The cDNA sequence and primary structure of the chicken transferrin receptor

G’ene, 102 (1991) 249-254 0

1991 Elsevier

GENE

Science

Publishers

249

B.V. 0378-I 119/91/$03.50

04065

The cDNA sequence and primary structure of the chicken transferrin receptor (Recombinant

Elizabeth

DNA;

deduced

amino acid sequence;

M. Gerhardt a, Lee-Nien

evolutionary

conservation;

primer extension)

L. Chan *, Shuqian Jing b, Meiying Qib and Ian S. Trowbridgeb

a Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550-2774 (U.S.A.), bDepartment of Cancer Biology, The Salk Institute for Biological Studies, San Diego, CA 92186-5800 (U.S.A.) Tel. (619)453-4100 Received by S.T. Case: 16 November Accepted: 24 January 1991

and

1990

SUMMARY

Recombinant cDNA clones encoding the chicken transferrin receptor (cTR) have been isolated and sequenced. Comparison of the deduced primary structure of cTR with those of the human transferrin receptor (hTR) and mouse transferrin receptor (mTR) shows that their size, hydropathy profile, location of sites for posttranslational modifications, and domain organization are highly similar. The cytoplasmic domain of cTR contains the motif Tyr-Xaa-Arg-Phe (YXRF) that is the recognition signal for high-efficiency endocytosis of hTR. The cTR has several highly conserved regions within its extracellular domain, including those flanking the putative N-glycosylation sites. Overall, however, the extracellular domain of cTR is only 53 % identical to the extracellular domains of hTR and mTR. The cTR also lacks three of the six Cys residues found in the extracellular domains of the mammalian TRs. These differences can account for functional and structural properties that distinguish cTR and m~m~ian TRs.

INTRODUCTION

Iron is required for a variety of vital cellular functions and is transported into cells by means of TR-mediated endocytosis of the iron-transferrin complex (Huebers and Finch, 1987). TR expression is regulated in a feedback fashion by the level of intracellular iron (Harford et al., 1991). The hTR has been extensively studied and is a dimeric transmembrane glycoprotein consisting of two identical disulfidebonded subunits of 95 kDa (Omary and Trowbridge, 198 1; Schneider et al., 1982). Biochemical studies of mTR and cTR indicate that they have a similar structure also

Correspondenceto: Dr. L.-N. L. Ghan, Department Chemistry

and Genetics,

TX 775.50-2774 (U.S.A.) Abbreviations: plementary

aa, ammo

University

ofTexas

of Human Biological Medical Branch, Galveston,

Tel. (409)761-2761; acid(s);

to RNA; cTR, chicken

Fax (409)761-5159.

bp, base pair(s);

cDNA,

DNA com-

TR; hTR, human TR; kb, kilobase

or 1000 bp; mTR, mouse TR; nt, nucleotide(s); TR, transferrin receptor; TR, gene (cDNA) aa sequence motif Tyr-Xaa-Arg-Phe.

RBC, red blood cell(s); encoding

TR; YXRF,

the

(Trowbridge et al., 1982; Schmidt et al., 1985). The aa sequences of the hTR and mTR have been deduced from the nt sequences of their cDNAs (McClelland et al., 1984; Schneider et al., 1984; Stearne et al., 1985; Trowbridge et al., 1988). The cytoplasmic domain of the hTR has been implicated in high efficiency endocytosis (Rothenberger et al., 1987) and, recently, the aa sequence within the cytoplasmic domain that determines rapid internalization has been identified (Alvarez et al., 1990; Jing et al., 1990; McGraw and Maxfield, 1990; Collawn et al., 1990). To define other functionally significant regions in TR, comparisons of the TR aa sequence of different species can be useful since such regions are usually conserved through evolution. This situation is especially true for avian and mammalian TRs since they have diverged to the extent that important conserved sequences can be ‘highlighted’ against a background of dissimilar sequences. The nt sequence of the 3’ noncoding region of the cTR cDNA and the identification of two highly conserved regions in this region of TR mRNA which are implicated in the post-transcriptional regulation of cellular TR expression have been reported

250 CCGCGFAGGGGGCGGCGGAGTGTG~AGCGGCGGGCG~CCTTCAGTC~GTT~TGGGTACTCCTCCGG~GGCA

77

ATG FAT CAT GCC AGA GCA GCA TTG TCT MC TTG TTC AGC GTC GAG CCG ATG TCG TAC ACA CGT TTC AGC ATT GCT CGG CAA ACA GAT GGA 1 Mst Asp His Ala Arg Ala Ala Leu Ser Asn Leu Phe Ser Val Glu Pro Met Ser Tyr Thr Arg Phe Ser Ile Ala Arg Gin Thr Asp Gly

167

GAC AAC AGC CAC GTG GAG ATG A4G CTG TCT 31 Asp Asn Ser His Val Glu Met Lys Leu Ser * CAG CCA GAG AGA AAT GGC AAG AGA CTC TGC 61 Gin Pro Gin Arg Am Gly Lys Arg Leu CYS

GCT GAT GAT GAG GAA GGA GGG GAC ATT GA.4 HGG CCA GAG CAC ATG CAT GTC AGT ATG GCT Ala Asp Asp Glu Glu Gly Gly Asp Ile Glu Arg Pro Glu His Met Hxs Val Ser Met Ala

257

TTC TTG GTC ATT GCA GCT GTT CTC CTC CTT TTG ATT GGG Phe Leu Va 1 Ile Ala Ala Val Leu Leu Leu Leu Ile Gly

TTT CTT ATT GGC TAC TTG AGT Phe Leu Ile Gly Tyr Leu Ser

347

TAT CFT GM 91 Tyr Arg Gly

T& CYS

GAG ATA ACT CCT ACT GCG TCG TAC TTA GTG GAT GGT Glu Ile Thr Pro Thr Ala Ser Tyr Leu "al Asp Gly

437

GA.4 GGA ACT GTG GA.4 GAA GAG ATT CAA GGA CCG CCT GTC ATC TTC TGG CCT GAA CTC AAA GCC ATG CTG TCA ,UA AAG CTG TCA Gee AAG 121 Glu Gly Thr "al Glu Glu Glu Ile Gin Gly Pro Pro Val Ile Phe Trp Pro Glu Leu Lys Ala Met Leu Ser Lys Lys Leu Ser Ala Lys

52,

AAT CTT GTA GAC AK 151 Asn Leu Val Asp Asn

TTG AGG LPU Arg

TGG AGG GTA GGT GTG GAC TCC TTT GAG GCT GGT GAG GCT GA.4 GAT ACA AK ATG GCC ACC TAC ATT CAT Trp Arg Val Gly Vnl Asp Ser Phe Glu Ala Gly Glu Ala Glu Asp Thr Asn Met ALa Thr Tyr Ile HIS

617

GAG FAA TTC AGG AK 181 Glu GLu Phe Arg Asn

TTC TTG GAT AAA GTG TGG AX FAT GAA CAC TAT ATC AAG TTG CAA GTC AGA GGC AGC ACC AAG AAC CA.4 GTG TCC Phe Leu Asp Lys Val Trp Asn Asp Glu HIS Tyr Ile Lys Leu Gin Val Arg Gly Ser Thr Lys AS,, GLn Val Ser

707

ATT TCG ATC AAT GGT AA.4 GAG GAG ATC TTG GAG ACT CCT GAT WA TAC GTT GCA TAC AGC GAG AGT GGC TCT GTT TCT GGC u.4 CCT GTC Ile Ser Ile Asn Gly Lys Glu Glu Ile Leu Glu Thr Pro Asp Ala TYK Val Ala Tyr Set Glu Ser Gly Ser Val Sex Gly Lys Pro "al

79:

211

TAT GTG MC Tyr Val Asn

TAC GGG CTG AAA AA.4 GAT TTT GAG ATC ATA CAG AAG GTC GTG GCT TCA CTG AAT GGA ACC ATA GTC ATT GTC AGA GCT GGA Tyr Gly Len Lys Lys Asp Phe Glu Ile Ile Gin LYS VaL Val Ala Se= Leu Asn Gly Thr Ile Val Ile Val Arg Ala Fly

887

241

AU, ATA ACA CTT GCT GAG AAG GTT GCA AAT GCC AAA GAG GCA GGA GCA GCT GGA GTC CTC ATG TAC GTG GAT TCA CTC AAG TAT GGA ATA 271 Lys Ile Thr Leu Ala Glu Lys Val Ala Asn Ala Lys Glu Ala Gly Ala Ala Gly Val Leu Mnt Tyr Val Asp Ser Leu Lys Tyr Gly Ile

977

CGA ATG CAG CTG GCT GCC AGG Arg Met Gin Leu Ala Ala A=&

CAA SAT GGA AGT GGC GGG T& GLn Asp Gly Se= G1.y Giy Cys

AC.4 GAT ACA CTT A-C CCA TTC GGA CAT GCC CAC CTT GGA ACT GGA GAC CCT TAC ACC CCA GGC TTC CCT TCG TIC AK 301 Thr Asp Thr Leu Iie Pro Phe Gly h‘is Ala His it?= Gly Thr Gly Asp Pro Tyr Thr Fro Gly Phe Pro Ser Phe Am CC.4 CCA GTT GAA TCT TCA GGA CTA CCC CAC ATT GCT GTT CAG Pro Pro Val Glu Ser Ser Gly Leu Pro His Ile Ala Val Gin * GAC ACA T:C TCT GAA GGT TGG AAA GGT GCG ATC CAT TCC TGT 361 Asp Thr Cys Ser Glu Gly Trp Lys GLy Ala Ile Nls Ser Cys 331

P&C AAT TCC ATG 391 Asn Asn Ser Met

CAC ACC CAG TTT 1067 HIS Thr Gin Phe

ACC ATC TCT AGC AGT GCA GCA GCC AGG CTG TTC AGI: AAA ATG GAT GGA 1157 Thr Ile Ser Ser Ser Ala ALa ala Arg Leu Phe Ser Lys Met asp Gly AAG GTG ACA ACA AAG CAC GAG AGC CAG ATA ATG GTG A&A CTA GAT GTG Lys VaL Thr Thr Lys Hxs Glu Ser Gin Ile Met Val Lys Leu Asp Val

1247

AA.4 GAC AGG AAG ATT CTG h4C ATC TTC GGT GCT ATC GAG GGA TTT GA.4 GPA CCA GAT CGG TAT GTT GTG ATT GGA FCC 1337 Lys Asp Arg Lys ILe Len Am Ile Phe Gly Ala 110 Gin Gly Phe Glu GLu Pro Asp Arg Tyr "a,. Val Ile Gly .&la

CAG AGA GAC TCC TGG GGC CCA GGA GTG GCT AA!, GCT GGC ACT GGA ACT GCT ATA TTG TTG GAA CTT GCC CGT GTG ATC TCA GAC ATA FTG 421 Gin Arg Asp Ser Trp Gly Pro Fly Val Ala Lys Ala Gly Thr Fly Thr Ala Ile Leu Leu Glu Leu Ala Arg Val ile Ser Asp Ile Val

142,

AA.4 AK GAG GGC 451 Lys Asn Glu Gly

1517

TAC AA.4 CCG AGG CGA AGC ATC ATC TTT GCT AGC TGG AGT GCA GGA GAC TAC GGA GCT GTG GGT GCT ACT GA.4 TGG CTG Tyr LYS Pro Arg Arl: Se= Ile Ile Phe Ala Se= Trp Ser Ala GLy Asp Tyr Gly Ala Val Gly ALQ Thr Glu Trp Leu

GAG GGG TAC TCT GCC ATG CTG CAT GCC AAA GCT TTC ACT TAC ATC AGC TTG GAT GcT CCA GTC CTG GGA GCA AGC CAT GTC AAG ATT TCT 1607 481 Glu Gly Tyr Ser Ala Met Leu His Ala LYS Alu. Phe Thr Tyr Ile Ser Leu Asp Ala Pro Vnl Leu Gly Ala Ser His Val Lys Ile Sex GCC AFC CCC TTG CTG TAT ATG CTG CTG GGG AGT ATT ATG f&G GGG GTG AAG AA? CC.4 GCA GCA GTC TCA GAG AGC CTC TAT AAC AGA CTT 169, 511 Ala Ser Pro Leu Leu Tyr Met, Leu Leu Giy Sex Ile Met Lys Gly VaL Lys Asn Pro Ala ALa Val Ser Glu Ser Leu Tyr AS* Arg Leu GGC CCA GAC TGG GTA AAA GCA GTT GTT CCT CTT GGC CTG GAT AAT GCA GCG TTC CCT TTC CTG GCG TAC TCA GGA ATT CCA GTG TTG TCT 278, 541 Gly Pro Asp Trp Val LYS Ala Val Val Pro Leu Gly Leu Asp As" Ala Ala Phe Pro Phe Leu ALa Tyr Ser Gly Il,e Pro "al ~eu Ser TTT GCT TTC TAC AAT AAA FAT GAG GAA TAT CGC 571 Phe Gly Phe Tyr Asn LYS Asp Glu Glu Tyr Arg

TTC CTG GAC ACT AAG GGT GAC ACA CTG GAG AAC CTG AGG AAA ATT GAT MT CTG GAT Phe Leu Asp Thr Lys Gly Asp Thr Leu Glu Asn Leu Arg Lys ILc Asp Asn Leu Asp

1877

GCT CTT CTG GCT GCT GCT GCA GA.4 GTA GCT GGA CAA GCA GCT CTC AGG CTG KC CAT GAT CAT GAG CTC TTC CTG GAC AK GGG AGA TAC 601 Ala Leu Leu Ala Ala Ala Ala Glu VaL Ala Fly Gin Ala Ala Leu A% Leu Thr HE Asp His Flu Leu Phe Leu Asp Ile Gly AXE ~yr

196,

AGT GAA GA.4 TTA CTG GCA 631 Ser Glu Glu Leu Leu Ala GCC CGT GGT GAC 661 Ala Arg Gly Asp

691

TAC CAG GAG GAG TTT TTG CC? TAC ATT AAG F&4 GTG CGG GAG CTG GGG TTG ACC TTG GAC TGG CTG TTT TTT 205, Tyr Gin Glu Glu Phe Leu Pro Tyr Ile LYS Glu Val Arg Glu Leu Gly Leu Thr Leu Asp Trp Leu Phe Phe

TTC CAG CGA GCT GTA ACT GCA CTG AGA AGA GAC ATT GCA AK AGT GAC GGG GAG AK AGG GTC AK CGC AGG GCC CTG 2147 Phe Gin Arg Ala Val Thr Ala Leu Arg Arg Asp Ile Ala As" Ser Asp Gly Glu As,, Ark "al IL@ Arg Arg Ala Leu

AAT GAC AGG ATG ATG AAG GTG GAG TAT GAC TTC CTG TCC CCG TAT CTC TCA CCA AAA GAT GTC CCT TTT CGC CAC ATC TTC TTT GGC AAA 2237 Asn Asp Arg Met Met LYS Val Glu Tyr Asp Phe Leu Ser Pro Tyc Leu Ser Pro Lys Asp "al. Pro Phe Ar& His Ile Phe Phe Gly ~ys

CGC CCC CAC ACC CTG CGG AGT CTG GTG GAG CAT CTG CAG CTG TTG AA4 ACC AX AGG AGC AGC GTG GA? CTG MC TTG CTF AGG GAG CAC- 2327 721 GLy Pro His Thr Ler: Ar8 Ser Leu Val Glu His Leu Gin Leu Lru Lys Thr Asn Arh Ser Ser "al Asp Leu ~sr> Leu Leu /,rg flu Gin CTG CCC CTA GCA ACG TGG ACC ATT AAA GGG GCG GCC AAT GCC TTG GGA GGT GAT ATC TGG GAA ACT GAC MT G.&A TTC TAG ACACTGCMGC 751 Leu Ala Leu Ala Thr Trp Thr Ile LYS Gly Ala Ala Asn Ala Leu Gly Fly Asp Ile Trp Glu Thr Asp Asn Glu phi> END ACGTGGTTAAGGTAACAGGGT

Fig. 1. The nt sequence domain

2440

of the 5’noncoding

is singly overlined

2419

and coding regions of CTRcDNAl,

and the potential

N-linked

glycosylation

a full-length

sites are underlined.

cDNA clone encoding

Cys codons

are marked

the cTR. The putative with asterisks,

transmembrane

and the internalization

recognition signal is marked by bold-face letters and doubly overlined. A Igtl 1 chicken cDNA library prepared from chicken erythroblasts was screened by standard methods (Maniatis et al., 1982) using CTRZ, a cTR genomic clone (Chan et al., 1989), as probe. A S-kb Eli-length cTR cDNA clone, CTRCDNA

1,wasisolated. Three EcoRI fragments

span the 5’-noncoding of overlapping

fragments

and coding regions were prepared

(0.6, 1.2 and 0.7 kb in size) and two EcoRI partial-digestion

were subcloned

using the Cyclone

into bacteriophage kit (International

M13mp19

for single-stranded

Biotechnologies,

fragments

(1.8and 1.9 kb in size) which

DNA sequencing

Inc., New Haven,

in both directions.

CT) and sequencing

was performed

Sets by

the dideoxy method (Sanger et al., 1977) as previously described (Chan et al., 1989). The sequence data were analyzed by the IBI/Pustell DNA Sequence Analysis Program (Pusteil and Kafatos, 1986) with an AT&T PC. The CTRcDNAl sequence has been deposited in the EMBL Database (accession No. X55348). This sequence

was obtained

in one laboratory

(E.M.G.

and L.-N.L.C.).

The complete

nt sequence

of cDNAs

spanning

region of the cTR was independently obtained in the other laboratory (S.J., M.Q. and I.S.T.). cDNAs encoding the C-terminal screening a chicken primary bursal LL6 lymphoma cDNA library, kindly provided by Dr. Carol Nottenburg (Fred Hutchinson WA), with an hTR cDNA probe at low stringency. from the same &I 1 chicken erythroblast library

A cDNA containing the nt region encoding as CTRcDNAl. The nt sequence encoding

the entire coding

447 aa were obtained by Cancer Center, Seattle,

the remaining 329 N-terminal aa of the cTR was obtained this region of the cTR obtained in both laboratories was

identical. Four confirmed sequence differences that may represent polymorphic variations were found in the coding region obtained from cDNAs the bursal lymphoma library: A’8’” + G, C’“@‘+ T, A*‘“‘-+ C and AZ3’* + G. This leads to two aa changes, Arg5s’ ---+His and Lys7jh -+Gln.

from

251 previously (Chan et al., 1989; Koeller et al., 1989; Harford et al., 1991). In this study, we describe the nt sequence of the coding region of the chicken cDNA and the deduced primary structure of cTR. Comparison of the aa sequence of cTR, mTR and hTR reveals structural features that are likely to be important in various aspects of TR function.

EXPERIMENTAL

1234

nt

AND DISCUSSION

(a) The nt sequence of chicken TR cDNA The nt sequence of the coding and 5’-noncoding regions of CTRcDNAl, a recombinant cTR cDNA clone, is shown in Fig. 1. In the 5’-noncoding region 77 nt have been sequenced. The coding sequence contains 2328 nt corresponding to 776 aa. A single start codon, ATG, and one stop codon, TAG, are present. The sequence of the 3’-noncoding region of cTR cDNA has been described already (Chan et al., 1989). The complete cTR cDNA contains 5019 nt, which includes a second polyadenylation site. To define the 5’-terminus of cTR cDNA, primer extension experiments were performed (Fig. 2). When the 20-nt primer that is complementary to nt 19-39 (Fig. 1) was hybridized to RNA from chick embryonic RBC, a primer extension product of 85 nt was produced (Fig. 2, lane 4). Negative controls, namely, hybridization of complementary primer to globin mRNA and hybridization of noncomplementary primer to chick-embryonic RBC RNA, gave no discernable extension products (Fig. 2, lanes 2 and 3, respectively). These results indicate that the cTR 5’-noncoding region contains 124 nt and that cTR mRNA from embryonic RBC has another 47 nt upstream from the 5’-end of CTRcDNAl.

Fig. 2. Analysis

of the 5’ terminus

from globin

mRNA

from

embryonic

chick

4, extension mentary

and complementary

product

RBC from

(b) Primary structure of cTR The aa sequence of cTR deduced from the cDNA sequence is shown in Fig. 1 also. The hydropathy plot of cTR (data not shown) indicates that the only region of high hydrophobicity, which likely corresponds to the transmembrane domain of the receptor, is located close to the N terminus of the polypeptide. cTR contains five Cys residues, one at the junction of the cytoplasmic domain and transmembrane region (Cys”) and four others (Cys residues 101, 108, 363, 374) in the external domain. Four potential N-linked glycosylation sites are located at Asn residues 261, 326, 391, and 738. The M, of the unglycosylated cTR predicted from its primary sequence is 85 72 1. This M, is consistent with what was previously reported for cTR isolated from chicken erythroblasts transformed with avian erythroblastosis virus (Schmidt et al., 1985). Northern-blot analyses show that cTR mRNA is consistently 4.9 kb in size in several different chick embryonic tissues (Chan et al., 1989; Gerhardt and Chan, 1990).

(Chan

were synthesized Peptide

and

noncomplementary

embryonic

RBC

product product primer;

and comple-

analyzed

in the same gel. Total RBC as previ-

et al., 1989). Oligodeoxyribonucleotide

Laboratory.

thereof.

RNA

from 12-day chick embryonic of Texas Medical

The primers

contain

region of the cTR cDNA sequence

complement

3, extension

on the right margin show the size of DNA

by the University

Synthesis

noncoding

by primer extension.

primer;

against DNA markers

cellular RNA was prepared ously described

RNA

chick

primer. The numbers

(in nt) as measured

of cTR mRNA

are: 1, primer (20-mer) alone; 2, extension

The lane designations

Primer extension

primers

Branch

DNA and

nt 19-38 in the 5’-

(see Fig. 1) and the reverse

analyses

were performed

accord-

ing to established procedures (Ausubel et al., 1989). The primers were end-labeled with [y-32P]ATP using T4 polynucleotide kinase. Hybridization of labeled primers to globin mRNA, which was used as a control, to RNA from

12-day chick embryonic

Reverse transcriptase the primers. and sequence

from avian myeloblastosis

The reaction

a 6% polyacrylamide

RBC was carried

products

sequencing

virus was used to extend

were purified gel together

and

out at 30°C.

and then analyzed

with DNA of known

on size

as markers.

(c) Evolutionary conservation of TR aa sequence and organization Comparison of the deduced primary structure of cTR with hTR and mTR (McClelland et al., 1984; Schneider et al., 1984; Trowbridge et al., 1988) reveals several common structural features (Fig. 3). The cTR is 776 aa compared with 760 and 763 aa for the hTR and mTR, respec-

252 t 1 96 HUMAN NNDQARSAFS NLFGGEPLSY TRISLARQVD GDNSHVFMKL AVDEEENAD. ...NNTKANV TKPKRCSGSI CYGTIAVIVF FLIGFMIGYL GYCKGVEPKT l

MOUSE CHICKEN consensus

t

MNDQARSAFS NLFGGEPLSX TRTSLARQVD GDNSHVEMKL AADEEENAD. ...NNMKASV RKPKRFNGRL CFAAIALVIF FLIGFMSGYL GYCKRVEQKE .MDHARAALS NLFSVEPMSY TIVSIARQTD GDNSHVEMKL SADDEEGGDI ERPEHMHVSM AQPQRNGKRL CFLVIAAVLL LLIGFLIGYL SYRGRMQLAA -MDqARsAfS NLFggEPlSY =SlARQvD 'GDNSHVEMKL a-DEEEnaD- ---nn-ka-v -kPkr--g-- C---IA-V-f fLIGFm-GYL g'lck-ve-kt

186 LWENQFREF KLSKVWRDQH YYIENQFHEF KFSKVWRDEH TYIHEEFRNF .LDKVWNDEH -Y-e-qF-eF kL-KVWrD-H

MOUSE CHICKEN Consensus

ECERLAGTES ECVKLAETEE RCQDGSGGCE eC__la_te_

PV .... ...R TD .......K ITPTASYLVD __________

TDFTSTIKL. 1EFADTIK.Q KNLVDNLRWR --f-tik--

LNENSYVPRE LSQNTYTPRE VGVDSF...E 1--n-y-prE

HUMAN MOUSE CHICKEN Consensus

FVKIQVKDS. YVKIQVKSSI YIKLQVRGST YvKiQVk-S-

.AQNSVIIVD KNGRLVYLVE NPGGYVAYSK AATVTGKLVH ANFGTKKDFE .GQNMVTIVQ SNGNLD.PVE SPEGYVAFSK PTEVSGKLVH ANFGTKKDFE KNQVSISING KEE ....ILE TPDAYVAYSE SGSVSGKPVY VNYGLKKDFE --Qn-v-Iv- -ng---- vE -P-gYVA-Sk ---V-GKlVh aNFGtKKDFE

...DLYTPw ...ELSYSm IIQKVVASM ----1---vN

TSIVIVRAGK ~LVIVRAGE ~IVIVRAGK Gs-VIVRAG-

HUMAN MOUSE CHICKEN Consensus

YMoQTKFPIV NAeLSFFGHA YMDKNKFPW EADLALFGHA Y'JDSLKYGIT DTLIP.FGHA YmD--Kfp-v -a-l--FGHA

HLGTGDPYTP HLGTGDPYTP HLGTGDPYTP HLGTGDPYTP

GFPSF-F GFPSF-F GFPSF-F GFPSFNHTQF

PPSRSSGLPN PPSQSSGLPN PPVESSGLPH PPs-SSGLPn

IPVQTISRAA IPVQTISRAA IAVQTISSSA IpVQTISraA

AEKLFGNMEG AEKLFGKMEG AARLFSKMDG AekLFg-MeG

D.CPSDWKTD .STCRMVTSE .SCPARWNNID .SSCKLELSQ DTCSEGWKGA IHSCKVTTKH --Q--W--d -s-C----s-

SKN..VKLTV NQN..VKLIV ESQIMVKLDV --n--VKL-V

HUMAN MOUSE CHICKEN Consensus

SNVLKEIKIL NIFGVIKGFV KNVLKERRIL NIFGVIKGYE Bl,iMKDRKIL NIFGAIQGFE -NvlKe--IL NIFGvIkG--

EPDHYVWGA EPDRWVVGA EPDRYWIGA EPD-YWvGA

QRDAWGPG.A AKSGVGTALL QP.DALGAGVA AKSSVGTGLL QRDSWGPG.V AKAGTGTAIL QRDa-G-G-a AKs-vGT-1L

LKLAQMFSDH LKLAQVFSDM LELARVISDI LkLAq-fSDm

VLKDGFQPSR ISKDGFRPSR VKNEGYKPRR --kdGf-PsR

SIIFASWSAG SIIFASWTAG SIIFASWSAG SIIFASW-AG

DFGSVGATEW DFGAVGATEW DYGAVGATEW DIG-VGATEW

476 LEGYLSSLHL LEGYLSSLHL LEGYSAMLHA LEGYlssLHl

HUMAN MOUSE CHICKEN Consensus

KAFTYINLDK KAFTYINLDK KAFTYISLDA KAFTYInLDk

AVLGTSNFKV WLGTSNFKV PVLGASHVKI -VLGtSnfkv

SASPLLYTLI SASPLLYTLM SASPLLYMLL SASPLLYtL-

EKTMQNVKHP GKIMQDVKHP GSIMKGVKNP -k-Mq-VKhP

VT.GQFLYQ. VD.GKSLY.R AAVSESLYNR v--g --LY--

.DSNWASKVF, KLTLDNAAFP .DSNWISKVE KLSFDNAAYP LGPDWVKAW PLGLDNAAFP -dsnW-skVe kL--DNA&-P

FLAYSGIPAV FLAYSGIPAV FLAYSGIPVL FLAYSGIPav

SFCFCE.DTD SFCFCE.DAD SFGFYNKDEE SFcFce-D-d

572 YPYLGTTMDT YPYLGTRLDT YRFLDTKGDT YpyLgT--DT

HUMAN MOUSE CHICKEN Consensus

YKJZLIERIPE LNKVARAAAE YEALTQKVPQ LNQMVRTAAE LENL.RKIDN LDALLAAAAE y-L----Ln---r-AAE

VAGQFVIKLT VAGQLIIKLT VAGQAALRLT VAGQ--lkLT

HDVELNLDYE HDVELNLDYE HDHELFLDIG HDvELnLDye

RYNSQLLSFV MYNSKLLSFM RYSEELLAYQ -Yns-LLsF-

RDLNQYRADI KDLNQFKTDI EEFLPYIKEV -dlnq ---di

KEMGLSLQWL RDMGLSLQWL RELGLTLDWL -emGLsLqWL

YSARGDFFRA YSARGDYFRA FFARGDFQRA ysARGD-fRA

TSRLTTDFGN TSRLTTDFHN VTALRRDIAN tsrLttDf-N

672 AEKTDRFVMK AEKTNRFVMR SDGENRVIRR aekt-Rfvm-

HUMAN MOUSE CH1CKF.N Consensus

KLNDRVMRVE EINDRIMKVE ALNDRMMKW --NDR-M-VE

KESPFRHVFW RESPFRHIFW KDVPFRHIFF -esPFRHIFW

GSGSHTLPAL GSGSHTLSAL GKGPHTLRSL GsGsHTL-aL

LENLKLRKQN NGAFWLFR VENLKLRQW mFmLFR VEHIQLLKTbI XSVDLNLLR -EnLkLr--N --afnetLFR

NQLALATWTI NQLALATWTI EQIJUATWTI nQLALATWT1

QGAANALSGD QGVANALSGD KGAANALGGD qG-ANALsGD

760 VWDIDNEF IWNIDNEF IWETDNEF -W-IDNEF

HUMAN

EEPG..EDFP SETMETEDVP GEGTVEEEIQ -E----Ed-p

AARRLYWDDL TSSRLYWADL GPPVIFWPEL ---rlyW-dL

KRKLSEKLDS KTLLSEKLNS KAMLSKKLSA K--LSeKL-s

AGSQKDENLA AGSQKDESLA AGEAEDTNMA AGsqkDe-lA

+ ++

+++

ITFAEKVANA ITFAEKVANA ITLAEKVANA ITfAEKVANA

c

+++

Fig.3. Comparison

of the aa sequences

of cTR, mTR, and hTR. Dots represent

gaps introduced

31-l

l

l

YHFLSPYVSP YHFLSPYVSP YDFLSPYLSP YhFLSPYvSP

281 ESLNAIGVLI QSFNAIGVLJ KEAGAAGVIJ4 ---nAiGVLI

to align sequences.

Capital

l

letters in the consensus

sequence represent aa common to all sequences, lower-case letters represent aa present only in hTR and mTR sequences, dashes indicate positions at which hTR and mTR sequences differ. Cys residues in the hTR are marked with asterisks and conserved Cys are shown in bold-face letters. Putative glycosylation

sites are underlined

and their positions

in the hTR sequence

marked

with plus signs. The position

and the internalization recognition signal sequence is shown in bold-face letters and doubly overlined. hTR. The mTR sequence has been deposited in the EMBL Database (accession No. X57349).

tively. All three proteins lack a leader sequence and the overall hydropathy profiles of avian and mammalian TRs are essentially superimposable. Each receptor is organized into a small N-terminal cytoplasmic domain, a single transmembrane region, and a large C-terminal extracellular domain that are 68,23, and 685 aa, respectively, in the case of the cTR. Although the cytoplasmic domain of the cTR has only 62% identity with the hTR compared with 93% identity between the cytoplasmic tails of mTR and hTR, the YXRF tetrapeptide recognition structure for high-efficiency

of transmembrane

The numbers

correspond

regions

is overlined

to the aa residues

in

endocytosis of the hTR (Collawn et al., 1990) is conserved in the cTR. This structural conservation is consistent with the rapid internalization of hTRs expressed in chicken embryo fibroblasts (Jing et al., 1990). The phosphorylation site at Ser24 and the acylation site at CYSTSin the hTR are both conserved in the cTR (Schneider et al., 1982; Rothenberger et al., 1987; Jing and Trowbridge, 1990). However, both the mTR and cTR lack CyP that is another lipid attachment site in the hTR (Jing and Trowbridge, 1987; 1990). Several features of the extracellular domain of

253 the cTR are significant.

Only three of the six Cys residues

found in the extracellular domains of the mTR and hTR are conserved in the cTR. CYSTS and CYSTS form two intermolecular disullide bonds between subunits of hTR (Jing and Trowbridge, 1987) but only Cys9* is conserved in the cTR. This finding is consistent with the observation that a fraction of cTRs, similar to mutant hTRs in which either CYSTSor Cys 98 has been modified to Set-, are not disulfidebonded dimers (Schmidt et al., 1985; Jing and Trowbridge, 1987). The three glycosylation sites found in mammalian TRs are conserved in the cTR and are located in, or adjacent to, regions of relatively high sequence similarity. Some regions-of similarity ‘may be important for receptor dimerization which is known to involve noncovalent interactions between the external domains of TR subunits (Omary and Trowbridge, 1981). The region of approx. 150 aa of the extracellular domain of the TRs proximal to the cell membrane are poorly conserved, and both the mTR and cTR lack the tryptic cleavage site at Arg’“’ in the hTR (Turkewitz et al., 1988). Overall, the cTR extracellular domain has 53 y0 identity to the extracellular domain of the hTR compared with 77% identity between the mTR and hTR. The sequence divergence of the cTR extracellular domain accounts for the fact that cTR does not bind most mammalian TRs, whereas most mammalian TRs can utilize other mammalian transferrins but not ovotransferrin (Shimo-Oka et al., 1984). The construction and functional analysis of human-chicken hybrid TRs in which different regions of the hTR are replaced by cTR sequences may provide information that will aid in the localization of the transfer+ binding site.

Ghan,

L.-N.L.,

Chicken

Grammatikakis,

transferrin

sequences

N., Banks,

receptor

and expression

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conservation

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E.M.:

of 3’ noncoding

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(1989) 3763-3771. Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S. and Tainer, J.A.: Transferrin receptor internalization sequence

YXRF implicates

motif for endocytosis. Gerhardt,

EM.

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transferrin

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Harford, J.B., Casey, J.L., Koeller, D.M. and Klausner, R.D.: Structure, function, and regulation oftransferrin receptor: insights from molecular biology.

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T., Miller, K., Hopkins,

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T.E. and Maxfield,

nalization

is partially

cytoplasmic

We thank Dr. B. Vennstrom for the chicken erythroblast cDNA library and Dr. Carol Nottenburg for the chicken bursal lymphoma cDNA library. We also thank Ms. N. Tovar and Joan Stewart for their assistance in the preparation of this manuscript. This work was supported in part by a grant from the American Heart Association, a University of Texas Medical Branch Small Grant, a grant from the John Sealy Memorial Endowment Fund, and grant CA34787 from the National Cancer Institute.

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