(Received for publication, February 25, 1997, and in revised form, April 28, 1997)
From the Department of Pathology, Oita Medical University, Hasama-machi Oita 879-55 Japan
The murine cell surface antigen mCD156 is a
glycoprotein that is expressed in monocytic cell lines and consists of
a metalloprotease domain, a disintegrin domain, a cysteine-rich domain,
and an epidermal growth factor-like domain in the extracellular region.
The mCD156 gene is composed of 24 exons and 23 introns and spans
approximately 14 kilobases. The first exon encodes most of the signal
peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain
and a disintegrin domain, respectively. Sequence analysis of the
5-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that
nucleotides at positions
183,
334, and
623 contained
cis-acting enhancing elements in a mouse monocytic cell
line, aHINS-B3. Nucleotides at positions
183 and
390 contained
elements responsible for lipopolysaccharide (LPS) inducibility,
although several other 5
-flanking regions were also involved in LPS
responsiveness. Regions
202,
507, and
659 play a role in
interferon-
inducibility. Some of the potential regulatory motifs
and other unknown cis elements may be involved in the
constitutive expression, and LPS and interferon-
inducibilities. The
mCD156 gene was mapped to chromosome 7, region F3-F4.
The mouse CD156 (mCD156)1 (MS2) and human CD156 are type I transmembrane glycoproteins found on myelomonocytic cell lineage (1, 2). The mCD156 cDNA is 3.2 kb long with an open reading frame encoding 826 amino acids. The extracellular region consisting of 644 amino acids has a metalloprotease (MTP) domain containing the zinc-binding consensus histidine and protease catalytic glutamic residues and a disintegrin domain containing a platelet aggregation inhibitor-like structure, whereas the cytoplasmic region consisting of 143 amino acids contains a proline-rich amino acid sequence containing consensus SH3 (Src homology 3) binding sequence. mCD156 plays a role in infiltration of leukocytes by leukocyte adhesion to endothelial cells and degradation of vascular basement membrane (3).
CD156 is a member of a family of proteins (ADAM) which are
characterized by a conserved structure in hemorrhagic snake venom proteins. Following our report of mCD156 (ADAM 8), a number of ADAMs
have been reported (1, 4-14). ADAMs 1-7 are expressed in reproductive
organs, mainly the testes, and are implicated in sperm-egg fusion
and spermatogenesis. ADAMs 8-12 (meltrin-) and 15 (metargidin) are of non-reproductive cell origin, including myeloid,
muscle, brain, and breast cells. ADAM 12 has been shown to mediate
myocyte cell fusion. Recently, a new family member designated TACE has
been reported to function as a tumor necrosis factor-
-converting
enzyme that liberates 17-kDa tumor necrosis factor-
from 26-kDa
membrane-bound pro-tumor necrosis factor-
(15, 16). TACE activity is
also found in ADAM 10, which is originally separated as an enzyme for
degradation of myelin basic protein (17). Another new family protein
designated ADAMST-1 appears to contain thrombospondin-like structures
and play a role in tumor cell migration (18).
We report here the isolation and characterization of the mCD156 gene
and the presence of the cell type-specific, lipopolysaccharide (LPS)
and interferon (IFN)--inducible promoter activities in its 5
upstream sequence. We have also determined the mCD156 gene location.
Restriction enzymes were purchased from Takara
(Kyoto, Japan), Toyobo (Osaka, Japan), Life Technologies, Inc.
(Gaithersburg, MD), and Wako Pure Chemicals (Osaka, Japan). Agarose
ultrapure DNA grade was from Takara. The DNA ligation kit, reverse
transcriptase, S1 nuclease, and mung bean nuclease were obtained from
Takara, and bovine alkaline phosphatase was obtained from Boehringer
Mannheim (Tokyo, Japan). LPS (Escherichia coli serotype
055:B5) was obtained from Sigma Chemical Co. (St. Louis, MO). Mouse
IFN- was purchased from Genzyme (Cambridge, MA). Radioactive
nucleotides [
-32P]dCTP (3,000 Ci/mmol) and
[
-32P]ATP (6,000 Ci/mmol) were obtained from NEN Life
Science Products. Chemicals used for DNA sequencing were obtained from
Toyobo. X-ray film (XAR-351) was obtained from Kodak (Rochester,
NY).
Murine macrophage cell line aHINS-B3 and murine glioblastoma cell line G203 were grown in Dulbecco's modified Eagle's medium containing fetal calf serum.
Production of Genomic Libraries and Isolation of mCD156-associated ClonesThe bacteriophage EMBL-3 murine liver
library was produced as described previously (19, 20). Briefly, genomic
DNA prepared from BALB/c murine liver was partially digested with
MboI, followed by size fractionation via sucrose gradient
ultracentrifugation. The DNA fragments in the size range of 12-20 kb
were inserted into the BamHI site of EMBL-3 arms. The
annealed DNA was packaged using a commercial packaging extract
(Stratagene Cloning Systems, La Jolla, CA), and then the phage was
grown in E. coli Q359 and screened as described. Radioactive
probes were prepared by 32P labeling cDNA inserts from
an mCD156 clone (1) using nick translation (21), with a specific
activity of 108 cpm/µg of DNA. Filters containing
recombinant plaques were screened according to the procedure of Benton
and Davis (22), prehybridized at 42 °C in 6 × SSC, 5 × Denhardt's solution for 4 h, and hybridized with 106
cpm/ml 32P-labeled insert at 42 °C for 20 h. The
filters were exposed to Kodak XAR-5 film with a Fuji intensifying
screen at 80 °C for 2 days. Phage clones were subsequently
purified by repeated screening cycles. Large scale phage DNA and
minipreparations were obtained as described by Maniatis et
al. (23).
DNA fragments originating from the genomic DNA inserts of phage clones were subcloned into pUC118/119. The pUC118/119 vectors were linearized and ligated for 12 h at 16 °C using a ligation kit purchased from Takara. The ligated DNA was used to transform MV1184 cells. White bacterial colonies were selected. Nucleotide sequences were determined by dideoxy chain termination (24, 25) using single-stranded plasmid DNA with modifications as described in the Sequenase technical manual (Toyobo). Nucleotide sequences were determined on DNA subcloned in pUC118/119 vectors using pUC primers. Other oligonucleotides used as primers for sequencing were synthesized using an automated DNA synthesizer (model 8700, Biosearch). Sequences were determined on both strands.
S1 Nuclease MappingS1 nuclease mapping was performed
according to a modified method of Berg and Sharp (26). A
single-stranded DNA corresponding to the
SalI-BamHI fragment containing the region from
the 5 upstream to intron 1 of the mCD156 gene was prepared using
M13KO7 phage as a vector and annealed with an end-labeled
oligonucleotide primer, a synthetic 30-base single-stranded oligomer
(3
-TGGTGTCCATAAGACGCTGAGCAGCCAGAG-5
) corresponding to bp +143 to +172
of the first exon of the mCD156 gene. The annealed DNA was incubated
with 400 µM dNTPs and 10 units of the Klenow fragment of
E. coli DNA polymerase for 30 min at 37 °C. The mixture
was heated to 65 °C for 5 min to inactivate the Klenow fragment and
then chilled on ice. After digestion with PstI and alkali
denaturation, the extended primer was separated on alkaline gel
electrophoresis. Total RNA, 50 µg from aHINS-B3 cells, was then
hybridized for 16 h at 50 °C with the extended primer in 40 mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM
EDTA, and 80% formamide. Following hybridization, the reaction was
diluted 10-fold with S1 nuclease buffer (300 mM NaCl, 30 mM sodium acetate, pH 4.5, and 3 mM
ZnCl2), and 10 µg of salmon sperm DNA. S1 nuclease (130 units) was added. The reaction mixture was then incubated for 1 h
at 37 °C. The reaction mixture was terminated by the addition of
termination buffer (2.5 M ammonium acetate and 50 mM EDTA), and the DNA·RNA hybrids were extracted with
phenol, precipitated with ethanol, resuspended in 80% formamide,
heated to 90 °C, and resolved on a 6% acrylamide, 8 M
urea gel.
The probe for primer extension analysis was the end-labeled synthetic 30-base single stranded oligomer used in the S1 nuclease protection. Total cellular RNA was isolated from aHINS-B3 cells. The primer was annealed to 100 µg of total RNA by heating the reaction mixture for 15 min at 80 °C in 20 µl of 80% formamide, containing 400 mM NaCl, 40 mM Pipes, pH 6.4, and 1 mM EDTA. The resulting DNA·RNA hybrid was ethanol precipitated and dissolved in reverse transcriptase buffer (50 mM Tris-HCl, pH 8.0, 20 mM 2-mercaptoethanol KCl, 10 mM MgCl2) in the presence of 1 mM deoxynucleotides, 100 units of reverse transcriptase, and 60 units of RNase inhibitor. After 90 min at 42 °C, the DNA·RNA hybrids were phenol extracted, ethanol precipitated, dissolved in loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF), heated to 90 °C, and resolved on a 6% acrylamide, 8 M urea sequencing gel.
Construction of mCD156-CAT PlasmidsmCD156G1 clone
containing the 2776-bp nucleotides corresponding to bases 2279 to 498 of the 5
upstream, exon 1 and intron 1 sequence of the mCD156 gene was
digested with XbaI and SphI. The insert in the
linealized DNA was 3
deleted with exonuclease III. Aliquots (2.5 µl)
were removed 1-min intervals and mixed with 50 µl of 2 × mung
bean nuclease buffer. After heating for 5 min at 65 °C, the samples
were incubated with mung bean nuclease for 60 min at 37 °C. The
3
-deleted samples were then digested with the Klenow fragment followed
by T4 DNA ligase treatment. To determine the extent of deletion, each
mutant was sequenced as described previously (19). Selected mutants was
digested with HindIII and inserted into the
HindIII site of the multicloning site containing pSVmCAT.
This plasmid containing the 2033-bp nucleotides corresponding to bases
1943 to 90 was digested with KpnI-SmaI and
subjected to successive treatment with exonuclease III, mung bean
nuclease, and Klenow fragment as above. Each 5
-deleted DNA was ligated
with T4 DNA ligase. Plasmid DNA was prepared by alkaline lysis followed
by two centrifugation steps through CsCl to isolate supercoiled plasmid
DNA.
Plasmid DNA was transfected
into the mouse macrophage line aHINS-B3 and the mouse glioblastoma line
G203 by calcium phosphate coprecipitation as described previously (19).
Cells were seeded at 2 × 106 cells/plate. For
transient transfections, a mixture of the test CAT hybrid gene (20 µg
equivalent) and the transfection control plasmid (20 µg) pCH110
containing the -galactosidase gene was precipitated in
Hepes-buffered saline, pH 7.1, and then added to the plates. After
4 h, the cells were given a 1.5-min glycerol shock followed by a
wash with ice-cold phosphate-buffered saline. Fresh medium was then
added and the incubation continued for 24 h. 24 h after
transfection, the cells were stimulated with LPS (100 ng/ml) or IFN-
(200 units/ml) for 16 and 24 h, respectively. Cells were washed
three times with phosphate-buffered saline, incubated with 40 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA for 5 min on ice, harvested by scraping and
centrifugation, and resuspended in 180 µl of 0.25 M
Tris-HCl, pH 8.0. Extracts were prepared by freeze-thaw and
centrifuged. Supernatants were collected and assayed for protein
according to the method of Bradford (27). CAT was assayed as described
previously (19). The extract was incubated with
[14C]chloramphenicol and 0.2 mg of acetyl-CoA for
1.5 h at 37 °C, and the products were separated by thin layer
chromatography. The CAT activity for each construct was normalized to
-galactosidase activity from the same sample. The relative CAT
activity for each deletion mutant was expressed as a percentage of that
of wild type
390/+90CAT stimulated with LPS.
Mouse chromosomes
were prepared according to a published procedure (28). Briefly,
lymphocytes were isolated from mouse spleen and cultured at 37 °C in
RPMI 1640 medium supplemented with 15% fetal calf serum, 3 µg/ml
concanavalin A, 10 µg/ml LPS, and 5 × 105
M 2-mercaptoethanol. After 44 h, the cultured
lymphocytes were treated with 0.18 mg/ml bromodeoxyuridine (Sigma) for
an additional 14 h. The synchronized cells were washed and
recultured at 37 °C for 4 h in
-minimal essential medium
with thymidine (2.5 µg/ml) (Sigma). Chromosome slides were made by
the conventional method used for human chromosome preparation
(hypotonic treatment, fixation, and air drying).
The genomic DNA of mCD156G2 containing a 15-kb insert spanning nearly
the entire coding regions except the most 5 end (see Fig. 1) were
biotinylated with dATP using the Life Technologies, Inc. BioNick
labeling kit (15 °C, 1 h) (29). The procedure for FISH
detection was performed according to the method of Heng et al. (30) and Heng and Tsui (29). Briefly, slides were baked at
55 °C for 1 h. After RNase A treatment, the slides were
denatured in 70% formamide in 2 × SSC for 2 min at 70 °C,
followed by dehydration with ethanol. The probe was denatured at
75 °C for 5 min in a hybridization mix consisting of 50% formamide,
10% dextran sulfate, and human cot I DNA, and prehybridized at
37 °C for 15 min. The probe was then loaded on the denatured slides.
After overnight hybridization, slides were washed and detected as well
as amplified using a published method, FISH signals and the DAPI
banding pattern were recorded separately by taking photographs,
and the assignment of the FISH mapping data with chromosomal
bands was achieved by superimposing FISH signals on DAPI-banded
chromosomes (30, 31).
The mCD156
mRNA was 3.1 kb (Fig. 1), from which mCD156 cDNA
was cloned and characterized (1). A genomic library, prepared from an
MboI partial digest of BALB/c mouse DNA inserted into EMBL-3, was screened using randomly labeled mCD156 cDNA. Two
positive clones, designated mCD156G1 and mCD156G2, were selected for
further analysis. Mapping and sequence analysis of these inserts
established that clone mCD156G1 contained nearly the entire 5 half of
the message for mCD156 but not for the 3
half of the gene, whereas clone mCD156G2 contained a 15-kb insert spanning nearly the entire coding regions except the most 5
end.
The mCD156 gene is composed of about 14 kb, 24 exons and 23 introns.
Fig. 1 is a line drawing of the intron-exon organization of the gene.
The introns range in size from 71 (intron 11) to 2,687 (intron 22) bp.
Large introns (intron 1, 907 bp; intron 2, 1,340 bp; and 22) occur on
the 5 and 3
side of the gene, and a cluster of exons is found between
them. Analysis of splice junction sequences revealed that all
intron-exon junctions followed the normal consensus sequence rules
(32), except at intron 11 where the acceptor splice site AG was
replaced by TA (Table I). Exons comprise approximately
22.8% of the gene; about 18% of the total gene actually codes for
amino acids.
|
Exons are often associated with separate structural or functional
domains of a protein (32), such as in the immunoglobulin (33). However,
the exon organization of the mCD156 gene is partially predictable based
on the predicted protein structure (Figs. 1 and 2). The
mCD156 protein is composed of a signal peptide, a pro-MTP, an MTP, a
disintegrin, a CR, an EGF, a transmembrane, and a cytoplasmic domain.
The first exon encodes most of the signal peptide sequence, and the
transmembrane domain is encoded by a single exon (25). In contrast,
other regions are comprised of multiple exons. Exons 2-6 encode a
pro-MTP domain; exons 7-12, an MTP domain; exons 12-14, a
disintegrin domain; exons 14-18, a CR domain, including an EGF domain
within exons 17-18. The cytoplasmic region shows a proline-rich amino
acid stretch that is encoded by exons 20-23. The carboxyl-terminal
residues that have little proline and the total 3-untranslated
region are encoded by exon 24.
The exon-intron organization of the ADAM 11 gene has been reported (34). The numbers of its exons and introns were 25 and 26, respectively, resembling those of the mCD156 gene. Therefore, we compared amino acids encoded by each exon between mCD156 and ADAM 11 (Fig. 2). It is of note that 12 exons out of 24 of mCD156 correspond to the homologous regions of exons of ADAM 11; for example, the boundary between exons 3 and 4 of the mCD156 gene corresponds to that between exons 1 and 2 of the ADAM 11 gene.
Determination of Transcription Initiation SiteThe
transcription start sites of the mCD156 gene were analyzed by S1
nuclease protection. An antisense single-stranded DNA probe (231 bp)
corresponding to the region spanning from site (+172) to an upstream
PstI site (183) (see "Experimental Procedures") was
constructed. The results suggested the presence of a major transcription start site approximately 114 nucleotides upstream of the
translation start site (Fig. 3A). The
transcriptional initiation site demonstrated by S1 nuclease protection
was confirmed by primer extension (see "Experimental Procedures").
An antisense synthetic oligonucleotide was used for primer extension
using RNA isolated from aHINS-B3 cells and reverse transcriptase. A
single transcriptional initiation site 134 nucleotides upstream of the
translation start site was identified (Fig. 3B), which
confirmed the S1 nuclease result.
Characterization of the 5
The 5 upstream
region contained a number of potential regulatory sequences, as shown
in (Fig. 4). TATA box-related elements (TATAGA) were
identified at positions
25 to
20, possibly directing transcription
from the indicated position +1 (Fig. 4). Although it differs from the
canonical sequence by a G in the 5th position and although G and C are
rare in TATA sequences and are associated with down-mutations in some
promoters, the human
-globin promoter TATA box contains a G (35).
Other TATA box-related sequences, TATAA and AAATTT, are found further
upstream, although these sequences appears to be too distant to direct
transcription from the cap site. Another regulatory element, the
atypical GC box-related motif GGGAGG (CCTCCC), is located at positions
295 to
290 and
231 to
226. Commonly, binding sites for Sp-1 are
located near the TATA box, but in some cases, such as
hydroxymethylglutaryl-CoA reductase, they are further upstream (36).
The CAAT (ATTG) box and its reverse sequences are found at positions
225 to
222,
217 to
214,
168 to
164, and
112 to
109
(37). The PU box GAGGAA (TTCCTC) that is recognized by the macrophage
and B cell-specific transcription factor PU.1, which is related to the
ets oncogene (38), is located at positions
484 to
479 and
279 to
274. An NF-1 binding site exists at positions
49 to
37 (39). The interleukin (IL)-6 response element motif, CTGGGA (TCCCAG), is found at
positions
621 to
616 and
200 to
195. The IL-6 response element
is present in many acute phase response genes such as mouse serum
amyloid A, rat
2-macroglobulin, and C-reactive protein (40), and in the complement component 5 gene. The NF-IL-6 binding site
and its reverse sequence are located at positions
591 to
588,
422
to
415, and
324 to
318. A minimal motif of IFN-stimulated response element, GAAANN, and its reverse sequence exists in the region
between
358 and
338 (41). An interferon regulatory factor-1 binding
sequence is located at positions
38 to
33 (42-44). A heat-shock
element, GAANNTTC, shares elements with AAATTT at positions
320 to
315 (45, 46). The further upstream sequence (
531 to
691) has no
consensus regulatory elements.
CAT Analysis of Transcriptional Regulatory Regions Upstream of the mADAM 8 Gene
To examine the regions essential for transcription
of the mADAM 8 gene, we fused various portions of the 5-flanking
region to the bacterial CAT gene as a heterologous reporter gene (see "Experimental Procedures" and Fig. 5). The
constructs were transfected into aHINS-B3 cells, which express mADAM 8, and G203 cells, which express no mADAM 8. The region including 44 bp
immediately upstream of the transcription start site (
44CAT) was
incapable of directing CAT synthesis in aHINS-B3 cells, whereas
183CAT clone containing further 139 bases upstream of the
44CAT
clone had a significant CAT activity. Stretch of the upstream sequence
greatly diminished the CAT gene expression. However, the CAT gene
expression increased by including position up to
334. Further
elongation of the 5
upstream sequence reduced the reporter gene
expression, but the
623CAT clone showed a significant CAT synthesis.
Very little or no CAT synthesis was observed in G203 cells.
We showed previously that expression of mMS2 mRNA in mouse
macrophage cell line is enhanced by LPS and mouse IFN- (1). We
examined the effect of LPS and IFN-
on CAT expression. The CAT
gene-transfected aHINS-B3 and G203 cells were stimulated with LPS (100 ng/ml) for 16 h (Fig. 5A). CAT synthesis in aHINS-B3 cells transfected with
183CAT and
390CAT clones after stimulation with LPS was high.
202CAT,
270CAT,
350ACT,
419CAT,
432CAT,
507CAT, and
559CAT clones were also capable of mediating the LPS-induced CAT synthesis. Fold induction of
183CAT and
334CAT was
relatively low because of high basal activity. The CAT gene-transfected aHINS-B3 cells were stimulated with IFN-
(200 units/ml) for 24 h (Fig. 5B). CAT activities in aHINS-B3 cells transfected
with
202CAT,
507CAT, and
659CAT clones were significantly
enhanced after treatment with IFN-
. CAT synthesis in G203 cells was
not enhanced by LPS and IFN-
treatment.
Under the
conditions used, the hybridization efficiency was 90% for the probe
(among 100 checked mitotic figures, 89 showed signals on one pair of
the chromosomes). Since the DAPI banding was used to identify the
specific chromosome, the assignment between the signal from the probe
and the mouse chromosome 7 was obtained. The detailed position was
determined further based on the summary from 10 photos (Fig.
6A). According to the summary, this gene is
located at chromosome 7, region F3-F4. An example of the mapping results is presented in Fig. 6B. The mouse chromosome 7 broadly distributes in human chromosomes including chromosomes 10, 11, 15, 16, and 19. The human and mouse ornithine aminotransferase genes
have been mapped to human chromosome 10q26 and mouse chromosome 7, respectively, and the human and mouse cytochrome P450, subfamily IIE
genes have been mapped to human chromosome 10 and mouse chromosome 7, respectively (47). Thus, compared with human CD156 locus, there is a
strong site relationship between human (chromosome 10q26.3) (2) and
mouse genome.
We have characterized the structure and determined the chromosomal
location of the mCD156 gene. The gene spans approximately 14 kb and
consists of 24 exons and 23 introns. The exon of this gene encode the
mCD156 (MS2) cDNA that had been cloned from mouse myelomonocytic
cells (1). The site of transcription initiation site, as determined by
S1 nuclease protection and primer extention, agreed well with the
location of a TATA box-like element TATAGA at nucleotide position 25
to
20 within the 5
-flanking sequence of the mCD156 gene. Other
sequences that might interact with factors that regulate transcription
were also present within the 5
-flanking region of the gene. Indeed,
CAT analysis suggested the involvement of several regions that
regulated the expression of the mCD156 gene in aHINS-B3 cells in the
presence or absence of LPS or IFN-
.
We also localized the mCD156 gene to mouse chromosome 7, region F3-F4. We localized the human CD156 gene to the distal region of chromosome 10q26.3 (2), an area syntenic to mouse chromosome 7, region F3-F4, the region of chromosome 7 on which the mCD156 gene is found. ADAM 11 has been mapped to human chromosome 17q21(12), where many genes including the myeloperoxydase and homeo box region 2 genes are mapped (47, 48). However, the mouse homologous loci of these genes reside in chromosome 11, which also accommodates homologous loci of human chromosomes 2, 5, 7, 16, and 22. Two forms of transcripts generated by alternative splicing MDC-542 and MDC-769 are identified for ADAM 11: transcripts for MDC-542 encode 542 amino acids, which lack a transmembrane and cytoplasmic region, whereas that for MDC-769 encodes a protein of 769 amino acids which contain full regions from pro-MTP to the cytoplasmic region (34). The size and number of exons in the MDC-769 gene are strikingly similar to those of the mCD156 gene, although exon 1 of MDC-769 has splice variants. They consist of 25 exons and 24 introns: for MDC-769, a pro-MTP domain is encoded by multiple exons including 1b, 1c, 1d, and 2-7; an MTP domain by exons 7-13; a disintegrin domain by exons 14-17; a CR domain including an EGF-like repeat by exons 18-22; a transmembrane domain by exon 23; and a cytoplasmic domain by exons 24 and 25. Alignment of amino acids between mCD156 and ADAM 11 revealed that 12 exons out of 24, in particular, in central region of mCD156 completely corresponded to those of ADAM 11. Thus, the ADAM family genes are probably derived from the same ancestral gene. Since the mouse ADAMs 1, 2, 4, and 5 genes have been mapped to chromosomes 5, 14, 9, and 8, respectively (49), ADAM genes appear to be distributed to different chromosomes after duplication.
ADAM family proteins are a group of proteins exhibiting a hemorrhagic snake venom-like structure, with a widespread cell distribution including sperm, epididymal epithelium, placenta, ovary, breast, skeletal muscle, lung, heart, liver, kidney, small intestine, colon, brain, thymus, spleen, and leukocytes, and a high degree conservation throughout evolution (8, 50). Transmembrane and cytoplasmic domains have been added on the COOH-terminal side of mammalian ADAMs with the process of evolution, and new functions have been acquired. It might be possible to classify ADAMs into five subgroups on the basis of structure and function. The group 1 family of ADAMs (ADAM 10, TACE, KUZ, and possibly CD156) plays a role in the protein degradation by conservation of MTP activity (11, 15, 16, 51). The group 2 family (ADAMs 1 and 12) has a short hydrophobic region similar to viral fusion peptide and is mostly involved in membrane fusion (4, 13). The group 3 family (ADAM 2) induces cell adhesion. The group 4 family (ADAMs 9, 10, 12, and 15) mediates intracellular signaling or cytoskeletal attachment via proteins containing SH3 domains (10, 11, 13, 14). mCD156 could also belong to this class because the cytoplasmic tail of mCD156 has short proline-rich sequences resembling to SH3 ligand motifs. The group 5 (KUZ, ADAMs 7 and 12) facilitates differentiation and maturation of cells including nerve, sperm, and myogenic cells (7, 13, 51). Other ADAM family proteins will be subdivided into these groups in the future.
Some regions 183,
334, and
623 contained CAT active elements.
Since the position up to
334 contains a PU box, the box would
contribute to enhanced CAT expression. PU.1 has been shown to play a
critical role in myeloid cell-specific expression of CD11b
(Mac-1
chain) (52), macrophage colony-stimulating factor receptor
(53), IL-1
(54), Fc
.RIIIA (55) and Fc
.RIb (56). These results,
therefore, suggest that the regions
183,
334, and
623 are
involved in the cell type-specific expression of the mCD156 gene. The
TATA box-like sequence, IL-6 response element, and NF-IL-6 binding
motif are present in the region
623 to
507, whereas CAAT and
inverted CAAT boxes are found in the region
183 to
94.
The regions 183 and
390 efficiently regulate the response to LPS,
although many other regions also appear to contain elements responsible
for LPS inducibility. Several elements including NF-
B (57-59),
Oct-2 (60), AP-1 (61), and NF-IL-6 (59) binding sites have been shown
to mediate response to LPS in several systems, although the role of
NF-
B and NF-IL-6 binding site is controversial (62, 63). Of these,
the 5
-flanking region contains three NF-IL-6 binding sites. NF-IL-6
binding sites may be involved in LPS inducibility, although the regions
183 and
390 have no such elements. Therefore, other cis
regions and/or novel elements may confer LPS responsiveness.
A few restricted regions play a role in IFN--mediated CAT synthesis,
in particular, the distal two regions
507 and
659 being important.
The 5
upstream region of the mCD156 gene contains elements responsible
for IFN-
inducibility such as NF-IL-6 binding sites (58, 63) and
IFN-stimulated response element (64). However, none of these elements
is found in the distal regions. Further analysis is needed to localize
cis regions for IFN-
-stimulated responsiveness.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D10911.
We thank Dr. M. Setoguchi for useful discussions and T. Iwao for the photography.