100 mglml soybean trypsin inhibitor, 50 mglml aprotonin, and 1 mM phenylmethylsulfonyl fluoride. Crosslinked material was resolved by 7.5% SDSPAGE and exposed to XAR film (Kodak) at -7O’C.

RNA Blot Analysis mRNA (2 vg) prepared by the proteinase KlSDS method (Gonda et al., 1982) was resolved byelectrophoresis on 1% agarose-2.2 M formaldehyde gels before being blotted onto nylon membranes (Biotrans, ICN). As a probe, the porcine partial cDNA clone P2-3F insert of 0.9 kb was labeled with [32P]dCTP by random priming (Sambrook et al., 1989). Hybridizations were performed in buffer containing 50% formamide at 42OC for 16 hr, and blots were washed at 55OC in 0.2x SSC, 0.1% SDS, and exposed to XAR film at -7OOC. Fusion Proteln Construct The Hpal-Hindlll fragment (-2.8 kb) of H2-3FF was end-filled and ligated to the Smal site of the vector pGEX-3X (Smith and Johnson, 1988). This resultant construct encoded a 66 kd fusion protein that consists of an N-terminal GST of 26 kd and a C-terminal ~40 kd cytoplasmic domain of the type II receptor (Asn-192 to Lys-565). After expression of the fusion product in E. coli, a one-step purification of the fusion protein was carried out as described by Smith and Johnson (1988). Kinase Reaction Kinase reactions were carried out by adding fusion proteins still bound to glutathione-agarose beads into buffer that contained 50 mM Tris (pH 7.5), 10 mM MgCI,, 1 mM CaC12, and 200-300 mCi of y[“P]ATP (-7000 Cilmmol) in a volume of 30 ~1. Reaction mixtures were incubated at room temperature for 30 min before the samples were analyzed on 7% SDS-PAGE. Phosphosmlno Acid Analysis One microgram of fusion protein labeled in vitro with y[32P]ATP was resolved by SDS-PAGE and then transferred to lmmobilon PVDF Membrane (Millipore). The membrane containing the fusion protein was then incubated with 200 ml of HPLC grade 6N HCI in a 1 ml glass reaction vial for 4 hr at llO°C. After a brief centrifugation. the supernatantcontainingsolubilized materialwasremoved, dried undervacuum, and resuspended in pH 3.5 buffer (containing 10 ml of pyridine and 100 ml of glacial acetic acid per liter). A sample with *3,000 cpm was spotted onto thin layer cellulose paper (Kodak) and resolved by electrophoresis in pH 3.5 buffer using a Pharmacia FBE-3000 electrophoresis unit (800 V, 1 hr). The phosphoamino acid spots were identified by spraying the sheet with a 0.25% ninhydrin solution in acetone. The sheet was then exposed to an X-ray film. Acknowledgments The contributions of the first two authors should be considered as equal. We thank R. Derynckfor providing recombinant human TGF-bl; J. Kyriakis and J. Avruch for preparing Figure 78; L. S. Mathews and A. M. Quinn for preparing Figure 6A; M. Estevez and D. L. Riddle for identifying the RSK motif in the extracellular domain; B. Seed and A. Aruffo for providing COS-M6 cells: L. F. Kolakowski, Jr., and P. Matsudairaforsequenceanalysis; S. Mittnacht for helpwith the kinase assay in Figure 7A; A. Moustakas for help with gel electrophoresis in Figure 3; A. Geiser, A. Roberts, and M. Sporn for communications of unpublished results; and members of the Weinberg and Lodish laboratories for their support, advice, and encouragement. This work was supported in part by National Cancer Institutegrant R35-CA39826 (R. A. W.) and a National Heart, Lung, and Blood Institute Centers of Excellence grant HL-41484 (H. F. L.). R. A. W. is a Research Professor of the American Cancer Society. H. Y. L. was supported by National Institutes of Health predoctoral training grant T 32 GM07287-16. X.-F. W. was supported by a postdoctoral fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

December

24, 1991; revised

January

10, 1992

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Accession

The accession M85079.

number

Number for the sequence

reported

in this

paper

is