the human and mouse genomes now finished the complete EGF-TM7 family comprises human and mouse CD97, mouse F4/80, human
EMR1 (EGF-module-containing mucin-like hormone receptor 1), human EMR2, EMR3
mEMR4, and ETL. These predominantly leukocyte-restricted glycoproteins are
defined by their unique chimaeric structures which consist of varying numbers of
N-terminal epidermal growth factor (EGF)-like repeats coupled to a family B
G-protein coupled receptor (GPCR)-related moiety via a mucin-like spacer region.
The spacer region contains several potential N- and O-linked glycosylation sites
and a GPCR proteolytic site (GPS), which has been demonstrated to be involved in
heterodimer formation. Due to their hybrid a dual adhesion and signalling function
has been suggested. Indeed, several
cellular ligands have already been characterised, however signalling has not yet
been formally demonstrated. The
genes of CD97 and EMR1-4 form a gene family clustered on the short arm of human
chromosome 19 and syntenic regions of the mouse genome suggesting the evolution
from an ancestral gene through gene duplication and exon shuffling other EGF-TM7
genes have also been identified in cows, pigs and zebrafish.
Non-vertebrate EGF-TM7 molecules have not yet been discovered.
ETL is slightly anomalous in that it located on human chromosome 1 and is
predominantly expressed on cardiomyocytes.
Within our laboratory we have now cloned and characterised F4/80, EMR2
and EMR3 and mouse EMR4, which are described below.
comprises seven EGF-like domains, a spacer region and a hydrophobic seven-span
transmembrane region. As a result of its macrophage-restricted expression F4/80
The founding member of the EGF-TM7 family, CD97 is a B
and T cell activation antigen expressed constitutively by human granulocytes,
monocytes, at low levels by resting T and B cells and is markedly and rapidly
upregulated upon T and B cell activation. In addition to its predominantly
leukocyte-restricted nature, CD97 expression is also detected on non-leukocytes
such as smooth muscle cells. CD97 occurs on the cell surface as three major
isoforms resulting from alternative RNA splicing. The CD97 isoforms have been
shown to bind CD55 through the EGF domains, however the isoform containing three
EGF-like domains binds with the highest affinity to CD55. More recently BiaCore
studies have demonstrated that the association mirrors that of other leukocyte
protein-protein interactions. The interaction has both fast kinetics and is of
low affinity with an off-rate and Kd of 0.6s-1 and 86µM respectively. CD55 is a
member of the Regulators of Complement Activation (RCA) family. These proteins
contain short consensus repeats (SCRs), a protein module commonly found in~0.3%
of all proteins, mostly but not exclusively in proteins of the complement
system. The module sconsist of <60 amino acids which form an all b-strand
structure stabilised by two conserved disulphide bonds. CD55 is known to protect
host cells from complement mediated damage by binding to C3b and C4b and
preventing the formation of C3 convertases. It has a widespread tissue
distribution and is expressed at high levels on many different cell types,
especially in those exposed to plasma. The protein consists of an N-terminal
extracellular portion of 4 SCR domains linked via a heavily O-glycosylated
spacer to a C-terminal GPI anchor. The most N-terminal SCR, which is known to
bind CD97 also provides the site of interaction for some viruses. SCR domains
2-4 are involved in regulation of complement activation and also in binding to a
variety of viral and bacterial pathogens. The interaction between CD55 and CD97
has been shown to be relatively species specific. Human CD97 does not bind to
mouse or rat CD55, however it does bind to monkey CD55. The cloning of mouse
CD97 also shows the reverse to be true. The expression profile of the murine
CD97 appears to be similar to that of human, but is not upregulated by the
activation of B and T cells. The
structure of the murine counterpart differs slightly in that the 3rd EGF domain
is replaced by a stretch of peptide with no known structural motifs. Although
the precise role of the CD55-CD97 interaction is still unknown, the unique
hybrid structure, the leukocyte-restricted expression pattern of CD97 and the
presence of both CD97 and CD55 in arthritic joints suggest possible roles for
the interaction in adhesion and signalling within the inflammatory and immune
cloned in our laboratory shows all the distinct characteristics of existing
EGF-TM7 proteins. It shares extremely high homology with CD97, differing by only
six amino acids within its EGF domains and has been mapped proximal to the CD97
gene on human chromosome 19. As with CD97 it occurs as various alternative
splice forms containing different numbers and arrangements of membrane-bound and
soluble EGF domains. However,
unlike CD97 its expression profile is restricted to neutrophils, DCs and
culture-derived macrophages and despite its similarity it has been shown not to
bind CD55. The longest isoform of
EMR2 and CD97 have recently been shown to bind certain forms of the
glycosaminoglycan, chondroitin sulphate. Chondroitin
sulphate is a major component of extracellular matrix and is often found on cell
surface proteoglycans. The binding
of EMR2/CD97-bearing neutrophil and macrophage suggests a mechanism whereby
these phagocytes migrate during inflammation or wound repair.
Extensive studies of EMR2, in addition to EMR3/4 have shown that they occur as hetrodimers. The nascent polypeptide chains of the receptors are cleaved in the ER and reassociate non-covalently to be presented as heterodimers on the cell surface. The proteolytic cleavage has been shown to occur at a conserved site known as the GPS site. We have shown that the conserved cleavage site (L-S/T) is necessary but not sufficient for cleavage to occur. Indeed the entire stalk region of EMR2 is required for cleavage, suggesting that the stalk region may form a functional domain that presents the peptide bond for cleavage. The significance of the GPS is not yet understood, however mutants in the GPS site of the human gene polycystin-1 are known to cause polycystic kidney disease.
standard molecular techniques another EGF-TM7 molecule, designated human EMR3
was cloned by our laboratory. Again it consists of EGF repeats coupled to a
G-protein coupled receptor moiety and its gene has now been mapped proximal to
CD97 and only 30kb from the EMR2 gene. In addition to its chromosomal
localisation EMR3 also shares a high degree of homology to EMR2 within its
transmembrane suggesting that the EGF-TM7 molecules have evolved from recent
gene duplication and exon shuffling events. RNA data have shown EMR3 to be
restricted to polymorphonuclear cells and culture derived macrophages. Using
sensitive FACS based assays involving multivalent fluorescent probes we have now
shown EMR3 to possess a cell surface ligand on macrophages and activated
neutrophils. The exact nature of the ligand is still under investigation.
The full-length mouse EMR4 cDNA encodes a predicted 689 amino acid protein containing two EGF-like modules, a mucin-like spacer domain and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localised in the distal region of mouse chromosome 17 in close proximity to F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level is also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-a treated resident peritoneal macrophages while IL-4 and IL-10 dramatically reduce the expression. mEMR4 is found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated non-covalently as a heterodimer. The proteolytic cleavage site has been identified by N-terminal amino acid sequencing and located at the conserved GPS site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.
GW, Stacey M, Kwakkenbos MJ, Hamann J, Gordon S, Lin HH.
”Proteolytic cleavage of the EMR2 receptor requires both the extracellular stalk and the GPS motif.”FEBS Lett. 2003 Jul 17;547(1-3):145-50.
Stacey M, Chang GW, Davies JQ, Kwakkenbos MJ, Sanderson RD, Hamann J, Gordon S, Lin HH. “The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulphate glycosaminoglycans” Blood 2003, October 15, 2003; 102 (8)
Stacey M, Chang GW, Sanos SL, Chittenden LR, Stubbs L, Gordon S,
H.H., M. Stacey, C. Saxby, V. Knott, Y. Chaudhry, D. Evans, S. Gordon, A.J.
McKnight, P. Handford, and S. Lea. 2001. "Molecular Analysis of the
M., Lin, H. H., Hilyard, K. L. and Gordon, S. 2001.
M., H.H. Lin, S. Gordon, and A.J. McKnight. 2000. "LNB-TM7, a group of
seven-transmembrane proteins related to family-B G- protein-coupled
receptors." Trends Biochem
Lin, H.-H., Stacey, M., Hamann, J., Gordon, S., McKnight, A.J. 2000. "Human
EMR2, a Novel EGF-TM7 Molecule on Chromosome 19p13.1, is Closely Related to
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