Original Contributions
TWO HUMAN TNF RECEPTORS HAVE SIMILAR
EXTRACELLULAR,
BUT DISTINCT
INTRACELLULAR,
DOMAIN SEQUENCES
Zlatko Dembic,’ Hansruedi Loetscher,’ Ueli Gubler,2
Yu-Ching E. Paq2 Hans-Werner Lahm,’ Reiner Gentz,’
Manfred Brockhaus,’ Werner Lesslauerl,*
Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in
inflammatory and immunologic responses. These activities are mediated by specific cell surface
receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was
isolated using partial amino acid sequence information and the polymerase chain reaction (PCR).
When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with
characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve
growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of
the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif
LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF
receptors are entirely unrelated, suggesting different modes of signaling and function.
o 1990 by W.B. Saunders Company.
Tumor necrosis factor (TNF) is a highly potent
cytokine. Its wide range of biological activities in inflammatory and immunologic responses have triggered many
studies of the specific cell surface receptors that mediate
TNF function.lV1O TNF receptors of significantly different molecular masses in the range of 50 to 140 kDa
were reported in protein cross-linking studies by various
investigators; the possibility that more than one receptor
existed therefore had to be considered. We have identified and purified from human cell lines and placenta two
distinct human TNF receptors of 55 kDa and 75 kDa
that are simultaneously expressed to different extents by
various cells.8311~12Both receptors bind TNF-ol and
TNF-P with high affinity”,13 (also, Schoenfeld and
Loetscher, unpublished data). A third TNF-binding
protein of 65 kDa was found by SDS-polyacrylamide gel
electrophoresis (PAGE) and ligand blotting to copurify
‘Central
Research
Units,
F. Hoffmann-Laroche
Ltd, 4002 Basel,
Switzerland.
lRoche
Research
Center,
Hoffmann-Laroche
Inc., Nutley,
New
Jersey 07 110, USA.
*To whom correspondence
should be addressed
at: Central Research
Units, Building
69, Room 14, F. Hoffmann-LaRoche
LTD, CH4002 Base], Switzerland.
o 1990 by W.B. Saunders
Company.
1043-4666/90/0204-0008$05.00/O
KEY
WORDS:
TNF
receptor gene family
CYTOKINE,
receptors/Cytokine
Vol. 2, No. 4 (July),
receptors/NGF-TNF-
1990: pp 231-237
with the 75-kDa receptor fraction from HL60 cells.
Both the 75-kDa and 65-kDa proteins in Western blots
bind the same monoclonal antibody, utr-I.” We therefore assume the 65-kDa protein to be a derivative or
fragment of the 75-kDa receptor and refer to the two
proteins as the 75-kDa receptor.
The cDNA cloning of the 55-kDa receptor has been
reported14,‘5; the open reading frame of the cDNA
predicts a receptor protein with extracellular, transmembrane, and intracellular regions. A surprisingly high
degree of sequence similarity to the nerve growth factor
(NGF) receptor extracellular region was discovered
which is most clearly delineated by a repetitive cysteine
residue pattern. Recently, the cDNA of the 75-kDa
TNF receptor was identified in a eukaryotic expression
cloning system.16 We have independently isolated a
75-kDa TNF receptor cDNA using peptide sequencing
and PCR techniques which confirms the sequence reported for the cDNA isolated by expression cloning.16
When expressed in COS-1 cells, the cDNA transfers
specific TNF-binding properties comparable to those of
the native receptor. The predicted extracellular region
contains four domains with characteristic cysteine residues highly similar to that of the 55-kDa TNF receptor
and to that of the NGF receptor,17318 CDw40,” and
OX40 antigen” extracellular domains. The intracellular
domains of the two TNF receptors, however, are entirely
unrelated. We therefore propose that the two TNF
231
232
/ Dembic
CYTOKINE,
et al.
receptors may address distinct intracellular signal transmission pathways.
RESULTS
Isolation
of the 75-kDa
TNF Receptor cDNA
The 75kDa and 65kDa protein bands of the
75-kDa TNF receptor from a preparative SDS-polyacrylamide gel were blotted onto PVDF membrane and
subjected to NH,-terminal amino acid sequencing by
gas phase sequenation as reported elsewhere.‘* Briefly,
two parallel sequences were obtained with the 65-kDa
band; since one sequence matched the ubiquitin sequence, the unique sequence could be identified as
LPAQVAFTPYAPEPGSTC.”
Furthermore, the amino
acid sequences of a total of seven internal peptides
LPRDURFTPYRPEPGST1
Vol. 2, No. 4 (July
1990: 231-237)
prepared by tryptic and proteinase K digests of the
75-kDa receptor fraction were determined. The four
peptide sequences used in the isolation of the cDNA
clone are indicated in Fig. 1; the remaining three
peptides, i.e. L114-P ‘r7, P123-V 137and G’**-P 302,
match the predicted amino acid sequence and thus
confirm that the cDNA encodes the receptor. To prepare
a probe for the isolation of cDNA clones a short DNA
fragment was amplified by polymerase chain reaction
(PCR) from human genomic DNA with the use fully
degenerate primer oligonucleotides derived from the
Q412-L428amino acid sequence (see Fig. 1 and Materials and Methods). A DNA fragment of the predicted
size was found to be amplified by PCR. Oligonucleotides
were synthesised according to the sequence of this DNA
fragment and used to identify the cDNA shown in Fig. 1
, ***
ID21
341
GCCRGCRCCGGGAGCTCRGRTTCTTCCCCTGGTGGCCRTGGGRCCCflGGTCflRTGTCRCC
RSTGSSDSSPGGHGTQUNUT
61
21
RGRGRRTRCTRTGRCCRGRCRGCTCRGRTGTGCTFCRGCRflflTGCTCGCCGGGCCflflCflT 1081
361
RE',YDQTRQflCCSKCSPGQH
TGCRTCGTGRRCGTCTGTRGCflGCTCTGRCCRCRGCTCRCRGTGCTCCTCCCflRGCCflGC
CIUNUCSSSDNSSQCSSQRS
121
41
I141
!L.iLLi
GCRRRRGTCTTCTGTRCCRRGRCCTCGGRCRCCGTGTGTGRCTCCTGTGRGGRCRGCRCfl 381
RKUFCTKTSDTUCDSCEOST
TCCRCRRTGGGRGRCRCRGRTTCCRGCCCCTCGGRGTCCCCGflRGGRCGRGCRGGTCCCC
STMGOTDSSPSESPKDEQUP
181
61
TRCRCCCRGCTCTGGRRCTGGGTTCCCGRGTGCTTGRGCTGTGGCTCCCGCTGTRGCTCT 1201
401
$'TQLUNUUPECLSCGSRCSS
241
El
GRCCRGGTGGRRRCTCRRGCCTGCRCTCGGGRRCRGRRCCGCRTCTGCRCCTGCflGGCCC
1261
DQUETQRCTREQHRICTCRP
421
GGCTGGTRCTGCGCGCTGRGCRAGCRGGRGGGGTGCCGGCTGTGCGCGCCGCTGCGCRRG
1321
GUVCRLSKQEGCRLCRPLRK
1381
TGCCGCCCGGGCTTCGGCGTGGCCRGRCCREGRRCTGRRRCRTCRGRCGTGGTGTGCRRG1441
1501
CRPGFGUARPGTETSDUUCK
***
1561
CCCTGTGCCCCGGGGRCGTTCTCCRRCACGRCTTCRTCCRCGGRTRTTTGCRGGCCCCRC 1621
1681
PCRPGTFSNTTSSTDICRPH
1741
,***
CRGRTCTGTRRCGTGGTGGCCRTCCCTGGGRRTGCRRGCRTGGRTGCRGTCTGCRCGTCC 1601
I861
plCNUURlPGNASllDRUCTS
1921
RCGTCCCCCRCCCGGRGTRTGGCCCCRGGGGCAGTRCRCTTflCCCCRGCCRGTGTCCRCflIv31
2041
TSPTRStlRPGRUHLPQPUST
2lOl
CGRTCCCRRCRCRCGCRGCCRRCTCCRGRRCCCRGCRCTGCTCCflRGCRCCTCCTTCCTG2161
2221
RSQHTQPTPEPSTRPSTSFL
2261
CTCCCRRTGGGCCCCRGCCCCCCRGCTGRRGGGRGCRCTGGCGRCTTCGCTCTTCCRGTT2341
2401
LPnGPSPPREGSTGDFRLPU
2461
2521
GGRCTGRTTGTGGGTGTGRCAGCCTTGGGTCTRCTRATRGGRGTGGTGflRCTGTGTC
2581
GLIUGUTRLGLLIIGUUNCU
2641
2701
2761
RTCRTGRCCCRGGTGRRRRRGRRGCCCTTGTGCCTGCRGRGRGRRGCC~RGGTGCCTCRC
2821
JnTQUKKKPLCLQRERKUPH
2881
2941
LPRD
3001
TTGCCTGCCGRTRRGGCCCGGGGTRCRCRGGGCCCCGRGCRGCRGCRCCTGCTGRTCRCR3061
LPRDKRRGTQGPEQQHLLIT
3121
3181
GCGCCGRGCTCCRGCRGCAGCTCCCTGGRGAGCTCGGCCRGTGCGTTGGRCRGRRGGGCG
3241
RPSSSSSSLESSRSRLDRRR
3301
3361
3421
CCCRCTCGGRACCRGCCRCRGGCRCCRGGCGTGGRGGCCRGTGGGGCCGGGGRGGCCCGG
3481
PTRNQPQRPGUERSGRGERR
I
301
101
361
I21
421
141
481
161
541
181
601
201
661
221
721
241
781
261
841
281
901
301
961
321
GGGRGCRCRTGCCGGCTC
GSTCRL
Figure 1. Amino acid sequences of the NH2 terminus
predicted amino acid sequences of the 75/65-kDa
TNF
Amino acid sequences determined
by protein
65-kDa
receptor
NH, terminus.
The predicted
glycosylation
sites are marked by asterisks.
and internal
receptor.
SDLETPETLLG
TTCTCCRRGGRGGARTGTGCCTTTCGGTCRCRGCTGGRCRCGCCRGRGRCCCTGCTGGGG
FSKEECRFRSQLETPETLLG
S
a
*
AGCRCCGRRGRGRRGCCCCTGCCCCTTGGRFTGCCTGRTGCTGGGflTGRRGCCCflGTTflR
STEEKPLPLGUPDAGflKPS
CCRGGCCGG;GTGGGCTGTGTCGTRGCCRflGGTGGGCTG~GCCCTGGCR~GRTGRCCCT~
CGRRGGGGCCCTGGTCCTTCCRGGCCCCCRCCRCTRGGRCTCTGRGGCTCTTTCTGGGCC
RRGTTCCTCTRGTGCCCTCCRCRFCCGCCGCRGCCTCCCTCTGflCCTGCflGGCCRRGRGCRGR
GGCRGCGRGTTGTGGRRRGCCTCTGCTGCCRTGGCGTGTCCCTCTCGGflRGGCTGGCTGG
GCRTGGRCGTTCGGGGCRTGCTGGGGCRRGTCCCTGRCTCTCTGTGRCCTGCCCCGCCCR
GCTGCRCCTGCCRGCCTGGCTTCTGGRGCCCTTGGGTTTTTTGTTTGTTTGTTTGTTTGT
TTGTTTGTTTCTCCCCCTGGGCTCTGCCCCRGCTCTGGCTTCCRGRRRRCCCCflGCRTCC
TTTTCTGCRGRGGGGCTTTCTGGRGRGGRGGGRTGCTGCCTGRGTCRCCCRTGRRGRCRG
GRCRGTGCTTCRGCCTGRGGCTGRGRCTGCGGGRTGGTCCTGGGGCTCTGTGCRGGGRGG
RGGTGGCRGCCCTGTRGGGRRCGGGGTCCTTCRRGTTRGCTCflGGRGGCTTGGflflRGCRT
CRCCTCRGGCCRGGTGCRGTGGCTCRCGCCTRTGRTCCCRGCRCTTTGGGflGGCTGRGGC
GGGTGGRTCRCCTGRGGTTRGGRGTTCGRGRCCRGCCTGGCCRRCRTGGTflRflRCCCCRT
CTCTRCTRRARRTRCAGRRATTRGCCGGGCGTGGTGGCGGGCRCCTRTflGTCCCRGCTRC
TCRGRAGCCTGRGGCTGGGRRRTCGTTTGRRCCCGGGRRGCGGRGGTTGCflGGGRGCCGR
GRTCRCGCCRCTGCRCTCCRGCCTGGGCGRCRtAGCGRGCGRGflGTCTGTCTCRRRRGRRRRRR
RRRRRGCRCCGCCTCCRRRTGCTRRCTTGTCCTTTTGTRCCflTGGTGTGRRRGTCRGRTG
CCCRGRGGGCCCRGGCRGGCCRCCATRTTCRGTGCTGTGGCCTGGGCRRGRTRRCGCRCT
TCTRRCTRGRRRTCTGCCRRTTTTTTRRRRRGTRRGTRRGTRCCRCTCflGGCCflflCflRGCCRR
CGRCRRRGCCRRRCTCTGCCRGCCRCRTCCRRCCCCCCRCCTGCCRTTTGCRCCCTCCGC
CTTCRCTCCGGTGTGCCTGCRGCCCCGCGCCTCCTTCCTTGCTGTCCTRGGCCRCRCCflT
CTCCTTTCRGGGRRTTTCRGGRRCTRFRFRTGRTGRCTGRGTCCTCGTRGCCRTCTCTCTRCT
CCTRCCTCRGCCTRGRCCCTCCTCCTCCCCCRGRGGGGTGGGTTCCTCTTCCCCRCTCCC
CRCCTTCRRTTCCTGGGCCCCRRflCGGGCTGCCCTGCCRCTTTGGTRCflTGGCCflGTGTG
RTCCCRRGTGCCAGTCTTGTGTCTGCGTCTGTGTTGCGTGTCGTGGGTGTGTGTRGCCRR
GGTCGGTRRGTTGRRTGGCCTGCCTTGRRGCCRCTGRRGCTGGGRTTCCTCCCCRTTRGR
GTCRGCCTTCCCCCTCCCRGCCRGGGCCCTGCflGflGGGGRRflCCflGTGTRGCCTTGCCCG
GRTTCTGGGRGGRRGCRGGTTGRGGGGCTCCTGGRRRGGCTCRGTCTCflGGflGCRTGGGG
RTRRRGGRGRRGGCRTGRRRTTGTCTRGCAGRGCRGGGGCRGGGTGRTRRRTTGTTGRTR
RRTTCCRCTGGRCTTGRGCTTGGCRGCTGRRCTRTTGGRGGGTGGGRGRGCCCRGCCRTT
RCCRTGGRGRCRRGRAGGGTTTTCCRCCCTGGRRTCAAGRTGTCflGflCTGGCTGGCTGCR
GTGRCGTGCRCCTGTRCTCRGGRGGCTGRGGGGRGGRTCRCTGGRGCCCRGGRGTTTGRG
GCTGCAGCGRGCTRTGRTCGCGCCRCTRCRCTCCRGCCTGRGCRRCRGRGTGflGRCCCTG
TCTCTTRRRGRRRRRRRRRGTCRGRCTGCTGGGRCTGGCCRGGTTTCTGCCCRCRTTGGR
CCCRCRTGRGGRCRTGRTGGRGCGCRCCTGCCCCCTGGTGGRCRGTCCTGGGRGRRCCTC
RGGCTTCCTTGGCRTCRCRGGGCRGRGCCGGGRRGCGRTGRRTTTGGRGRCTCTGTGGGG
CCTTGGTTCCCTTGTGTGTGTGTGTTGRTCCCRRGRCRRTGRRRGTTTGCRCTGTRTGCT
GGRCGGCRTTCCTGCTTRTCRRTRRRCCTGTTTGTTTTRCRCGTCGRRRRRRflR
tryptic
sequencing
are underlined.
transmembrane
domain
peptides,
and the cDNA
nucleotide
and
The amino acid sequence starts at the
is doubly underlined.
Potential
N-linked
Human TNF receptors / 233
Figure 2. Schematic representation of the domain
structure of the extracellular regions of the two TNF
receptors and of the NGF receptor.
The domains are boxed. Cyst&e residues are represented by vertical lines. The domain boundaries correspond to amino acid residues of Fig. 1: residues 17 to
54 (domain I), 55 to 97 (II), 98 to 140 (III), and 141
to 179 (IV). TNFR-A, 75-kDa TNF receptor;
TNFR-B, 55-kDa TNF receptor; NGFR, NGF
receptor “,“. , L and TM, predicted leader and transmembrane regions, respectively.
L
Cysteine-rich
I
1.
II
III
TM
IV
il III I k!J III I k--II II IHI II IH
I
II
111
TNFR-A
IV
III I IHI Ill I f--II Ill I IHI Ill I II
TNFR-8
Fl
jJ
I
111 1 IHI
II
111 1 /j-./l
111
111 1 it/j
IV
m 1 w
NGFR
in cDNA libraries prepared from HL60 and placenta.
This cDNA has an open reading frame that predicts a
439-amino acid membrane protein with extracellular
(235 residues), transmembrane (26 residues), and intracellular (178 residues) regions. Three basic amino acids
are located in the intracellular region sequence adjacent
to the putative inner membrane face.
In the predicted amino acid sequence of the extracellular region of the 75-kDa TNF receptor four conserved
domains were discovered which are most clearly delineated by a repetitive pattern of cysteine residues schematically represented in Fig. 2. The first two domains
contain six cysteine residues in a CX12.,4CX,,CX,,CX,9CX, C pattern which is highly homologous to that of the
four domains of the previously reported 55-kDa TNF
receptor extracellular region with the consensus sequence CX,,,sCX,,CX,CX,,,CX,,C.14,15
In the third
and fourth domains of the 75-kDa receptor this cysteine
pattern is less well conserved, but the alignment of the
total extracellular regions of the two TNF receptors
scores significantly above the random score with the
Mutation Data Matrix.21 This alignment score establishes a significant sequence similarity between the
extracellular domains of the two TNF receptors as well
as to those of the NGF receptor and the CDw40 and
OX40 antigens.‘7-20 Furthermore, we note that this
sequence motif has some similarity to the cysteine-rich,
putative metal-binding motif referred to as LIM.22
In sharp contrast to the high degree of homology
between the extracellular domains, the intracellular
regions of the two TNF receptors do not exhibit any
recognizable sequence similarity. A search of amino
acid sequence data banks with the 75-kDa receptor
TABLE
repeats
intracellular domain sequence revealed no significant
similarity to other known mammalian sequences,
The intracellular regions of both TNF receptors are rich
in proline and serine residues (75-kDa receptor: 18%
Ser, 9% Pro; 55kDa receptor: 8% Ser, 12% Pro).
Similar proline/serine-rich structures have been found
in the intracellular regions of several growth factor
receptors.23,24
TNF Binding in COS-1 Cell Transfectants
To confirm that the cDNA presented in Fig. 1
encodes a TNF-binding cell surface protein, the cDNA
was recloned in the pLJ268 expression vector25 and
transfected into COS- 1 cells; transient transfectants
were analysed for 1251-TNF binding. Specific TNFbinding properties were conferred to the COS-1 cells by
the transfected cDNA (Table 1). Expression of the
75-kDa receptor was confirmed in cell lysates of transfectants with the specific monoclonal antibody utr-411
(Table 1). TNF binding was also studied with COS-1
cell transfectants at various ligand concentrations and
the binding data were analysed according to Scatchard
(Fig. 3). The transfected cells were found to express a
TNF-binding protein characterized by a Kd of about 0.1
nM, which is clearly distinct from the endogenous
lower-affinity TNF receptor of COS-1 cells.i4 An analysis of the COS-1 cell transfectants in the fluorescence
microscope after staining with the 75-kDa TNF receptor-specific monoclonal antibody utr-1 revealed that
only a very small percentage of the cells expressed
receptor. The cause of the apparently low transfection
TNF binding and expression of TNF receptor protein in COS-1 cell transfectants
Specific cell surface
bound TNF-LY
Transfectant
Specific DNA COS- 1 cell transfectant
Control DNA COS- 1 cell transfectant 18
Control DNA Cos- 1 cell transfectant 2s
cpm/dish*
5,170
1,230
1,010
Relative
expressionof
cpm/ 106 cells
890
210
185
75/65-kDa
receptor
versus 55-kDa
in cell lysatet
1.39
0.05
0.15
‘All values are the average of two independent experiments.
$The quotient of specific 75-kDa and 55-kDa receptor ‘*‘I-TNFa
binding measured in sandwich assays (see Materials and Methods).
IControls
1 and 2 refer to parallel transfectants
in which constructs in which the cDNA was ligated into the expression vector in a false reading frame were used.
234
/ Dembic
CYTOKINE,
et al.
0.15
0
200
400
600
800
1000
Vol. 2, No. 4 (July 1990: 231-237)
B
0
1000
2000
3000
4000
5000
BOUIldiCell
TN= (PM)
Figure 3. “‘1-TNF-cr binding to transient CO&l cell transfectants.
(A) Specific binding at various TNF-ol concentrations. Measurements at higher concentrations confirmed
saturation of TNF binding (data not included in figure). (B) Plot of the binding data according to Scatchard. The
mean and standard deviations of triplicate experiments are given. The assays with transfected and control cells
contained 2.2 x lo6 and 4.3 x lo6 cells/assay, respectively. 0, 75kDa TNF receptor transfectants; n ,
non-transfected control cells. The K,‘s of transfected and control cells from Scatchard analysis are about 0.1 and 0.2
nM, respectively.
yield remains unknown, but it explains the low receptor
copy number in the pool of transiently transfected cells.
TNF Receptor Expression
in Cell Lines
The expression of the TNF receptors was studied in
human cell lines by Northern analyses (Fig. 4). Previous
flow cytometric analyses of cells stained with receptorspecific monoclonal antibodies had shown that HEp2
cells stain for the 55-kDa receptor only, while HL60
cells stain for both the 55kDa and 75-kDa receptors.”
In agreement with previous reports, Raji cells were
found to be devoid of TNF receptors.“‘26 These findings
were supported by the Northern blot analyses shown in
Fig. 4. We note, however, that the lack of 75-kDa TNF
receptor expression appears not to be a stable property
Actin
a b c
c=1 0 El-
75 ret
55 ret
a b
000
c
a b c
000
-II
‘1..
-28s
Figure 4. Northern analysis of TNF receptor expression in Raji (a),
HL60 (b), and HEp2 (c) cell lines.
By cell surface staining with specific monoclonal antibodies, no TNF
receptors are detected on Raji cells, low amounts of 55-kDa receptor
(55 ret) are detected on HEp2 cells, and both 55-kDa and 7%kDa
receptors (75 ret) are detected at relatively higher levels on HL60
cells.
of Raji cells, since other investigators detect 75-kDa
TNF receptor mRNA in these cells.16 From preliminary
studies of 55-kDa and 75-kDa TNF receptor expression
HL60 cells appear to be more representative of the
average human cell than HEp2 or Raji cells, because
many cells were found to express both TNF receptors
simultaneously, albeit to very different extents.
Expression of Each TNF Receptor is
Independently
Regulated
To investigate the regulation of the two TNF
receptors, we have studied their expression in phytohemagglutinin-activated
peripheral blood lymphocytes
(PBL). By cell surface staining with the specific monoclonal antibodies utr-I (anti-75-kDa
receptor) and
htr-9 (anti-55-kDa receptor),” we find that the expression of the 75-kDa receptor is strongly induced from a
low resting level, whereas the 55-kDa receptor expression remains at a constant and low level after mitogen
activation (Fig. 5). The inducibility of TNF receptors in
several cell lines has been previously reported.27 The
finding that the induction is restricted to one type of the
two TNF receptors in stimulated PBL as well as
analogous findings in cell lines (Hohmann et al., submitted for publication) indicates that the two TNF receptors are functionally distinct.
DISCUSSION
Most human cells express two distinct TNF receptors simultaneously. The molecular cloning of the 55kDa receptor14,‘5 and of the 75-kDa receptor (reference
16 and this work) now allows a comparison of the
predicted amino acid sequences of both receptors. The
Human TNF receptors/ 235
(Fig. 5) it appears more likely that the two TNF
receptors are functionally distinct.
MATERIALS
AND
METHODS
Cells and Flow Cytometry
Figure 5. Flow cytometric analysis of 75/65-kDa (TNFR-A) and
55kDa
(TNFR-B) receptor expression on resting (dotted tine) and
activated (solid line) peripheral blood lymphocytes (PBL).
extracellular regions are found to be highly similar to
each other and to those of the NGF receptor and the
CDw40 and OX40 antigens. These cell surface molecules thus form a novel gene family. The functional
significance of the similarity to the LIM sequence
motiP2 remains to be established.
Two TNF-inhibitory
peptides of human serum and
urine have been described and partial amino acid
sequences have been reported.28-30 One of these inhibitors previously has been recognised as a fragment of the
55-kDa TNF receptor.14,‘5 We now find that the short
NH,-terminal sequence of the second inhibitor3’ matches
the V5-Pg peptide sequence of the 75kDa TNF receptor
(Fig. 1). The Northern blot analysis of cell lines (Fig. 4)
reveals a single 75-kDa TNF receptor mRNA species of
about 4 kb and provides no evidence for a second
message which might encode this inhibitor; analogous
conclusions are valid for the other inhibitor.14 Both of
these TNF inhibitory peptides therefore are NH,terminally truncated, soluble fragments, presumably of
the extracellular regions of the two TNF receptors, and
therefore are most likely the products of posttranslational processing of the receptor.
The predicted amino acid sequences of the intracellular regions of the two TNF receptors are unrelated
and, furthermore, no similarities to other known mammalian sequences were discovered. It might be concluded that the different intracellular domains transmit
distinct signals upon TNF binding to the receptors.
However, we cannot presently exclude the possibility
that the intracellular regions have no role in signal
transduction. A model analogous to that of the interleukin 6 (IL 6) receptor might be considered, where the
complex of IL 6 and IL 6 receptor can interact extracellularly with a non-ligand-binding
membrane glycoprotein, thus providing the IL 6 signah31 both TNF receptors might then address the same signal transducing
element. However, in view of the independently regulated expression documented at least in T-cell activation
The cell lines HL60 (ATCC CCL 240), HEp2 (ATCC
CCL 23), Raji (ATCC CCL86) and COS-1 (ATCC CRL
1650) were grown in RPM1 1640 or Dulbecco’s modified
Eagle’s medium supplemented with 10% inactivated horse or
fetal calf serum. Human PBL from a Ficoll gradient were
cultured in RPM1 1640, 10% fetal calf serum with or without 2
mg/mL phytohemagglutinin (Wellcome). Cells were stained
with biotinylated utr-1 (anti-75-kDa receptor) or htr-9 (anti55-kDa receptor) antibodies followed by streptavidin-phycoerythrin and analysed on a FACScan flowcytometer.
Reagents
Recombinant human TNF-a purified from Escherichia
coli was a gift from W. Hunziker, E. Hochuli, and B. Wipf
(Hoffmann-LaRoche LTD, Basel). TNF-LUwas radioiodinated
with Na’*jI (IMS30, Amersham) and Iodo-Gen (Pierce) to
0.3 x IO*-1.0 x IO* cpm/pg as described.32
cDNA Cloning and Northern Analysis
The 75-kDa and 65-kDa TNF receptors were purified
from HL60 cells,and tryptic digests and gas phase sequencing
were performed as reported elsewhere.‘*A DNA fragment was
prepared from the peptide sequence Q412-L 428by PCR on
human genomic DNA using 2 low-stringency annealing cycles
(95OC7 min / to 37OCin 2 min / 37°C 1 min / to 72OCin 2.5
min / 72OC1.5 min / to 95OCin 1 min / 95°C 1 min / to 37OC
in 2 min) followed by 38 standard cycles(95OC 1 min / 55OC2
min / 72°C 2 min); the forward and reversePCR primers were
ctcgaattcCARCTNGARACNCC
and CtcgaattcNARNGGYTTYTCYTC, respectively. The DNA band of predicted
size from a polyacrylamide gel of the PCR product was
recloned, sequenced, and found to encode the Q4’*-L 428
peptide. A 48-mer oligonucleotide derived from this DNA was
used as a probe to screencDNA libraries. Several overlapping
clones were identified in a human placenta cDNA library in
Xgtl 1 (Clontech) and in a HL60 cDNA library in Xgtll that
was prepared with the use of cDNA synthesisand cloning kits
(Amersham). All recloning and nucleotide sequencing was by
standard protocols.33For Northern analysis, 12 pg aliquots of
Raji-, HL60-, or HEp2-cell total RNA were electrophoresed
through an agarose gel containing formaldehyde. RNA was
transferred to a Zeta Probe (BioRad) filter, and hybridized to
actin, 55-kDa receptor (full length), and 75-kDa receptor
(170-bp 5’-fragment) cDNA probes as indicated.
Expression and TNF Binding in COS Cell
Transfectants
The cDNA shown in Fig. 1, truncated at the 3’-end was
cloned into a pLJ268 vector (gift of B. Cullen*‘) containing the
IL 2 receptor signal sequence under the control of the RSV
long terminal repeat promoter and polyadenylation signals
236
/ Dembic
CYTOKINE,
et al.
derived from the rat preproinsulin II genomic gene. DNA was
transiently transfected into COS-1 cells with DEAE dextran
following standard protocols.33 Specific 12’I-TNF-~ binding on
transfectants was measured in the absence and presence of
excess unlabeled TNF-ol after 3 days in culture as previously
reportedI
and Scatchard analysis was carried out. Briefly,
COS-1 cellswere detached with EDTA (GIBCO), washed and
incubated with “‘I-TNF-(U for 2 hr at 4°C; cell-bound and free
radioactivity was then counted. Aliquots of transfected cells
were lysed by 1.0% Triton X-100. The expression of the
75-kDa TNF receptor and of the “55-kDa-type” endogenous
COS- 1 cell receptor was measured in transfectant cell lysates
in a solid phase sandwich assayusing the 75-kDa and 55-kDa
receptor-specific monoclonal antibodies utr-4 and htr-20,
respectively, and with ‘251-TNF-a in the absenceand presence
of unlabeled TNF-or. The relative receptor expression in the
cell lysate in Table 1 is defined as the quotient of specific
75-kDa and 55-kDa TNF receptor 12’I-TNF-o( binding measured in the two sandwich assays. Controls 1 and 2 refer to
parallel transfectants in which constructs where the cDNA
was ligated into the expression vector in a false reading frame
were used.
Acknowledgments
We thank M. Steinmetz for stimulating discussions,
W. Bannwarth for synthetic oligonucleotides, and W.
Eufe, N. Grau, A. Hayes, J.D. Hulmes, C. Kocyba, C.
Kuerschner, H.P. Kurt, K. McCune, M. Ott, U. Roethlisberger and L. Stehrenberger for help and excellent
technical support.
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