Molecular Clon;ng, Expression, and Chromosomal
Localization of the Human Earliest Lymphocyte
Activation Antigen AIM/CD69, a New Member of
the C-Type Animal LecCi'n Superfamily of
By Manuel L6pez-Cabrera, Ana G. Santis, Elena Fern~indez-Ruiz,
Russ Blacher,* Fred Esch,* Pali3ma S~inchez-Mateos,
and Francisco S~lnchez-Madrid
From the Servicio de Inmunologfa, Hospital de la Princesa, Universidad Aut3noma de Madrid,
28006 Madrid, Spain; and *Athena Neurosciences, San Francisco, California 94080
The activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69.
This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during
lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting
receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis
for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based
strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated
cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino
acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane,
and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was
rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid
degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded
by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native
CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both
somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding
(G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is
a new member of the Ca2+-dependent (C-type) lectin superfamily of type II transmembrane
receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families
of NK cell-specific genes. CD69 also has structural homology with other type II lectin cell
surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor
(CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional
characteristics with most members of this superfamily, which act as transmembrane signaling
receptors in early phases of cellular activation.
pression of a wide number of known genes (1). The genes
that are induced during the early phase of cell activation are
also known as immediate-early genes. To this group belong
several protooncogenes, such as c-fos, c-jun, or c-re?r, and genes
encoding molecules involved in signaling events, induding
triggering molecules, growth factors, and some cytokine
receptors (1, 2).
The CD69 molecule (3), also designated as activation in-
ducer molecule (AIM) (4), early activation antigen (EA-1)
(5), Leu-23 (6), or MLR-3 (7) antigens, is a phosphorylated
disulfide-linked 27/33-kD homodimeric protein (8). This an-
he activation ofT lymphocytes by antigens or mitogens
initiates a coordinate up- and downregulation of the ex-
tigen seems to be the earliest inducible cell surface glycopro-
tein and is synthesized de novo and expressed upon T lym-
phocyte activation with a wide variety of stimuli, such as
anti-CD3/TCR and anti-CD2 mAbs, activators of protein
kinase C, or PHA (4, 5, 9, 10). The expression of CD69
by IL-2-activated NK cells parallels the acquisition of lytic
activity by these cells (6). CD69 is undetectable on the plasma
membrane of most circulating PBL; however, this molecule
is expressed by a large fraction of T cells in the inflammatory
infiltrates of several human diseases (11, 12), as well as by
a small percentage of resident T and B cells in different normal
lymphoid tissues (13). In this context, CD69 expression on
thymocytes has been associated with the acquisition of im-
J. Exp. Med. y The Rockefeller University Press i 0022-1007/93/08/0537/11 $2.00
Volume 178 August 1993 537-547
munocompetence during T cell maturation (14, 15). Other
cell types, including platelets and epidermal Langerhans cells,
also express the CD69 molecule (16, 17).
The CD69 glycoprotein functions as a signal-transmitting
receptor in different cells. In T cells, the signals triggered
by CD69 antibodies include rise ofintracellular calcium con-
centration, expression of the IL-2R c~ subunit (CD25), syn-
thesis of different cytokines, such as IL-2, TNF-ot, and IFN-%
and cell proliferation (4, 9, 18-20). Furthermore, the trig-
gering of T cells through the CD69 activation pathway en-
hances the binding activity of transcription factor AP-1, which
plays a key regulatory role in the initial steps of cell activa-
tion (21). Likewise, CD69 also acts as a triggering molecule
on NK cells and platelets: CD69 antibodies are capable of
inducing target cell lysis by IL-2-activated NK cells (22, 23),
and of triggering platelet aggregation, Ca 2 + influx, and hy-
drolysis of arachidonic acid (16). The possible coupling of
CD69 with a GTP-binding protein has also been reported (24).
In an attempt to understand the molecular basis for the
CD69 functions, the cDNA encoding this protein was cloned
and its nucleotide sequence determined. We herewith report
that CD69 protein is a type II membrane protein with a C-type
animal lectin domain. Comparative protein sequence ho-
mology indicates that CD69 is a member of a gene super-
family of type II membrane proteins that includes a wide
number of mouse, rat, and human NK cell-specific genes
that, like CD69, function as signal-transmitting receptors.
Materials and Methods
mAbs. The different anti-CD69 TP1/8, TPl/55, and FAB/1
mAbs, directed to extracellular antigenic determinants of CD69,
have been previously described (8). The CH/2 mAb is specific for
an intracellular CD69 epitope (8). Monodonal Ig (Igl,K) from the
myeloma line P3X63 was inchded as negative control in some assays.
Purification of CD69 Protein. The anti-CD69 FAB/1 mAb
(IgG1,K) (8) was purified from ascites fluids by protein A af~nity
chromatography and coupled to CNBr-activated Sepharose Cb4B
(Pharmacia, Uppsala, Sweden) at 2 mg/ml.
PBL were obtained from buffy coats by Ficoll-Hypaque (Phar-
macia) centrifugation. Activation of PBL was carried out by cul-
turing 2 x 10 ~ cells/ml in the presence of PMA (10 ng/ml) and
soluble anti-CD3 SPV-T3b mAb for 24 h (4). Activated PBL (2
x 10 s cells/m1) were lysed in a buffer containing 20 mM Tris-
HC1, pH 8.0, 1% Triton X-100, 5 mM iodoacetamide, 1 mM PMSF,
0.2 U/ml trypsin inhibitor of aprotinin, and 0.025% NAN3. After
30 min at 4~ the lysate was clarified by centrifugations at 1,000 g
for 10 rain followed by 50,000 g for 1 h. Preparative scale im-
munoaffinity isolation was performed by passing 50 ml of lysate
over sequential glycine-quenched Sepharose CL-4B and anti-CD69
FAB/1- Sepharose Cb4B columns at a flow rate of 1 ml/min. The
FAB/1 column was washed with a five-column bed volume of 25
mM Tris-HC1, pH 9.5, 1.5 M NaC1, 0.5% Triton X-100, 1 mM
iodoacetamide, 0.5 mM PMSF, and 0.025% NAN3. The column
was then washed with 3 vol of 100 mM ammonium acetate, pH
6.0, 25 mM N-octyl-/~-glucopyranoside (OG). The CD69 protein
was eluted with 3 vol of 100 mM ammonium acetate, pH 2.5,
25 mM OG at a flow rate of 0.3 ml/min. Fractions (1 ml) were
collected into tubes containing 50 #1 of 1 M ammonium bicar-
bonate. Homogeneity of immunoafl:mity-purified protein was as-
sessed by SDS-PAGE and silver staining.
Protein Digestion, Peptide Purification, and Sequencing. Approxi-
mately 200 pmol (1 ml) of purified protein was dissolved with 200
#1 of 1 M Na2HPO4 and denatured at 100~ for 5 min. After
cooling, N-glycanase enzyme (Genzyme Corp., Cambridge, MA)
was added and incubated for 16 h at 37~
sample was concentrated, washed three times with 0.5 ml of 0.1%
SDS, and reduced in volume to near dryness. The sample was then
diluted up to 50 #1 with sample buffer containing 0.5% 2-ME
without SDS and alkylated with the addition of I/zl of 4-vinyl-
pyridine and run on SDS-11% PAGE.
In situ digestion of the protein in the polyacrylamide gel matrix
was done by incubating the gel pieces with endoproteinase Lys-C
(Promega Biotec, Madison, WI) for 20 h at 37~
The resulting Peptides were then subjected to narrow-bore,
reverse-phase HPLC. The first step of peptide purification was made
on a Vydac C4 (2.1 x 250-mm) column run in 0.1% TFA. Frac-
tions of pure peptide were subjected to automated Edman sequencing
on a protein sequencer (477A; Applied Biosystems, Inc., Foster City,
CA) on line to the 120A Separation system.
cDNA Cloning. Degenerated sense and antisense oligonucleo-
tides, deduced from the amino acid sequences of two Lys-C Pep-
tides (Fig. 1), were used as primers to achieve PCK (25) on a pool
of reversely transcribed KNA from human PBL activated with 10
ng/ml of PMA at various time points (30 min to 48 h). Amplification
was carried out for 35 cycles (l-rain denaturation at 95~
annealing at 50~ and 1.5-min elongation at 72~ using an RNA
PCR kit (Perkin Elmer Cetus, Norwalk, CT). Amplification of
the 3' end of CD69 cDNA was achieved by PCK on reverse-
transcribed RNA from PMA-activated PBL essentially as described
(26), using the internal primer AIM 1 (5'-AGGAACCTGGTC-
ACCCATGG-Y, nucleotides [nt] 1 521-540). To isolate the 5' end
of CD69 cDNA, random-primed first-strand cDNA was synthe-
sized from PMA-activated PBL poly(A) + KNA and poly-
adenylated according to a protocol previously described (27), then
the 5' cDNA end was amplified as described (26), using the in-
ternal primer AIM 3 (5'-TTCCATGCTGCTGACCTCTGT-Y, nt
616-636 of the [-] strand). To verify the nucleotide sequence of
the CD69 open reading frame (OR.F), two independent PCR were
performed on PMA-activated PBL KNA using the primers AIM 6
(5'-GAGCTCCAGCAAAGACTTTC-3 ~ nt 12-31) and AIM 7
strand) from the 5'- and Y-untranslated regions. Amplification was
carried out for 35 cycles (1-min denaturation at 95~
nealing at 58~ and 1.5-min elongation at 72~
products of PCRs were purified from agarose gels by using the
Gene Clean kit (Bio 101 Inc., La Jolla, CA) and cloned into pCK
II plasmid (Invitrogen Corp., San Diego, CA).
DNA sequencing was performed by the dideoxy termination
method (28) either by subcloning restriction fragments into
Bluescript vectors or by direct oligonucleotide-primed DNA se-
quencing with internal primers when no convenient restriction sites
Northern Blot Hybridization Analysis. Total KNA was isolated
from human PBL, either untreated or treated with 10 ng/ml of
PMA at various time points (0.5, 1, 3, 6, 12, 24, and 48 h) by
the guanidine isothiocyanate method (29). A total of 10-20 #g of
RNA of each sample was run on 1% formaldehyde-agarose gels
and transferred onto nitrocellulose membranes. Hybridizations were
performed using the cloned 197-bp fragment, obtained by PCK
After incubation, the
nt 686-705 of the [-]
1 Abbreviations used in this paper: nt, nucleotide; ORF, open reading frame.
538 Molecular Cloning of the CD69 Activation Antigen
with oligonucleotides 3.1+ and 4.2- (Fig. 1), as a 32p-labeled
probe. To normalize by laser densitometry the CD69 mRNA ex-
pression, a 32p-labded 13-actin cDNA probe was used as control.
Chromosome Mapping of the CD69 Gene. Chromosomal local-
ization of the CD69 gene by PCR was performed on DNA from
interspecies hamster-human somatic cell hybrids (BIOSMAP TM
Somatic Cell Hybrids PCRable TM DNAs; BIOS Corp., New
Haven, CT) using the AIM 1 and 3 primers. The specificity of
PCR products was confirmed by hybridization with a genomic
CD69 probe, which included the sequence between the AIM 1
and 3 oligonucleotides.
The regional localization of the CD69 gene was assessed by
fluorescence in situ hybridization with an EMBL3 phage containing
a 15-kb genomic CD69 DNA. The X-h CD69-1 phage, containing
a 15-kb genomic insert, was isolated from a human genomic li-
brary constructed in the EMBL3 phage vector, as will be described
elsewhere. The genomic done was labeled by nick translation with
digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Ger-
many) and diluted in hybridization buffer (50% formamide, 10%
dextran sulphate, 2 x SSC, and 50 mM phosphate, pH 7.0). After
heat denaturation, 4 ng/gl of labeled probe was preannealed with
a 500-fold excess of sonicated human placental DNA for 1 h at
37~ and hybridized overnight to denatured metaphase chromo-
somes from a normal male. Hybridization, washings, and detec-
tion with rhodamine-conjugated antibodies were performed as de-
scribed (30). Chromosomes were counterstained with 75 ng/ml
of 4'-6-diamino-2-phenylindole (DAPI) in antifade medium. After
the fluorescent microscopy, GTG banding was performed as de-
FACS | Analysis. Fluorescence flow cytometry analysis was per-
formed on a FACScan | cytofluorometer (Becton Dickinson & Co.,
Mountain View, CA). Cells were incubated at 4~ with 100 gl
hybridoma culture supernatants, followed by washing and labeling
with fluorescein isothiocyanate-labeled goat anti-mouse Ig (Pierce
Chemical Co., Rockford, IL). Data were collected in a logarithmic
scale, and percentage of positive cells was obtained by substracting
the background given by the mouse myeloma P3X63.
Transfection of CO$ Cells, Indirect Immunofluorescence, and Immu-
no~ecipitation. To construct the CD69 expression vector (pAIMI-
neo), a recombinant pCRII plasmid harboring the complete CD69
ORF and part of the 5' and 3' adjacent sequences (nt 12-705) was
partially digested with BstxI and the resultant 700-bp fragment
ligated to the plasmid pcDNAI-neo (Invitrogen Corp.) downstream
of the cytomegalovirus promoter. The orientation of the insert was
tested by restriction enzyme analysis.
For transient expression of CD69, monolayers of COS-7 cells
were transfected with pAIMI-neo by the diethylaminoethyl-dextran
method (31). Plasmid pcDNAI-neo was also included in the trans-
fection as a control.
Transfected COS ceils were grown on glass coverslips and cells
were fixed in 3.7% formaldehyde in PBS for 10 min at room tem-
perature. Cells were incubated with the primary antibody for I h
at 37~ rinsed in Tris-buffered saline, and stained with the appro-
priate dilution of the secondary antibody (FITC-labded goat
anti-mouse Ig; Pierce Chemical Co.). TPl/8 and TPl/55 mAbs
were used as primary antibodies. Cells were observed using an Op-
tiphot photomicroscope (Nikon Inc., Instr. Group, Melville, NY).
Activated PBL, and transfected COS-7 cells harvested 72 h after
transfection, were radioiodinated in solution with chloroglycoluril
(Iodogen; Pierce Chemical Co.). For immunoprecipitation, equal
amounts of input radioactivity were mixed with 30/zl of different
purified anti-CD69 mAb directly conjugated to Sepharose (2
mg/ml). Immunoprecipitates were processed as previously described
(4) and samples were subjected to SDS-PAGE on 12% gds either
under reducing or nonreducing conditions.
Isolation and Characterization of CD69 cDNA.
glycoprotein was purified from PMA-activated human PBL
by mAb affinity chromatography. Homogeneous CD69 pro-
tein was obtained as determined by SDS-PAGE followed by
silver staining (Fig. 1 A). The purified protein was treated
with N-glycanase, digested with Lys-C endopeptidase, and
the resulting peptides were separated by reverse-phase HPLC
and subjected to amino acid micro-sequencing. A total of 38
residues were determined from four independent peptides (Fig.
1 B). Degenerated sense and antisense oligonucleotides (Fig.
1 C) based on two Lys-C peptides (Fig. 1 B, underlined) were
designed and used as primers to carry out PCR on reversely
transcribed poly(A) + RNA from a pool of PBL activated
with PMA at different times (30 min to 24 h) (see below,
Fig. 5 A). A DNA fragment of 197 bp was amplified using
the oligonucleotides 3.1 + and 4.2-, whereas no amplification
product was obtained with the oligonucleotides 4.1+ and
3.3-. The specificity of the 197-bp product was verified by
a second PCR performed on the first PCR products, using
the oligonucleotide 3.2 +, which overlaps oligonudeotide 3.1+,
and the oligonucleotide 4.2- (Fig. 1 C). The specific prod-
ucts of both PCRs were doned and sequenced. The full-length
cDNA was obtained by PCR after the rapid amplification
of cDNA 5' and 3' ends (RACE) protocols (Fig. 1 D). The
complete nucleotide sequence of the 1.7-kb CD69 cDNA
and the amino acid sequence of the translation product is
shown in Fig. 2.
To confirm the predicted amino acid sequence of the CD69
translation product, two additional independent PCRs were
performed using primers from the 5'- and 3'-untranslated se-
quences adjacent to the ORE No polymerase-induced mu-
tations were observed on these cloned PCR products. The
ORF extended from nt 82 to 678, and contained the amino
acid sequences of the four different Lys-C peptides. ORF was
preceded by a 5'-untranslated region of 81 bp and followed
by a Y-untranslated region of 1,023 bp, which contained three
poly(A) and multiple rapid degradation signals (Fig. 2).
Expression of CD69 Antigen in COS Cells. To confirm that
the cDNA isolated was encoding CD69 antigen, COS-7 cells
were transfected with the CD69 ORF, which was placed under
the control of the cytomegalovirus promoter. Transfected cells
reacted with several anti-CD69 mAbs as shown by im-
munofluorescence (Fig. 3 A). Furthermore, immunoprecipi-
tation with mAb directed against extracellular and intracel-
lular epitopes of the CD69 antigen (8) from 12sI externally
labeled transfected COS-7 cells yielded two bands under
reducing conditions, which corresponded to the 27- and 33-
kD bands of the native CD69 precipitated from activated PBL
(Fig. 3 B). Under nonreducing conditions, immunoprecipi-
ration with anti-CD69 mAb yielded a single band of 60 kD,
which is the expected molecular mass of the disulfide-linked
CD69 homodimer (Fig. 3 B). These results demonstrate that
539 L6pez-Cabrera et al.
Figure 1. cDNA cloning procedure. (A) Homogeneity of immunoatfinity-purified CD69 protein was analyzed by SDS--12% PAGE under redudng
condition followed by silver staining. (B) CD69 Lys-C peptide sequences. The peptides and their position in the cDNA-derived sequence are shown.
They have been represented in the same order as they are located in the CD69 OKE The peptide sequences used for the design of degenerated PCK
primers are underlined. (C) Degenerated oligonudeotides designed from CD69 Lys-C peptide sequences are shown. (N) G, A, T, or C; (Y) C or
T; (R) G or A; (D) G, A, or "1~ and (M) A or C; + and - indicate that the oligonucleotide sequences correspond to the sense or antisense DNA
strand, respectively. (D) Diagrammatic representation of the cDNA cloning procedure.
the cDNA-encoded CD69 protein possesses identical prop-
erties as native CD69 antigen.
Predicted Amino Acid Sequence of the CD69 Translation
Product. The deduced amino add sequence of the CD69 OILF
corresponded to a polypeptide of 199 residues with a predicted
molecular weight of 22,545, which is in agreement with the
previously reported molecular weight of deglycosylated CD69
protein (22,000-24,000) (6, 8). This polypeptide was devoid
of NH2-terminal hydrophobic signal peptide but contained
a transmembrane domain of 26 amino acids as predicted by
hidrophobicity analysis (32). (Fig. 2). These features on the
primary structure of CD69 indicated that it is a type II integral
membrane protein and is therefore composed of a NHz-
terminal cytoplasmic domain of 38 residues, a single trans-
membrane region of 26 residues, and an extracellular COOH-
terminal domain of 135 amino acids.
A putative site was found for N-linked glycosylation in
the extracellular domain of CD69 (Fig. 2). The existence of
this N-linked glycosylation site is in agreement with previous
results demonstrating, by both glycosidase and tunicamycin
treatments, the presence of N-linked glycosylation in the CD69
protein (6, 8). Several potential phosphorylation sites for
serine/threonine kinases, PKC, and casein kinase II were found
within the cytoplasmic tail (Fig. 2), in accordance with the
constitutive Ser/Thr phosphorylation of CD69 in both ma-
ture thymocytes and activated T lymphocytes (6, 14, 33).
CD69 Is a Member of the C-Type Animal Lectin Superfamily.
Comparison of the predicted amino acid sequence with the
SWISSPROT and EMBL databases using the search algorithm
FASTA (34) indicated that CD69 has homology with pro-
teins of the animal C-type lectin superfamily, which also
possess the unusual type II membrane orientation (Fig. 4).
Most markedly, computer-aided comparisons using the
BESTFIT program revealed that CD69 has strong amino acid
sequence similarity ranging from 44 to 48% with the human
NKG2 (35), and the rat and mouse NKR-P1 protein families
(36, 37). It was found that CD69 protein is also homolo-
gous to the human and mouse low affinity IgE receptor
(CD23) (38-40), the hepatic asialoglycoprotein receptor (41),
and the mouse T cell antigen Ly49 (42, 43). The highest
sequence similarity was confined mainly to the C-type lectin
domain, which covers a stretch of 116 amino adds of the CD69
polypeptide, the most striking feature being the presence of
12 invariant residues (Fig. 4). Particularly important is the
540 Molecular Cloning of the CD69 Activation Antigen
GT TTCTTCATGCT CTGAGGACTGGGTTGGCTACCAGAGGAAATGCTACT TTATTTCTACTGTGAAGAGGAGCT GGACTTCA
V 8 8 C 8 i D W V G Y Q R K C Y F I 8 T V K 1~ i W T 8
GCCCAAAATGCT TGTTCTGAACATGGTGCTACTCT TGCTGTCATTGATTCTGAAAAGGACATGAACTTT CTAAAACGATAC
A i N A C st i H G A T L A V I D fJ i K D M N F ~ K 1~ Y
A G 1~. II i H W V O L
8 N G K K K i P G Im P W K W i i N
AACT GGTTCAACGTTACAGGGTCT GACAAGTGTGTTTTT CTGAAAAACACAGAGGTCAGCAGCATGGAAT GTGAGAAGAAT
14 W i 14 V T G a D K C V F L
1( 14 T i V st it 14 i C i X N
L Y W Z C 14 Ir P Y X
Figure 2. CD69 cDNA nucleotide sequence and
predicted amino acid sequence. The transmembrane do-
main is double underlined, and potential polyadenylation
signals are single underlined. Potential rapid degradation
signals are in boxes. Putative N-linked glycosylation sites
are indicated by asterisks and phosphorylation rites are PKC
and casein kinase II (CK2). These sequence data have been
submitted to the EMBL Data Library under accession
conserved placement of six of the Cys residues (Fig. 4), which
appear to be involved in disulfide bonds (41, 44). Although
there are no long continuous stretches of invariant residues,
almost all of the essential residues conforming the C-type
carbohydrate recognition domain (41) are present at approxi-
mately the same spacing. CD69 also showed certain degree
of similarity with the transmembrane and cytoplasmic regions
of other C-type lectin cell surface receptors expressed by leu-
kocytes, such as the NKG2 and NKR-P1 members.
CD69 mRNA Expression. The cell surface expression of
CD69 is almost exclusively restricted to leukocytes and is tran-
siently induced upon treatment of lymphocytes with several
mitogenic reagents such as phorbol esters (4). Northern blot
analysis of RNA from untreated and PMA-treated PBL using
a specific CD69 cDNA probe showed a PMA-inducible RNA
species of 1.7 kb, in agreement with the size of the isolated
cDNA (Fig. 5 A).
The early expression of CD69 protein upon T cell activa-
tion together with the presence of rapid degradation signals
at the 3' untranslated region of the CD69 cDNA led us to
analyze the induction kinetics of the CD69 mRNA. There-
fore, PBL were stimulated with PMA at various time points,
and then the total RNA was subjected to Northern blot anal-
541 L6pez-Cabrera et al.
ysis with a CD69 cDNA probe (Fig. 5 B). In paraUel, the
surface expression of CD69 antigen was assessed by flow
cytometry (Fig. 5 C). CD69 mRNA showed a transient in-
crease after exposure of cells to PMA. It became detectable
by 30 min to 1 h, with maximal expression by 6 h, reaching
a 50-fold increase, and declined thereafter to undetectable levels
(Fig. 5 B). The surface expression of CD69 antigen displayed
a slower kinetics compared with that of the mRNA induc-
tion. It reached a peak by 12 h of PMA treatment, declined
partially by 24 h, and then the induced level persisted for
at least 48 h (Fig. 5 C). These results indicated that the level
of CD69 mRNA regulates CD69 protein expression. Simi-
larly, Northern blot analysis performed on RNA from different
cell lines, which were either unstimulated or stimulated with
PMA for 6 h, showed that the tissue-specific distribution of
CD69 antigen is regulated at transcriptional level (data not
Chromosomal Assignment of the CD69 Gene. The CD69
gene was located on chromosome 12 by PCR analysis of DNA
from interspecies hamster-human somatic cell hybrids (Fig.
6, and data not shown). As observed in Fig. 6 A, an
amplification product of 1.1 kb was obtained only in those
hybrids containing chromosome 12 (lane 4). The presence
Figure 3. Transient expression of cDNA-encoded CD69 in COS-7 cells.
(A) Immunofluorescence staining of CD69-tramfected cells with anti-CD69
TP1/8 and TP1/55 mAbs. The monoclonal Ig from P3X63 myeloma was
included as negative control. (B) Immunoprecipitation analysis of CD69
from tramfected COS cells. Lysates from ~2SI-labeled transf~cted cells with
pcDNA1-Neo (vector without an insert) (lanes 1-3), transfected with
pAIMI-Neo (containing the CD69 OR.F) (lanes 4-6), and PMA-activated
PBL (lanes 7-9) were precipitated with the following antibodies: P3X63
as negative control (lanes I, 4, and 7), the anti-CD69 TP1/8 (lanes 2,
5, and 8), and CH/2 mAbs (lanes 3, 6 and 9) specific for extracellular
and intracellular epitopes, respectively. The samples were analyzed by
SDS-12% PAGE under reducing conditions. Precipitates with the anti-
CD69 TP1/8 mAb from PMA-activated PBL (lane I0) and from pAIMI-
Neo-transfected cells (lane I1) were also analyzed under nonreducing con-
of 1.0-kb intron between oligonucleotides AIM 1 and 3 (data
not shown) is consistent with the size of the amplified frag-
ment. The specificity of the PCR product was further
confirmed by Southern blot hybridization (Fig. 6 B).
To determine the regional location of the human CD69
gene, we have performed fluorescence in situ hybridization
on human metaphase chromosomes using a CD69 genomic
probe. A total of 30 metaphase spreads were analyzed for the
KV~FC~G ~IyYFVMDR KT~SGCKQ~ QSSSiS~LK~! i,~D~DELK~ 187
I~E~IT~S NS~kY~GKER ~T~ESLL~ TSKNSS~LS!~ ~N~M~S ~
i~ .... sPss
YETGFK~P EQPDDWYGK~ 247
W..~S~ GEP~S~ .... 2~3
~.~W~ .......... ~
KD~IDNRP SKLALNT ..........
~ T v r ~ A ~KHE~I<D ...........
!~<.*<EPG ~S~<E ~
.SQGED~M RGSGRWNDAF L~DR~GA~ DRLAT
?9 .. S~K~F. ~KN~SSME ~EK~.~ I N~Y~
NIRDGG~ML. ~SK~LDNGN [~DQVFI .C~i I G~LD
NA. ELN~AV. ~QVNRLKSAQ [~GSSII.YH~ KHKL.
Figure 4. Sequence alignment of
the lectin-like domains of CD69
protein and several other members
of the supergene family. The amino
acid positions within each sequence
are shown. Filled circles indicates
positions where the residue is highly
conserved. The six invariant Cys
residues are enclosed within black
boxes. Identifies between CD69 and
other C-type lectins are shaded; (h)
human, and (ra) mouse.
542 Molecular Cloning of the CD69 Activation Antigen
Figure 5. Analysis of human CD69 mRNA. (A) Northern blot anal-
ysis of RNA from untreated (lane - ) and PMA-treated PBL (lane + )
with a 3zP-hbeled CD69-cDNA fragment spanning nt 482-678. Positions
of the ribosomal 28S and 18S RNA are indicated on the left. (B) Induc-
tion kinetics of CD69 mRNA. PBL vat-re stimulated with PMA (10 ng/ml)
at various time points (30 min, and 1, 3, 6, 12, 24, and 48 h), and the
total RNA of each sample was prepared and subjected to Northern blot
analysis with the same CD69 cDNA probe described above. A •-actin
cDNA probe was used as a control for loading amounts. (C) Diagram-
matic representation of the induction kinetics of CD69 mKNA and pro-
tein. Laser densitometry giving corrected scanning units of the CD69
mRNA expression normalized to the B-actin control gene is represented
by failed circles. Surface expression of CD69 protein analyzed by flow cytom-
etry is represented by open squares. (MFI), mean fluorescence intensity.
presence of rhodamine fluorescent spots. As illustrated in Fig.
6 C, hybridization was specific and the signal was clearly de-
tected on the short arm of chromosome 12, on bands p13-
p12 based on GTG banding. As summarized in Fig. 6 D,
64% of the total fluorescent spots found on chromosome 12
were clustered on the p13-p12 bands.
This study describes the cloning and sequencing of the
cDNA encoding the human early lymphocyte activation an-
tigen CD69. The molecular doning of CD69 antigen was
achieved by a combined strategy that included partial protein
sequencing and PCR techniques. Confirmation that the cloned
cDNA indeed represents CD69 is based on several lines of
evidence. (a) The predicted amino acid sequence encoded by
the OKF is a protein with a molecular weight of 22,545,
which is similar to the reported molecular weight (22,000-
24,000) of the deglycosylated native CD69 protein (6, 8);
(b) the four peptide sequences obtained from the CD69-
purified protein are found in the translated sequence of the
isolated gene; and (c) transfection of COS cells with the doned
CD69 cDNA shows that a disulfide-linked homodimer of
the expected molecular weight under both reducing and non-
reducing SDS-PAGE conditions can be precipitated by sev-
eral anti-CD69 mAbs that are directed against extracellular
and intraceUular epitopes of the molecule (8).
The ORF of CD69 cDNA predicts a 199-amino add poly-
peptide with a type II transmembrane topology. The extracel-
lular COOH-terminal domain possesses an available site for
N-linked glycosylation as proposed for the native protein (6,
8). Previous studies of cell protease treatments revealed the
presence of a CD69 pronase-resistant peptide of 16,000 Mr
that lost the external epitopes, suggesting that the native pro-
tein could be predominantly an intracellular protein (8). How-
ever, it is now evident that CD69 indeed possesses a large
protease-resistant fragment in the extracellular region of the
protein. This is in accordance with the observations that the
purified glycosylated native protein was refractory to protease
fragmentation, and that most of the currently used proteases
failed to cleave the deglycosylated protein without previous
SDS denaturation (8). Taken together, these findings show
that the majority of the antigenic sites recognized by different
CD69 antibodies, including those with agonistic properties,
would be located in the terminal fragment of the external
domain, likely into the lectin domain.
The regulation of the expression of CD69 in PBL appears
to be of remarkable interest. Like other immediate early genes
(45, 46), the CD69 gene displays a rapid and transient ex-
pression, followed by a rapid decay and degradation at the
RNA level. The presence of reiterate rapid degradation signals
(47) at the 3' end of CD69 mKNA may explain its rapid
turnover. This tight regulation ensures the transient activa-
tion of this gene and contributes to prevent the uncontrolled
expression of the CD69 receptor implicated in lymphocyte
proliferation. The pattern of CD69 gene expression closely
resembles that of proto-oncogenes and most cytokines in lym-
phocytes, and it is clearly distinct from that of other lym-
phocyte activation molecules such as the IL-2 and transferrin
receptors, which exhibit a slower kinetics of both KNA in-
duction and degradation (1, 48). However, the expression of
the CD69 protein on lymphocyte cell surface persisted for
longer periods of time. This may be attributed to the high
stability of the CD69 glycoprotein, and its refractoriness to
proteolytic mechanisms as discussed above. Whatever the
mechanisms regulating the expression of the CD69 gene are,
they remain unknown and deserve further investigation.
Comparison of CD69 cDNA with known sequences in-
dicates that the extracellular region of CD69 is characterized
by the presence of a C-type animal lectin domain with similar
membrane orientation and protein sequence as those found
in members of this lectin family. Hence, CD69 is related to
a growing family of type II transmembrane receptors, which
includes the gene families coding for human NKG2 (35), rat
543 L6pez-Cabrera et al.
Figure 6. Chromosomal local-
ization of the CD69 gene. (A) PCR
analysis of DNA from human, ham-
ster, and a set of human-hamster
hybrids from the panel of BIOS-
MAI rrM. PCK reactions were per-
formed using AIM I and 3 primers.
0ane M) molecular weight markers;
(lane 1) hamster; (lane 2) human;
(hnes 3-8) hybrids containing the
following human chromosomes:
(lane 3) chromosome 3; (lane 4)
chromosomes 3, 5, 12, 14, 20
(40%), and 22 (25%), Y; (lane 5)
chromosomes 5, 11, 12 (45%), 14,
19, 21, and 22; (lane 6) chromo-
somes, 1 (60%), 5, 13, 14, 18, and
19; (lane 7) chromosomes 1, DS, 13,
19, 21, and 22; (lane 8) chromo-
somes 5 and 20. (B) Southern blot
analysis of PCR products amplified
in A. The corresponding genomic
fragment of CD69 was used as a
specific random primer probe la-
baled with ~x-pzP]dCTP. A faint
specific band can be detected after
longer autoradiography exposure in
lane 5. (C) Fluorescence in situ hy-
bridization on human metaphases
with a digoxigenin-labeled 15-kb
genomic DNA probe for the human
CD69 gene. Arrows indicate the
specific site of hybridization of the
CD69 probe detected by rhodamine-
labded antidigoxigenin antibodies
on DAPI counterstained chromo-
somes. Fluorescent spots are located
at 12 p13-p12. (Inset) Examples of
DAPI and GTG-banding chromo-
some 12 from other ceils. (D) As-
signment of the CD69 gene to 12
p13-p12. The idiogram of chromo-
some 12 schematically indicates the
distribution of fluorescent spots for
the CD69 locus.
NKR-P1 (36), and mouse NKR-P1 (37, 49), which show
preferential expression on LAK and NK cells. Other type
II lectin cell surface receptors with structural similarities to
CD69 are the mouse Ly 49 (42, 43) expressed by activated
T cells, the CD23 low at~nity Fce receptor, present on acti-
vated B cells and monocytes (38-40, 50), and the hepatic
asialoglycoprotein receptors implicated in the clearance of
plasma glycoproteins (41). Interestingly, the intracellular do-
main of CD69 maintains a certain degree of sequence ho-
mology only with members of the lectin superfamily expressed
by leukocytes, but not with lectin receptors found in hepa-
Our studies by PCR analysis on somatic cell hybrid DNA
demonstrate that the human CD69 gene is located on chro-
mosome 12, in accordance with previous serological analysis
of human-mouse somatic cell hybrids (51). In addition, we
have regionally mapped the CD69 gene to the distal region
of the short arm of chromosome 12, bands p13-p12. Interest-
ingly, the genes coding for Ly 49 and NKI.1 antigens are
closely linked within the distal portion of mouse chromo-
some 6, which is the homologous region with human chro-
mosome 12p (52, 53). The possibility of a gene cluster en-
coding this family of C-lectin receptors expressed by activated
T lymphocytes and NK cells raises important questions in
terms of both the regulation of gene expression and their
function in leukocyte activation.
The CD69 protein shares structural and functional features
with NK-specific receptors encoded by human NKG2 and
rat and mouse NKR-P1 (53). All are disulfide-linked dimers
with a similar molecular size whose expression is either in-
duced or enhanced during activation of T lymphocytes and
NK cells. Although differentially expressed among leukocytes,
all these molecules act as triggering structures. The activating
effect of antibodies specific for the mouse and human CD69
antigen in stimulating redirected target cell lysis by IL-2-
activated NK cells (22, 23) is similar to that described with
544 Molecular Cloning of the CD69 Activation Antigen
antibodies specific for other members of the family such as
mouse NKI.1 and rat NKR-P1 antigens (22, 54). Further-
more, CD69 antibodies are capable of inducing intracellular
signals, including mobilization of intracellular calcium and
T cell proliferation in conjunction with suboptimal doses of
phorbol esters (4, 19). Similarly, NKR-P1 also functions as
an activating molecule on rat NK cells stimulating phos-
phoinositide turnover and rise of intracellular calcium (55).
CD69 also displays common functional properties with the
type II lectin Fce receptor CD23. The CD23 receptor, like
CD69, is involved in lymphocyte proliferation and can be
upregnlated by cytokines (56). Moreover, a proteolytic soluble
fragment of CD23 containing the lectin domain with mito-
genic activity for B lymphocytes has been described (56). CD23
acts as a receptor for the Fc portion of IgE and, additionally,
its interaction with the B cell antigen CD21 has recently been
reported (57). The region involved in the interaction with
IgE has been demonstrated to correspond almost exactly to
the domain homologous with animal lectins (58). The in-
tegrity of the invariant cysteines is absolutely required for
IgE binding (58).
The expression of a C-type lectin domain by the members
of this superfamily of transmembrane receptors suggests their
possible interaction with carbohydrates in a calcium-dependent
manner. Evidence for such a role has been described for the
CD23 member of the family (59). In addition, studies sup-
porting the possibility that carbohydrate structures are in-
volved in recognition events mediated by NK cells have been
reported (60). However, the identification of such putative
carbohydrate ligands is required to elucidate the functional
role of the lectin domain of CD69 and the other members
of this family of signal transmembrane receptors.
It is important to remark that although CD69 is absent
on circulating PBL, it is expressed at remarkably high levels
in the majority of T cells in the inflammatory infiltrates of
several human diseases. Thus, CD69 expression has been de-
tected in T lymphocytes of synovial membrane and synovial
fluid from rheumatoid arthritis patients (12), as well as on
CD8 + T ceils in infiltrates of virus-induced chronic inflam-
matory liver diseases (11). In addition, CD69 is also expressed
by certain CD4 + T cells in the germinal centers of normal
lymph nodes and by a small subpopulation of medullary
thymocytes (13, 14). Therefore, it could be hypothesized that
CD69 functions as an immediate early receptor that would
be required for tissue positioning and localization, and/or
for migration toward sites of inflammation. Conceivably, the
expression of a C-type lectin domain externally exposed on
CD69 may enable its interaction with carbohydrate moities,
allowing T cell migration across the endothelial cell layer and
different tissue components. The molecular characterization
of the CD69 protein and its adscription to this superfamily
of C-lectin signaling molecules provide new insights regarding
the possible functional role of these cell surface transmem-
We thank Drs. A. L. Corbi, J. M. Redondo, M. A. Vega, R. Gonz~llez-Amaro, M. O. de Lan~zuri,
and M. IApez-Botet for critical reading of the manuscript. We acknowledge the contribution of Dr.
A. L. Corbi in an early step of this work, Dr. M. A. Vega for his helpful interpretation of amino acid
sequences, and A. Rodriguez for his help in transfection and sequencing. We are also indebted to the
Spanish EMBnet node for the computing and nucleotide data base facilities. The superb typing assistance
of I. Moreno Montes is also acknowledged.
This work was supported by grants from INSALUD (FIS 91/0259 and PB92-0318), and by fellowships
from Spanish Ministry of Education and Science (M. IApez-Cabrera), and Comunidad Aut6noma Madrid
(A. G. Santis and P. S~chez-Mateos).
Address correspondence to F. S~nchez-Madrid, Servicio de Inmunologh, Hospital de la Princesa, c/Diego
de Le6n, 62, 28006 Madrid, Spain.
Received for publication 24 March 1993.
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