From the Medical Research Council Molecular
Hematology Unit, Institute of Molecular Medicine, Oxford OX3 9DS,
United Kingdom, the § Wellcome Trust Centre for Molecular
Mechanisms in Disease and the Department of Clinical Biochemistry,
University of Cambridge, Cambridge CB2 2XY, United Kingdom, the
¶ Hanson Centre for Cancer Research, Institute of Medical and
Veterinary Sciences, Adelaide, 5000 South Australia, Australia, and the
Peter McCallum Cancer Institute,
Melbourne, 3002 Victoria, Australia
Received for publication, August 31, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Functional analyses have indicated
that the human CD164 sialomucin may play a key role in hematopoiesis
by facilitating the adhesion of human CD34+
cells to the stroma and by negatively regulating
CD34+CD38lo/ The hematopoietic system is composed of a continuum of cells that
are hierarchically ordered on the basis of their proliferative and
differentiation potentials. At its apex is a rare population of slowly
cycling pluripotent hematopoietic stem cells that are not only capable
of extensive proliferation or commitment to one of nine lymphoid or
myeloid cell lineages, but also have remarkable versatility with their
ability to be reprogrammed into non-hematopoietic lineages (reviewed in
Refs. 1 and 2). Hematopoietic stem cells and their progeny migrate to
and colonize a series of hematopoietic sites during ontogeny. Under
steady-state conditions, adult hematopoiesis is restricted mainly to
the bone marrow, where hematopoietic and mesenchymal stem cells and
their progeny reside in specific microenvironmental niches composed of
phenotypically and functionally heterogeneous stromal cells and their
associated biosynthetic products, including cytokines, chemokines, and
adhesion ligands. Stem cell fate (i.e. migration, homing,
cell positioning, differentiation, survival, proliferation, commitment,
specific gene expression, reprogramming, and death or apoptosis) is
regulated by external cues provided by the associated
microenvironmental niche. Recent studies have implicated cooperative
interactions between cytokine, chemokine, adhesion, and signaling
receptors on hematopoietic stem cells and their progeny as key elements
in detecting, translating, and fine-tuning these extrinsic cues
(reviewed in Refs. 3 and 4). Despite this, the precise mechanisms of
hematopoietic stem cell trafficking and localization within
microenvironmental niches in adult and fetal development remain to be
determined. The identification of the full complement of
receptor/ligand interactions and the molecular mechanisms that
determine their involvement in regulating stem cell fate therefore
remains one of the major investigative areas in hematopoiesis.
We have recently identified and cloned human CD164, a novel 80-100-kDa
sialomucin expressed, although not exclusively, by CD34+
and CD34lo/ Our aim in this work was to define the size, diversity, and subcellular
distribution of the human CD164 family and to compare the structural
organization and chromosomal location of the human CD164
gene with its murine counterpart as a first step toward more precisely
exploring the functional role of this molecule in vivo in
murine model systems and in vitro. Toward this end, we
surveyed both human and murine RNAs from many different normal tissues
and transformed cell lines for alternatively spliced isoforms. We have
further defined the complete genomic structure of these human and
murine genes and show that they possess similar genomic structures and
similar chromosomal localizations. These results place CD164 within the
mucin subgroup that is composed of multiple exons and further
demonstrate the diverse chromosomal distribution of this family of
molecules. Structural features and subcellular localization studies
support the view that murine MGC-24v and rat endolyn genes are the
orthologs of human CD164. The new CD164(E1-6) isoform, the major
translated product, most closely resembles the described murine and rat
cDNAs. The finding of additional novel variants of human CD164
extends the repertoire of the CD164 transmembrane receptor isoforms to
four members and suggests that these may differentially interact with
cognate ligands to modulate the activity of CD164 in vivo on
a variety of cellular targets.
Cell Cultures--
The cell lines TF1, KG1A, KG1B, U937, K562,
Calu-1, A431, and KATO-III were maintained in RPMI 1640 medium
containing 10% (v/v) fetal calf serum. TF1 also required 2 µg/ml
recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom) for
growth. Human bone marrow was collected with informed consent and
appropriate ethical approval. Human bone marrow stromal reticular cells
and CD34+ progenitor subsets were isolated and cultured as
described previously (5).
Inhibition of Cell Growth--
The effects of engagement of the
CD164 molecule by the CD164 class II mAb 103B2/9E10 on recruitment of
cells into the cell cycle were determined in single cell assays
as described previously (5). To measure the effects of the CD164 mAbs
on nucleated cell production, CD34+ cells (103
cells/culture) were cultured in triplicate in serum-deprived medium
containing 10 ng/ml each purified recombinant IL-1 CD164 cDNA Probes--
Screening of the human PAC library
and subclones and the probing of Southern blots were carried out with
the human CD164 cDNA probes h1, h2, h3, and h4, derived
from the 105A5 cDNA clone (5, 6) (see the legend to Fig. 1) in the
pGEM T vector (Promega, Madison, WI). The murine CD164
cDNA was prepared by PCR amplification of BALB/c mouse heart first
strand cDNA (CLONTECH, Palo Alto, CA) using
primers mF164 and mCD164-SR or mR164 (see Table I) to the murine
MGC-24 cDNA sequence (13) in the
DDBJ/GenBankTM/EBI Data Bank (accession number AB028895).
Screening of the murine PAC library and subclones and Southern blotting
were carried out with the murine CD164 cDNA probes m1
and m2 (described in the legend to Fig. 6A). Each cDNA
probe (20-50 ng) was labeled with 50 µCi of
[ Southern Blots of Genomic DNA--
A Southern blot containing
human placental DNA digested with EcoRI, HindIII,
BamHI, PstI, and BglII was obtained
from CLONTECH. Mouse strain 129 genomic DNA (15 µg/digest) was similarly digested and Southern-blotted onto
Zeta-Probe membrane (Bio-Rad, Hertfordshire, UK) as described
(6). These blots were hybridized with Isolation and Subcloning of PAC Clones--
The human PAC
library derived from normal human male genomic DNA and the murine PAC
(RPC121) library derived from 129/Svev TACfBr mouse spleen genomic DNA
in the pCYPAC2N and pPAC4 vectors, respectively, and gridded onto
Hybond N filters were provided by the Human Genome Mapping
Project Resource Centre (Cambridge, UK). These filters were
probed with
These sequences and those described below were analyzed using
MacVector, Seqed, Assemblign, Analysis, and Sequencher software packages and aligned with each other and with the CD164
cDNAs, and a contig was generated. Sequence data were then analyzed
by accessing MacVector and the Prosite (Swiss Institute of
Bioinformatics (SIB)), GenBankTM, EMBL, and NCBI Databases
for alignment, homology, N-glycan, hydropathy, and motif
analyses. O-glycbase on the NCBI site was used for predicting
O-glycosylation sites. The signal, transmembrane, and
polyadenylation sites were predicted using Signal P (14), TMHMM (15),
and WebGene HC-PolyA (16) programs, respectively.
Chromosomal Localization of the Murine CD164
Gene--
Fluorescence in situ hybridization analysis was
carried out essentially as described by Buckle and Rack (17). Murine
digoxigenin-labeled PAC was co-hybridized with a commercial
biotin-labeled mouse chromosome 10 paint (Cambio, Cambridge) to
chromosomes prepared from a normal mouse splenic culture stimulated
with lipopolysaccharide for 48 h and detected as described (18).
Chromosomes were counterstained with 4,6-diamidino-2-phenylindole, and
images were captured using a CCD camera and MacProbe Version 4 software
(Perceptive Scientific Instruments, Inc., League City, TX).
RNA Preparation--
Total RNAs from the KG1A, K562, U937, TF1,
Calu-1, and KATO-III cell lines, and from human bone marrow stromal
cells, and from murine strain 129 testis were extracted using the
RNAzol or guanidinium isothiocyanate protocol (19, 20).
PCR Analysis of cDNAs--
PCR analyses were carried out
with Marathon-Ready cDNAs derived from human bone marrow, placenta,
spleen, kidney, and colon or with a murine cDNA panel derived from
adult mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney,
and testis and from day 7, 11, 15, and 17 mouse embryos
(CLONTECH) and with appropriate CD164, RT-PCR Analysis of RNA--
Total human RNAs (2 µg) were
reversed-transcribed by standard techniques. PCRs were then carried out
as described above on these transcribed cDNAs using CD164 reverse
oligonucleotide primers RD1-RD6, MGC-24-B7, MGC-GP-B2, and MGC-GP-B5
(within exons 1-6 and in the 3'-UTR) and the F164 forward primer at
the translational start site and GAPDH positive control primers
(GAPDH-F and GAPDH-R) (see Table I). The PCR products were subcloned
into the pMOS-Blue T vector and automatically sequenced using a
3 pM concentration of the appropriate forward or reverse primer.
3'-Rapid Amplification of cDNA Ends (3'-RACE)--
The
3'-ends of the human and murine CD164 mRNAs were
determined by 3'-RACE using Marathon-Ready cDNAs derived from
human bone marrow, placenta, spleen, kidney, and colon or from
murine testis as templates. The reaction mixtures contained 200 ng of
each cDNA sample and a 10 µM concentration of either
primer MGC-GP-F2 or MGC-GP-F4 for human or primer mCD164-E5F,
mCD164-E6F1, mCD164-3F8, or mCD164-3F10 for mouse and reverse adaptor
primer 1 or 2 (see Table I) together with 200 µM each
dNTP (Amersham Pharmacia Biotech), Taq/PWO (Pyrococcus
woesi) DNA polymerase mixture (Expand Long Template PCR
system), and Taq antibody (CLONTECH) in
Expand Long buffer using the Expand Long Template PCR system according
to the manufacturer's instructions and standard conditions. The PCR products were subcloned into the pMOS-Blue T or pGEM T-easy vector prior to automatic sequencing using the appropriate forward or reverse primers.
Northern Blot Analysis--
Human multiple hematopoietic and
non-hematopoietic tissue Northern blots containing poly(A)+
mRNAs were purchased from CLONTECH. The blots
were probed with RNase Protection--
Two human CD164 probes were constructed
for the RNase protection assay. The first probe, PE1, spanning exon 1 plus 390 bases of the 5'-UTR and 83 bases of intron 1, was prepared by
digesting the 2.73-kb BamHI fragment of the human PAC clone
(B2.73) with the restriction enzymes SalI and
SmaI prior to subcloning into the similarly digested pSPT19
vector (Roche Molecular Biochemicals). Probe PE2, containing exons
3-5, was generated by PCR amplification of the CD164
cDNA using the primer pairs FBAM3 and RECO1 (see Table I), followed
by insertion into the BamHI/EcoRI-digested pSPT19
vector. The long GAPDH universal control probe (GAPDH-L) was produced
by PCR amplification of bone marrow-derived Marathon-Ready cDNA
using primer pairs GAPDH-F and GAPDH-R, whereas the short GAPDH
universal control probe (GAPDH-S) was produced by PCR amplification of
the bone marrow-derived Marathon-Ready cDNA using primer pairs PGF
and PGR (see Table I) and inserted into the
HindII/HindIII-digested pSPT18 vector. PE1, PE2,
and GAPDH plasmids, digested with the KspI,
HindIII, and EcoRI restriction enzymes,
respectively, for linearization, were labeled with
[ Indirect Immunofluorescence and Confocal Microscopy--
Calu-1
and A431 cells were grown on glass coverslips. High density suspension
cultures of KG1B cells were settled onto
poly-D-lysine-coated glass coverslips for 30 min to obtain
a dense lawn of attached cells that could be fixed on the coverslips.
All three cell types were fixed with 4% (w/v) paraformaldehyde in
phosphate-buffered saline for 30 min at room temperature, incubated
with 0.25% (w/v) NH4Cl (3 × 5 min), and then blocked
and permeabilized with 1% (w/v) bovine serum albumin and 0.1% (w/v)
saponin in phosphate-buffered saline for 30 min. Cells were incubated
for 1 h simultaneously with two of the following primary
antibodies in 0.5% (w/v) bovine serum albumin and 0.025% (w/v)
saponin in phosphate-buffered saline: mouse anti-human CD164 mAbs N6B6
and 103B2/9E10 (6), fluorescein isothiocyanate-conjugated mouse
anti-human transferrin receptor antibody (Dakopatts, Glostrup,
Denmark), rabbit anti-early endosomal antigen-1 antibody (serum 243, a
gift from Michael J. Clague) (22), and rabbit antiserum to the
cytoplasmic domain of Lamp-1 (a gift from Colin R. Hopkins).2 For primary
antibody pairs derived from different species, fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies to mouse
and rabbit IgG (F(ab)2 fragments, developed in donkey; 10 µg/ml; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA)
were used. When both primary antibodies were from mouse,
isotype-specific fluorescein isothiocyanate- or Texas Red-conjugated
secondary antibodies (F(ab)2 fragments or whole IgG,
developed in goat; 10 µg/ml; Southern Biotechnology Associates,
Birmingham, AL) were employed. Control cells were labeled with the same
two primary antibodies and only one or the other secondary antibody to
ensure that no cross-reaction between isotypes was observed. Cells were mounted in Prolong Anti-fade (Molecular Probes, Leiden, The
Netherlands). Confocal images were taken with a Leica TCS SP system
equipped with a Leitz Plan-Apo 63× objective (NA 1.4) at a pinhole
setting of 1 airy unit and a resolution of 1024 × 1024 pixels
using a zoom of 2.0-4.0. Adobe Photoshop software was used for image processing.
Complete Sequencing of Human CD164 PAC Clones
To isolate genomic clones, a male peripheral blood leukocyte PAC
library was screened with human CD164 cDNA probes h1 and h2. Southern blotting with CD164 probe h3 demonstrated identical patterns of hybridized restriction enzyme-digested fragments from both
PAC clones and from human placental genomic DNA, indicating that the
PAC clones had not undergone gross rearrangements within the region of
the CD164 gene. A single hybridization band was observed
with each enzyme, the largest being the BamHI-restricted fragment (B1) of ~17 kb. When the blots were reprobed with cDNA probe h3, two additional BamHI fragments of 2 and 3 kb (B2
and B3, respectively) were identified. These three BamHI
fragments span ~21 kb of DNA and contain the whole coding sequence of
the human CD164 gene (Fig. 1
and data not shown). Thus, the human CD164 gene comprises
six exons that are interspersed with five introns of varying sizes
(2.540 kb (intron 1), 1.427 kb (intron 2), 1.788 kb (intron 3), 5.608 kb (intron 4), and 1.494 kb (intron 5)). All predicted splice donor and
acceptor sequences matched the G(T/A)G consensus sequence (23). As
described previously, this PAC clone was used to localize the human
CD164 gene to chromosome 6q21 (6).
cell proliferation. We
have identified three novel human CD164 variants derived by alternative
splicing of bona fide exons from a single genomic
transcription unit. The predominant CD164(E1-6) isoform, encoded by
six exons, is a type I transmembrane protein containing two
extracellular mucin domains (I and II) interrupted by a cysteine-rich
non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify
ligand binding characteristics, are located on the exon 1-encoded mucin
domain I. Three human CD164(E1-6) mRNA species,
exhibiting differential polyadenylation site usage, are differentially
expressed in hematopoietic and non-hematopoietic tissues. This study
provides additional evidence that human CD164(E1-6) represents the
ortholog of murine MGC-24v and rat endolyn. Comparative analysis of
murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two
potential splice variants and a similar genomic structure. Whereas the
human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By
confocal microscopy, human CD164 in CD34+CD38+
hematopoietic progenitor (KG1B) and epithelial cell lines appears to be
localized primarily in endosomes and lysosomes, with low concentrations
at the cell surface. However, in a minority of KG1B cells, CD164 is
more prominently expressed at the plasma membrane and in the recycling
endosomes, suggesting that its distribution is regulated in cells of
hematopoietic origin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
hematopoietic stem cells and associated
microenvironmental cells (5-12). It contains, in its extracellular
region, two mucin domains (I and II) linked by a non-mucin domain,
which has been predicted to contain intradisulfide bridges (12).
Functional analyses in vitro have indicated that this
receptor may play a key role in hematopoiesis by facilitating the
adhesion of human CD34+ cells to bone marrow stroma
and by negatively regulating CD34+CD38lo/
hematopoietic progenitor cell proliferation (5). These effects involve
the CD164 class I and/or II epitopes recognized by the monoclonal
antibodies (mAbs)1 105A5 and
103B2/9E10 (9). These epitopes are carbohydrate-dependent and are located on the amino-terminal mucin domain I (6, 7, 9).
Recently, Kurosawa et al. (13) and Ihrke et al.
(12) demonstrated that murine MGC-24v and rat endolyn share significant sequence similarities with human CD164 (5).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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, IL-3, IL-6,
G-CSF, GM-CSF, and Steel factor with 10 µg/ml mAb 103B2/9E10, 105A5, mIgG3, or mIgM. Additional cytokines and mAbs were added on days
7, 14, and 21, and nucleated cells were counted at weekly intervals. To
examine the effect of engagement of the CD164 molecule on the growth
and development of committed hematopoietic progenitor cells,
561-Dynabead (Dynal, Oslo, Norway)-purified CD34+
cells (103) were incubated with either purified mAb
103B2/9E10 or 105A5 or isotype-matched mIgG3 or mIgM control mAb at
concentrations ranging from 0.01 to 30 µg/ml for 1 h at 4 °C.
Cells were plated in 0.9% (w/v) methylcellulose (Dow Chemicals, Lake
Jackson, TX) supplemented with a 10 ng/ml concentration of each of the
recombinant human cytokines IL-1
, IL-3, IL-6, G-CSF, GM-CSF, Steel
factor, and erythropoietin (5). Erythroid burst-forming units
(BFU-E) and granulocyte/macrophage colony-forming cells
(GM-CFC) were counted at day 14 of culture (5).
-32P]dCTP (Amersham Pharmacia Biotech,
Buckinghamshire, UK) using the T7 Quickprime or Megaprime kit (Amersham
Pharmacia Biotech, Uppsala, Sweden) as described previously (6).
-32P-labeled human
CD164 probe h3 or murine probe m1.
-32P-labeled human h1 and h2 and murine m1
and m2 CD164 probes as described above. One positive PAC clone
containing human CD164 was selected and digested with
BamHI into three fragments (B3, B2, and B1 fragments), and
the B1 fragment was divided by the BglII restriction site
into four fragments (Bgl3.25, Bgl2.61, Bgl7.1, and Bgl4 fragments) and
then subcloned into similarly digested pCRScript(SK+) vectors (see Fig.
1). The CD164 gene inserts were sequenced directly using
oligonucleotide primers derived from the cDNA and genomic DNA.
Alternatively, using pCRScript SK(+) vectors containing the B2 and B3
inserts and the Bgl2.61, Bgl3.25, Bgl4, and Bgl7.1 inserts as
templates, nested deletions were generated using the Erase-a-Base
technique and the Erase-a-Base® kit (Promega) according to
the manufacturer's instructions prior to automatic sequencing using
M13 forward or reverse primers. To confirm the junctions between the
BamHI and BglII fragments, PCRs on the human PAC
clones were carried out using the Expand Long Template system (Roche
Molecular Biochemicals, Mannheim, Germany) and the following primer
pairs: B3-PCR-F1 and B2-PCR-B1, CD164-2KF2 and MGC-GP-B1, B3.5-R2 and
B2.5-F6, B2.5-RF7 and B7-R3, and B7-F2 and B4-FR3 (see Table I). The
PCR products were polyethylene glycol-precipitated and sequenced
directly using the same primers (6). One mouse PAC clone was selected,
digested with BamHI or EcoRI, subcloned into
similarly digested pCRScript(SK+) vectors, and PCR-amplified using M13
forward and reverse primers or primers to the cDNA, and the PCR
products were sequenced (6).
-actin,
or GAPDH forward and reverse primers (see Table I) using the Expand
High Fidelity PCR system (Roche Molecular Biochemicals) as described by
the manufacturer. The products were subcloned into the pMOS-Blue T
vector (Amersham Pharmacia Biotech) and sequenced.
-32P-labeled probes CD164(E1),
CD164(E1-4), CD164(E4), CD164(E5), CD164(E6), CD164-PolyA-2,
CD164-PolyA-3, and GAPDH, which were generated by PCR
amplification as detailed above from the human bone marrow-derived
CD164(E1-6) cDNA with the respective primer pairs (see
Table I and Fig. 3A): F164 and RD1, F164 and RD4, EXON4-F
and RD4, EXON5-F and RD5, CD164(E6)-TM-F and MGC-24-B7, MGC-GP-F4 and
MGC-GP-B4, MGC-F7 and CD164-PolyA-3, and GAPDH-F and GAPDH-R. Probes
were labeled with [
-32P]dCTP using the sequence-tagged
site method (21). The human
-actin (CLONTECH) or
GAPDH probe was labeled with [
-32P]dCTP using the
Megaprime kit. A murine multiple tissue Northern blot containing
poly(A)+ mRNAs from murine heart, brain, lung, liver,
skeletal muscle, kidney, and testis was probed with
[
32-P]dCTP-labeled murine CD164 probe m1 (described in
the legend to Fig. 6A). Murine strain 129 testis total RNA
was Northern-blotted onto Hybond N membranes (Amersham Pharmacia
Biotech) using standard techniques. Using GAPDH (see Table I) and
-actin probes, similar mRNA levels were found in the different
tissues (data not shown), indicating essentially even loading of
poly(A)+ RNA in the different lanes. The blots were probed
with 32P-labeled murine probe m1 or m3 (see Fig. 6,
A and E). The latter was generated by
sequence-tagged site labeling of the PCR products from mouse
CD164 cDNA using the mCD164-3F10 and mCD164-3R6
primers (see Table I).
-32P]GTP (50 µCi; Amersham Pharmacia Biotech) using
SP6 RNA polymerase (20 units; Roche Molecular Biochemicals) and the
SP6/T7 transcription kit (Roche Molecular Biochemicals) prior to DNase
I (20 units) digestion. Total RNAs (30 µg) from Calu-1 and KG1A cells
were mixed with the [
-32P[GTP-labeled PE1 or PE2 probe
or the GAPDH positive control probes (1 × 106
cpm/probe/tube) prior to digestion with ribonucleases A (160 ng/µl)
and T1 (2.8 units/µl) (Roche Molecular Biochemicals) and treatment
with a 2:1 mixture of 20% (w/v) SDS and 10 mg/ml proteinase K (ICN
Pharmaceuticals Ltd., Hampshire, UK) and then electrophoresed on
8% (w/v) polyacrylamide gels (Sequagel, National Diagnostics, Aylesbury, UK) using standard techniques. 32P-Labeled
pBR322 MspI-digested DNA (New England Biolabs Inc., Hertfordshire) at 104 cpm was loaded per marker
lane. The gels were dried on a Bio-Rad Model 583 gel dryer and exposed
to Kodak X-Omat film with intensifying screens at
70 °C.
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ABSTRACT
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Fig. 1.
Genomic structure of human
CD164. Shown is a map of the intron/exon
structure of the human CD164 gene. The relationship between
the genomic sequence (GenBankTM/EBI accession number
AF299340) and the predicted full-length human CD164(E1-6)
nucleotide sequence (accession number AF299341) is shown. Human
CD164 cDNA probes used to screen PACs and Southern blots
were prepared as follows. The SacI/SpeI probe h1
fragment contained bp 1-1907 of cDNA sequence (where bp 1 indicates the translational start site), encompassing the whole coding
sequence plus part of the 3'-UTR and part of the pRUF.neo
vector. The SpeI/ApaI probe h2 fragment
encompassed bp 1908 to 2872 of the 3'-UTR and part of the
pRUF.neo vector. Probe h3 comprised a 1.173-kb
EcoRV/HindIII CD164 probe from the 105A5 cDNA
spanning bp 1309-2487 of untranslated CD164 cDNA
sequence in the 3'-UTR. Probe h4 comprised a 612-bp
BstXI/NcoI probe derived from bp 42 to 653 of the
cDNA.
Identification of Four Splice Variants of Human CD164
Our previous data (5) described the partial sequence of two human
CD164 cDNA clones (103B2/9E10 and 105A5) isolated from a
human bone marrow stromal cDNA expression library. Complete sequencing of these two cDNA clones revealed cDNA inserts of
2873 bp linked to additional 5'- and 3'-sequences derived from the pRUF.neo vector. Both clones were identical except
for six base pair changes, one of which occurred in the coding sequence
and resulted in an amino acid change from serine to glycine at position 89 from methionine 1 of the translational start site in the 105A5 cDNA clone (Fig. 2).
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Comparison with the genomic CD164 DNA demonstrates that
these human CD164 cDNAs are encoded by exons 1-4 and 6. To identify other splice variants of CD164, cDNAs were
amplified from a variety of cell lines and tissues by RT-PCR using the
forward primer F164, upstream from and including the ATG codon of the
translational start site, and the reverse exon 1-6-specific primer
RD1, RD2, RD3 RD4, RD5, or RD6 or the 3'-UTR primer MGC-24-B7,
MGC-GP-B2, or MGC-GP-B5 (Table I). The
resultant products were Southern-blotted using exon-specific probes
(Fig. 3A) and sequenced. For
all except the latter four primers, single RT-PCR products
corresponding to the appropriate exons were generated with the
hematopoietic cell lines TF1, U937, KG1A, and K562; the epithelial cell
lines KATO-III and Calu-l; and normal human cultured bone marrow
stromal cells, normal bone marrow mononuclear cells, and placental,
spleen, kidney, and colon tissues (data not shown). With the exon
6-specific and 3'-UTR reverse primers, two different sequences were
obtained in most tissues and cell lines, one containing all exons
(CD164(E1-6)) and the other lacking exon 5 (CD164(E5)) (Figs. 2 and 3, B and 3C). In spleen, a third isoform was also found that
contained all exons but exon 4 (CD164(
E4))
(Figs. 2 and 3, B and C). A fourth splice variant
was generated by PCR amplification of spleen and colon cDNAs with
the F164 and MGC-GP-B5 primers (Table I). This CD164 variant
(
3'-UTR) contained the sequence derived from exons 1 to 6, but
lacked 356 bp of the 3'-UTR (Fig. 2). A homologous gene,
MGC-24, has previously been identified in the KATO-III cell line (24). The sequence for this cDNA is identical to our cDNA sequence from bp 1 to 523, but then splices onto the 3'-UTR from bp
1120 to the TGA stop codon at bp 1164-1166. This would splice within
the predicted transmembrane region and create a new 3'-amino acid
sequence (Fig. 2). Considerable efforts were made to identify the
MGC-24 transcript by RT-PCR using F164 and the reverse
primer MGC-GP-B2 (Table I) in our tissues and cell lines, including KATO-III, but these proved unsuccessful.
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Our recent studies (8) have shown that the human CD164 protein is
widely expressed in both hematopoietic and non-hematopoietic tissues.
To test whether the predominant splice variant of human CD164 contained exons 4 and 5 as in CD164(E1-6),
total RNAs from the colonic epithelial cell line Calu-1 (Fig.
4B) and from the hematopoietic
cell line KG1A (Fig. 4D) were analyzed by an RNase protection assay. The relative levels of the different transcripts were
analyzed using probe PE1 to protect exon 1 and probe PE2 to protect
exons 3-5 of human CD164 (Fig. 4, A and
C). The results in Fig. 4 (B and D)
clearly demonstrate major protected fragments of 109 and 171 bases for
exon 1 and exons 3-5, respectively. This indicates that
CD164(E1-6) is the major isoform in these cell lines.
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Northern Blot and 3'-RACE PCR Analyses Reveal Three Species of Human CD164 mRNA with Different Polyadenylation Sites
Composite poly(A)+ RNA Northern blots comprising
multiple hematopoietic (Fig. 5,
A and C) and non-hematopoietic (Fig. 5,
B and D) tissues were analyzed using exon- and
3'-UTR-specific cDNA probes (Fig. 3A). Similar results
were obtained whether probes to exons 1-4 (Fig. 5, A and
B, respectively) or to exon 1, exon 4, exon 5, or exon 6 were used (data not shown), confirming human CD164(E1-6) as
the major isoform in the tissues and cell lines examined. All the
tissues expressed at least two bands of ~3.2 and 2.8 kb, except
brain, which appeared to express the 3.2-kb mRNA only (Fig.
5B). Although the 3.2-kb band appeared to be the most
prominent mRNA species in all tissues, both the 3.2- and 2.8-kb
species were expressed in slightly variable amounts in the different
hematopoietic tissues examined. The 2.8-kb band was more prominent in
spleen, lymph node, peripheral blood leukocytes, and fetal liver, but
little was detected in thymus and bone marrow (Fig. 5A). The
variability in mRNA expression was more evident on the
non-hematopoietic blot, with the most prominent bands occurring in
heart and placenta (Fig. 5B). Moreover, some tissues
expressed another band of 1.2 kb (Fig. 5B). This band was
most obvious in placenta, but was also visible in pancreas after short
autoradiographic exposures. However, after long exposures, it became
evident in hematopoietic tissues and adult liver and skeletal
muscle.
|
These Northern analyses suggested that variability in mRNA size was due to usage of different polyadenylation sites. This was confirmed by 3'-RACE PCR of cDNAs derived from bone marrow, placenta, spleen, kidney, and colon using the MGC-GP-F2 or MGC-GP-F4 forward primer (Fig. 2 and Table I) and adaptor 1 as the reverse primer (Table I). Two PCR products of ~700 and 1500 bp were generated with the MGC-GP-F4 primer, and one of 600 bp with the MGC-GP-F2 primer. Sequencing confirmed that the human CD164 mRNAs terminated at three different polyadenylation sites, PolyA-1, PolyA-2, and PolyA-3, in human CD164 mRNA (Fig. 2). Computer analysis of these sequences using the WebGene polyadenylation site program indicated the existence of three signal sites associated with these three polyadenylation sites (Fig. 2). The first and second signal sites (25, 26), which serve as the binding site for the 160-kDa protein cleavage polyadenylation specificity factor, are AUUAAA, and the third signal site for the cleavage polyadenylation specificity factor is AAUAAA. The U(T)-rich elements, which serve as the binding site for the 64-kDa cleavage stimulation factor protein CstF (25, 26), were also identified 10-30 bp downstream from the first and second polyadenylation sites. All three polyadenylation sites were confirmed by hybridizing Northern blots with PCR probes between PolyA-1 and PolyA-2 and between PolyA-2 and PolyA-3 (Fig. 3A). One band (3.2 kb) was identified with the CD164-PolyA-3 probe (Fig. 5, C and D), and two bands (3.2 and 2.8 kb) with the CD164-PolyA-2 probe; and in tissues such as placenta, all three RNA species were detected using the exon 1-4- and exon 5-specific probes (Fig. 5B and data not shown).
Conservation of the Genomic Structure, Chromosomal Localization, and Differential Polyadenylated mRNAs of Murine CD164
For comparison, the murine CD164 cDNA was isolated
by PCR amplification from a murine heart cDNA library and found to
be essentially identical to that described for murine
MGC-24v by Kurosawa et al. (13) except for two
base pair changes at positions 72 and 73 from the ATG codon of the
translational start site. However, these base pair changes did not
alter the predicted amino acid sequence and may represent polymorphic
variation between mouse strains. Sequence analysis of the murine
CD164 PAC clone 488F16 revealed an intron/exon structure
identical to the human gene (Fig. 6,
A and B). PCR analysis of murine adult and
embryonic tissues and sequencing of the PCR products revealed that
murine CD164(E1-6) was the major isoform detected with only
one variant, CD164(E3), lacking exon 3, which
was found in a few cases in adult heart (data not shown). Comparative
analyses of murine tissue Northern blots probed with murine CD164 probe
m1 (Fig. 6A) revealed a band at ~3.2 kb in the tissues
examined, viz. heart, brain, lung, liver, skeletal muscle,
kidney, and testis (Fig. 6C). This band was particularly
prominent in liver, kidney, and testis. The 2.8-kb mRNA species was
not evident on the murine Northern blot. However, it was of interest to
note that in murine testis, the 1.2-kb band was also detected as a
major mRNA species (Fig. 6, C and D). As with
the human cDNA, 3'-RACE PCR of cDNA from murine testis
and Northern blot analyses identified the 3.2- and 1.2-kb bands as RNA
species with different polyadenylation sites (Fig. 6 (D and
E) and data not shown). The murine PAC 488F16 clone was used to localize the murine CD164 gene to chromosome
10B1-B2 (Fig. 7), a region syntenic with
human chromosome 6q21.
|
|
Functionally Important Domains and Motifs of CD164
Protein Structure--
With the intention of predicting
functionally important structural features of CD164, we compared the
conserved characteristics within the exon-defined amino acid sequence
of human CD164(E1-6) with those of murine CD164(E1-6) and the
predicted sequence of rat endolyn/CD164(E1-6) (12) (Fig.
8).
|
The highest amino acid identities occurred among the exon 6-encoded peptides, with 94.5 and 96.4% identities for the respective mouse and rat sequences compared with the human sequence. This high degree of homology was mainly due to the completely conserved transmembrane and cytoplasmic domains encoded by this exon. The carboxyl-terminal amino acids have been suggested to constitute a targeting motif of the type YXXø (with X being any amino acid and ø being a bulky hydrophobic amino acid) interacting with clathrin-adaptor complexes involved in vesicle traffic (12). Lower amino acid identities were observed for peptides encoded by exons 1 (50 and 44.8%), 2 (both 57.1%), 3 (41.7 and 37.5%), 4 (both 69.2%), and exon 5 (73.7 and 52.6%). The non-mucin domain encoded by exons 2 and 3 contains, in all these species, eight cysteines, four of which are predicted to be involved in the formation of intramolecular disulfide bridges (12). This domain links mucin domains I and II, which are encoded by exon 1 and exons 4 and 5 and part of exon 6, respectively. Using the Prosite scan protein analysis program, we confirmed the presence of sequences CFNVSVVNTTCFW, CVNATFTNNITCFW, and CVNATLTNNITCVW in the exon 2-encoded non-mucin domains of human, mouse, and rat CD164, respectively (12). These sequences belong to the consensus sequence C(LVFYR)X7,8(STIVDN)CXW, one of two cytokine receptor consensus motifs found in such molecules as the erythropoietin, G-CSF, GM-CSF, IL-4, IL-6, and thrombopoietin receptors (reviewed in Ref. 27). Both murine and human CD164(E1-6) possess 32 potential O-linked and 9 N-linked oligosaccharide attachment sites, whereas rat endolyn/CD164(E1-6) is predicted to contain 35 O-linked and 8 N-linked glycosylation sites. Some important characteristics of the predicted exon-defined domains of human CD164 isoforms are summarized in Table II. The similarities in the structures of human and murine CD164(E1-6) are illustrated diagrammatically in Fig. 8 (B and C).
|
Functional Importance of Mucin Domain I Epitopes--
Our previous
data indicated that CD164 is present at the surface of cells, where it
may function as an adhesion molecule for attachment of
CD34+ hematopoietic progenitor cells to the stroma
and as a negative regulator of CD34+CD38lo/
hematopoietic progenitor cell proliferation (5). The class I and II
epitopes of human CD164 defined by mAbs 105A5 and 103B2/9E10, respectively (9), mediate these functional effects. mAb 105A5 identifies a sialic acid epitope on O-linked
oligosaccharides, whereas mAb 103B2/9E10 identifies an
N-linked oligosaccharide-containing epitope, both on mucin
domain I (9) (Fig. 8B). Previous single cell analyses using
mAb 103B2/9E10 as an inhibitor of proliferation (5) demonstrated that
this mAb inhibits the recruitment of single
CD34+CD38lo/
cells, the more primitive
hematopoietic precursors, into the cell cycle in serum-deprived media
in the presence of the cytokines IL-6, IL-3, G-CSF, and Steel factor.
We have extended these experiments in Fig.
9 to further demonstrate the importance
of mucin domain I. Both mAbs 105A5 and 103B2/9E10 prevented the
generation of more mature nucleated cells from CD34+ cells
over 4 weeks of culture in serum-deprived liquid medium containing
cytokines (Fig. 9A). That this was most likely due to
inhibition of recruitment of CD34+CD38lo/
cells into the cell cycle was further substantiated by the finding that
the inhibition included nucleated cells from both the erythroid and
myeloid lineages (Fig. 9, B-E). The growth of day 14 GM-CFC (Fig. 9, B and C) as well as BFU-E (Fig. 9,
D and E) precursors from CD34+ bone
marrow cells was inhibited in a dose-dependent manner when clonogenic methylcellulose cultures containing cytokines were grown
after preincubation of the cells with the class I and II CD164
mAbs.
|
Subcellular Distribution of CD164--
We have demonstrated
previously (6, 8) that CD164 is more highly expressed on the surface of
primitive CD34+CD38lo/ cells than on the
surface of more mature CD34+CD38+ hematopoietic
progenitor cells. Furthermore, our preliminary results indicate that,
during differentiation, the majority of CD164 is lost from the cell
surface and becomes distributed inside CD34+CD38+
cells.3 In non-hematopoietic
cells, rat endolyn/CD164(E1-6) is primarily localized in endosomal and
lysosomal compartments (12, 28, 29). Thus, we examined in greater
detail the intracellular localization of human CD164 in
epithelium-derived cell lines (Calu-1 and A431) and in the
CD34+CD38+ hematopoietic cell line KG1B by
confocal microscopy using the class II mAb 103B2/9E10 (see above and
Fig. 8B) or the class III mAb N6B6, which preferentially
recognizes the peptide backbone encoded by exons 2 and 3 of human CD164
(Fig. 8B). Both antibodies generally gave a very similar
labeling pattern in all three cell lines examined (data not shown). In
all cases, the majority of CD164 was found in structures inside the
cells (Fig. 10, left
panels).
|
Cell-surface label was also observed, but, in fixed and permeabilized
cells, was clearly less pronounced than the intracellular label.
Interestingly, in agreement with previous data (8), surface CD164 was
somewhat easier to detect with mAb N6B6 than with mAb 103B2/9E10. In
general, the N6B6 antibody gave a slightly more homogeneous pattern
with regard to the labeling intensity of CD164-positive structures
within a single cell and also among cells. This was especially obvious
in KG1B cells, where a minority of cells did not appear to express the
103B2/9E10 epitope or expressed it only very weakly, whereas the level
of staining with mAb N6B6 was not significantly reduced. This indicated
that CD164 molecules carrying the 103B2/9E10 epitope were
down-regulated in a subset of KG1B cells. The common labeling pattern
in all three cell lines was consistent with CD164 being primarily
located in endocytic compartments. Immunoreactivity was found in
punctate structures of various sizes that were located throughout the
cells and that were slightly more concentrated in the perinuclear area.
Antibodies to various known marker proteins for endocytic compartments
were used in double labeling experiments in conjunction with mAbs N6B6 and 103B2/9E10 to confirm and to define the distribution of CD164 throughout the endocytic pathway. A good coincidence of CD164 and the lysosomal marker Lamp-1 was found in all three cell lines (Fig.
10, E/F and I/K, compare
white arrows). In both Calu-1 and A431 cells, there were
also significant numbers of strongly CD164-positive structures that
were Lamp-1-negative (Fig. 10, E and F,
black arrowheads; A431 cells not shown). These structures
were identified as an early endosomal population by the presence of the
marker early endosomal antigen-1 (Fig. 10, C and
D), but did not appear to be recycling endosomes since they
did not contain significant amounts of the transferrin receptor (data
not shown). This receptor was seen in smaller punctate structures,
which were enriched in two locations as expected, namely, in a distinct
perinuclear region and underneath or at the plasma membrane (Fig.
10B). Although some CD164 was also seen in small structures
similar in size and number to those containing the transferrin
receptor, both populations were clearly distinct from each other. In
the majority of KG1B cells, CD164 was seen primarily in the same large
structures as Lamp-1 (Fig. 10, I and K), although
there was some overlap with early endosomal antigen-1 (data not shown).
Interestingly, in a minority of KG1B cells, CD164 was found more
prominently at the cell surface or in smaller vesicular structures that
were positive for the transferrin receptor. This suggested that, in these cells, a significant fraction of CD164 molecules were recycling between the cell surface and early endosomes (Fig. 10, G and
H). In a few of these cells, there was almost no CD164 label
seen in other intracellular structures than transferrin
receptor-positive endosomes, whereas other cells contained
simultaneously varying amounts of label in larger structures typical of
lysosomes. Altogether, these microscopy results show that, in many
hematopoietic and non-hematopoietic cell lines, only a small amount of
CD164 is localized at the cell surface, whereas the majority is
distributed over endosomes and lysosomes. However, the variation seen
among KG1B cells, i.e. the spectrum from almost entirely
lysosomal to almost exclusively cell-surface and early and recycling
endosomes, indicates that a mechanism exists by which the localization
of CD164 can be regulated.
![]() |
DISCUSSION |
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The identification of cell-surface receptors that regulate
hematopoietic stem cell fate is critical to our understanding of the
molecular events that control the plasticity of such cells, their
proliferative capacity, their migration to the correct
microenvironmental niches, and their differentiation into one of nine
hematopoietic lineages. In turn, such knowledge is invaluable for
clinical applications relating to stem cell usage. Recently, we have
demonstrated that one such cell-surface receptor, CD164 (reviewed in
Ref. 7), potentially plays a key role in regulating the recruitment of primitive CD34+CD38lo/ hematopoietic
stem/progenitor cells into the cell cycle (8). In this study, we have
further substantiated that the two glycosylation-dependent class I and class II epitopes present on the exon 1-encoded domain are
important for mediating the inhibition of
CD34+CD38lo/
hematopoietic progenitor cell
proliferation by preventing the development of erythroid and myeloid
lineages and the subsequent generation of differentiated nucleated
cells in vitro. Both these and other recent (5, 30-33)
studies indicate that CD164 as well as other hematopoietic sialomucins
such as CD34, CD43, and P-selectin glycoprotein ligand-1 function as a
group of negative regulators by preventing hematopoietic
stem/progenitor cell proliferation and differentiation.
Moreover, in this work, we report the genomic cloning of human CD164. This human CD164 gene spans at least 22 kb of DNA and contains six exons. Although all the sialomucins mentioned above have similar structural and functional characteristics, their genomic structures and chromosomal localization diverge from each other to varying degrees. For molecules such as human CD43 and P-selectin glycoprotein ligand-1, which contain mucin domains only, the cDNA coding sequence is specified by a single exon. The CD43 and P-selectin glycoprotein ligand-1 genes encompass 4.6 and 11 kb of DNA, respectively, with each containing a single intron that interrupts the sequence specifying the 5'-untranslated region of the mRNAs. These genes are located on chromosomes 16p11.2 and 12q24, respectively (reviewed in Ref. 7). In contrast, genes such as CD34 that contain Ig-like or non-mucin cysteine-rich domains are composed of multiple exons. The human CD34 gene, for example, spans at least 26 kb of DNA, contains eight exons, and is situated on human chromosome 1q32. Thus, CD164 belongs to that subgroup of mucins that are composed of multiple exons, with human CD164 mapping to chromosome 6q21 and murine CD164 to the syntenic chromosome 10B1-B2. This supports the view that the function of molecules such as P-selectin glycoprotein ligand-1 is regulated primarily through post-translational modifications, whereas that of molecules such as CD164 with multiple exons may have more sophisticated regulatory mechanisms, which involve not only post-translational modifications of the oligosaccharide side chains, but also differential exon usage. The complete distribution and functional relevance of the different CD164 splice variants await further analysis.
As a prelude to generating CD164 knockout mice for
functional studies in vivo, we have also completely
sequenced the putative mouse ortholog of human CD164 and
demonstrated a similar distribution of exons. Although differences in
the intron and exon sizes are seen between the mouse and human genes,
the predicted proteins are similar in size and structure. Furthermore,
we have identified two and three isoforms differing in coding sequences
for murine and human CD164, respectively. The longest
isoform, CD164(E1-6), which is encoded by all six exons,
appears to predominate in the tissues and cell lines analyzed. This is
in contrast to the originally cloned human CD164 or
CD164(E5), which lacks the exon 5-encoded domain. An additional transcript was detected in human CD164 using RT-PCR amplification. This contained all six exons, but lacked 356 bp
of the 3'-UTR. In agreement with the recent studies by Kurosawa
et al. (13), the predominant mouse isoform is encoded by all
six exons. A soluble isoform of human CD164 termed MGC-24 has been
described previously (24). Despite extensive RT-PCR analyses, we were
unable to detect this soluble variant. From analysis of our genomic
sequence, this variant would be derived from exons 1 to 5 and part of
exon 6, which would be spliced within the transmembrane region onto a
sequence within the 3'-UTR.
On Northern blots, three human and two mouse CD164(E1-6)
mRNAs containing different polyadenylation sites were detected,
with the shortest transcript of ~1.2 kb most highly expressed in
placenta and testis, respectively. The importance of these short
transcripts remains to be determined. Interestingly, in a review of 33 genes, Edwalds-Gilbert et al. (26) identified the testis as
a "hot spot" for differential polyadenylation site usage.
CD164 is an example of a gene whose expression depends on
differential usage of polyadenylation sites within a single 3'-UTR. The
conserved distribution of the 3.2- and 1.2-kb CD164
transcripts between mouse and human suggests (i) that a mechanism may
exist to regulate this tissue-specific polyadenylation and (ii) that
differences in polyadenylation are important for the expression and
function of CD164 in different tissues. Other research has suggested
that genes containing multiple tandemly arrayed polyadenylation sites will select the most 5'-site preferentially. This does not seem to be
the case in our studies, where the longest transcript was prevalent in
all tissues. Furthermore, studies suggest that the 3'-UTR regulates
both the stability and translatability of the mRNA transcript
(reviewed in Refs. 34 and 35) as well as controls its nuclear export
and subcellular targeting (36). In the human and murine 3.2-kb
CD164 mRNA transcripts, there are 15 and 7 AUUUA sequences, respectively. These cis-acting AU-rich elements
are thought to be destabilizing elements in such genes as GM-CSF, c-fos, and interferon- (reviewed in Refs. 26 and 36),
although the optimal destabilizing sequence (UUAUUUAU) is not found in either mouse or human CD164. The choice of polyadenylation
sites also depends on the activities and amounts of tissue-specific polyadenylation factors. Alternative polyadenylation usage, if it
affects the stability and translatability of mRNA transcripts, would also affect the amount of protein transcribed and thus be of
functional relevance. For example, eukaryotic initiation
factor-2
contains multiple 3'-UTR polyadenylation sites. Two of the
mRNA species of 1.6 and 4.2 kb that are transcribed from this gene in activated T cells show differences in stability and translatability. The 1.6-kb transcript is less stable, but more readily translated in vitro. Thus, an increase in polyadenylation enzyme
activity as T cells move from the G0 to S phase of the cell
cycle leads to a shift to the shorter transcript and a concomitant
increase in protein production (reviewed in Ref. 26). A similar
scenario may apply for CD164. However, situations in which
the short transcript is up-regulated are likely to be underrepresented
in the "steady-state" blots used in this study.
In addition to similarities in the long and short mRNA transcript sizes and the predominance of the CD164(E1-6) isoforms in both mouse and human, our studies provide further evidence to confirm that the murine gene described here is the ortholog of human CD164. This is evidenced by the highly conserved amino acid similarities and protein structure as a type I integral transmembrane sialomucin containing two extracellular mucin domains linked by a non-mucin domain as well as the conserved chromosomal localization. Thus, murine CD164 provides an ideal model for the function of the human CD164 gene in vivo in mouse gene knockout/knockin studies. This is particularly important if the effects of CD164 engagement by its cognate or surrogate ligands on the proliferation and adhesion of hematopoietic stem/progenitor cells in vitro demonstrated in this and previous (5) studies are to be reproduced in vivo.
Two other aspects of the structure of CD164 are of particular interest
with respect to putative regulatory events. First, CD164 shares one of
several conserved features of a cytokine-binding pocket (12). In this
respect, it is notable that evidence exists for a class of cell-surface
sialomucin modulators that directly interact with growth factor
receptors to regulate their response to physiological ligands. One such
example is the membrane-bound ASGP2 subunit of rat sialomucin
MUC4 (37). This molecule interacts in cis through its
epidermal growth factor-1 domain with the extracellular region of
ErbB2, a cell-surface tyrosine kinase receptor implicated in embryonic
cardiac and neural tissue development. ErbB2 does not appear to bind
diffusible ligands directly, but instead acts as an auxiliary
co-receptor to enhance signaling via the ErbB receptor network. Growth
factor ligands such as neuregulin-1 bind to the related epidermal
growth factor receptor to promote heterodimerization with ErbB2 and
consequently to potentiate activation and signaling of the ErbB2
tyrosine kinase. Interactions of the sialomucin ASGP2 subunit with
ErbB2 are thought to increase the degree of activation of ErbB
receptors or the number of receptors available for activation (37).
This in turn would regulate the specificity and strength of receptor
signaling. In the case of CD164, the cytokine receptor motif in its
non-mucin domain is reminiscent of the non-mucin ASGP2 subunit of the
MUC4 sialomucin, although it is unclear if CD164 contributes to
cytokine binding or signaling. However, we are now addressing this question.
Second, the cytoplasmic tail of CD164 contains a carboxyl-terminal YHTL
motif of the type YXXø found in many endocytic membrane proteins or receptors (12, 38, 39). These tyrosine-based motifs bind to
the µ-subunit of adaptor-protein complexes, which mediate the
sorting of membrane proteins into transport vesicles from the plasma
membrane to the endosomes and between intracellular compartments (38,
39). The cytoplasmic tail of rat endolyn/CD164(E1-6) has been shown to
direct the targeting of the protein to endosomes and lysosomes in
non-hematopoietic cells (12). Here, we have demonstrated that human
CD164 is not always primarily located in late endocytic compartments.
Although this was the usual distribution found in human epithelial cell
lines and in the majority of KG1B cells used in this study, CD164 was
more prominently expressed at the cell surface and in recycling
endosomes in a minority of KG1B cells. The KG1B cell line is considered
equivalent to CD34+CD38+ hematopoietic
progenitor cells. Our earlier studies showed that CD164 is more highly
expressed at the cell surface of primitive CD34+CD38lo/ progenitor cells than on the
more mature CD34+CD38+ progenitors (6, 8). The
current results are consistent with the notion that a minority of KG1B
cells have a more primitive phenotype. It may be that CD164 present at
the cell surface binds its cognate ligand, which is internalized
together with its receptor into endocytic compartments, where it is
possibly released while CD164 returns via recycling endosomes back to
the cell surface. The distribution of CD164 in the majority of KG1B
cells indicates that, in later stages of CD34+ cell
maturation, the distribution of CD164 is shifted toward lysosomal
compartments, where it is unlikely to bind its ligand. The mechanisms
whereby sorting CD164 to different endocytic compartments at different
stages of maturation of hematopoietic precursor cells is accomplished
are not clear. However, a mechanism might be operating by which the
binding affinities of the YXXø motif in the cytoplasmic tail for different adaptor protein complexes can be regulated, e.g. by cytokine receptor interactions in cis.
This would lead to changes in the surface and intracellular
distribution of CD164. We have previously observed that the class II
103B2/9E10 epitope is more rapidly down-regulated from the cell surface
than the class III N6B6 epitope (6, 8). Interestingly, a second small population of KG1B cells, presumably representing more mature cells,
did not express the class II epitope at all or expressed it at very low
levels. This suggests that another regulatory mechanism exists that
influences the glycosylation pattern of CD164 and thus the binding
characteristics regarding its ligand(s). Whether different
intracellular trafficking routes are regulated by differential glycosylation of CD164 or vice versa remains to be determined. However,
it is possible that both events are interconnected and contribute to
the regulation of CD164 function.
In this study, we have demonstrated that human and murine CD164/endolyn
are highly conserved with respect to their glycoprotein and gene
structures and their chromosomal locations. Furthermore, we show that,
in both species, the major isoform is encoded by all six
CD164 exons and that this maintains functionally important motifs that regulate cell proliferation or subcellular distribution. Thus, this is the first step in our aim to define the nature, expression, and regulated expression of the natural cellular ligands and binding partners for CD164. The availability in a variety of
species of the genomic structures and of a defined set of
CD164 cDNAs and antibodies plus the identification of
functionally important modules and motifs will permit the critical
definition of CD164 functions and will aid in determining structure,
function, and signaling relationships both in vitro and
in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Professors P. Sissons and A. C. Minson for access to the Leica confocal microscope, Dr. A. J. Thomson for advice on the RNase protection assay, Dr. K. Clark for advice on sequencing, and Dr. M. J. Clague and Professor C. R. Hopkins for providing antibodies. We also thank Professor D. J. Weatherall and Dr. J. P. Luzio for support.
![]() |
FOOTNOTES |
---|
* This work was supported by the Leukaemia Research Fund, the Medical Research Council, an Overseas Research Student award, and the Wellcome Trust, United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF299340 (human genomic CD164), AF299341 (human CD164(E1-6) cDNA), AF299342 (human CD164(E5)), AF299343 (human
CD164(
E4)), AF299344 (mouse genomic CD164), and AF299345 (mouse
CD164(E1-6)).
** To whom correspondence should be addressed: MRC Molecular Hematology, Inst. of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. Tel.: 44-1865-222-632; Fax: 44-1865-222-500; E-mail: Swatt@molbiol.ox.ac.uk.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007965200
2 C. R. Hopkins, unpublished data.
3 S. M. Watt, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: mAb, monoclonal antibody; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleukin; G-CSF, granulocyte colony-stimulating factor; mIg, murine Ig; PAC, P1 artificial chromosome; RT-PCR, reverse transcription-polymerase chain reaction; contig, group of overlapping clones; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; 3'-RACE, 3'-rapid amplification of cDNA ends; kb, kilobase pair(s); bp, base pair(s); BFU-E, erythroid burst-forming unit(s); GM-CFC, granulocte/macrophage colony-forming cells.
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