(Received for publication, July 28, 1995; and in revised form, September 26, 1995)
From the
F4/80 is a monoclonal antibody that recognizes a murine
macrophage-restricted cell surface glycoprotein and has been
extensively used to characterize macrophage populations in a wide range
of immunological studies. Apart from the tightly regulated pattern of
expression of the F4/80 antigen, little is known about its possible
role in macrophage differentiation and function. We have sought to
characterize the molecule at the molecular level, through the isolation
of cDNA clones, and now describe the sequence of the F4/80 protein. The
primary amino acid sequence demonstrates homology to two protein
superfamilies. The NH-terminal region consists of seven
epidermal growth factor-like domains, separated by approximately 300
amino acids from a COOH-terminal region that shows homology to members
of the seven transmembrane-spanning family of hormone receptors. The
potential role of these distinct domains is discussed with respect to
the possible function of the F4/80 molecule.
Macrophages play a crucial role in the initiation and effector
stages of both innate and adaptive immune responses. A number of
specialized cell surface molecules expressed by macrophages participate
in these responses, such as macrophage mannose receptor, macrophage
scavenger receptor, complement receptor 3 (CD11b/CD18), and opsonic Fc
receptors for antigen-specific immunoglobulins. One of the most highly
restricted macrophage membrane molecules is defined by the monoclonal
antibody F4/80, which recognizes a 160-kDa glycoprotein on the surface
of most mouse macrophage populations(1) . The F4/80 mAb ()has been extensively used in a range of
immunohistochemical studies of development in normal mice and in a
number of pathologic models as a macrophage-specific marker (2, 3, 4, 5, 6) . To date,
no defined function has been ascribed to F4/80, although its expression
is known to be down-regulated by interferon-
and in response to
Bacille Calmette-Guérin
infection(7, 8) , as well as being absent from
macrophages localized within T cell areas of lymph nodes and spleen.
This further suggests that T cells, or a T cell-derived product, may
play a role in the regulation of F4/80 expression. The lack of F4/80
expression on migrating veiled cells, derived from F4/80
Langerhans cells within the skin epidermis, and the low level of
expression on blood monocytes (6, 9) would suggest
that the molecule is in some way involved in cell adhesion within
certain tissues. As a means of delineating the physiologic role of
F4/80 on mature macrophages, we have successfully isolated F4/80 cDNA
clones. Herein we report that the primary amino acid sequence of F4/80
shows a degree of homology to two protein superfamilies: the
extracellular NH
-terminal region contains seven EGF-like
repeats, while the final third of the molecule shows homology to
members of the seven transmembrane (Tm7) hormone receptor family.
The macrophage cell line J774.2 has been shown previously to
express high levels of the F4/80 antigen(1, 10) , and,
as such, an unamplified cDNA library was constructed with
poly(A) RNA from this source in the
ZAPII vector
and screened using rabbit polyclonal antisera raised against purified
F4/80 antigen. Following a further two rounds of screening, 10 positive
clones were isolated and characterized by restriction enzyme analysis
and sequencing. None of the clones appeared to contain a full-length
cDNA coding sequence, as witnessed by the lack of an in-frame ATG at
their 5` end. A 5` rapid amplification of cDNA ends PCR strategy was
therefore applied, utilizing antisense oligonucleotide primers specific
for internal sequence within clone pF4/80(12.2) to amplify a cDNA
fragment that overlapped pF4/80(12.2) and encoded 46 bp of novel 5`
sequence including the ATG initiation codon. The overall composite
sequence consists of 3286 bp, including a stretch of 49 adenosines
corresponding to the poly(A) tail and an upstream AATAAA motif. The
open reading frame encodes a precursor of 931 amino acids with a
predicted signal peptide of 27 residues(12) , resulting in a
mature protein of 904 amino acids with a predicted mass of 98.9 kDa (Fig. 1A). On SDS-polyacrylamide gel electrophoresis
analysis, F4/80 appears as a smear of approximately 160 kDa, which,
together with the mass differential from the deduced sequence, suggests
extensive glycosylation of the molecule. The presence of 10 potential N-glycosylation sites (Asn-X-Ser/Thr) and a region
highly rich in Ser and Thr residues (Fig. 1A, amino
acids 399-642) suggests that the protein is heavily N-
and O-glycosylated in agreement with earlier studies. The
predicted amino acid sequence also contained four tryptic peptides
obtained from the purified protein, ranging from 64 to 96% similarity (Table 1). To confirm that the cloned cDNA encoded the F4/80
protein, Northern blot analysis was performed using RNA from a range of
mouse cell lines and screened with a 757-bp probe from clone
pF4/80(12.2). Fig. 2demonstrates that the transcript recognized
by this probe is expressed exclusively in cells of the macrophage
lineage, in accordance with the well documented macrophage-specific
expression pattern of F4/80. The level of mRNA expression in J774.2
cells is also higher than in RAW 264.7 cells, which correlates with the
increased level of F4/80 surface expression on the J774.2 line. The
approximate size of the mRNA species (3.2 kb) also correlated well with
the size of the cloned cDNA. As further evidence of the identity of the
cDNA, CHO-K1 cells were transfected with a full-length expression
construct in the pcDNA3 vector. Following selection with Geneticin,
cells were analyzed by fluorescence-activated cell sorting for F4/80
antigen expression using the noncompetitive mAbs F4/80 and 5C1 (Fig. 3). The 5C1 mAb was raised against purified F4/80 antigen
and has not been described previously. Compared with cells transfected
with a control pcDNA3/
-galactosidase construct, the pcDNA3/F4/80
transfectants demonstrated detectable surface expression of the F4/80
antigen.
Figure 1: A, deduced amino acid sequence of F4/80. The region comprising seven EGF-like repeats (residues 32-367) is indicated by horizontal arrows, and the seven putative transmembrane-spanning regions are underlined. Three potential protein kinase C phosphorylation sites, located intracellularly, are highlighted by asterisks. The F4/80 cDNA sequence is available from EMBL/GenBank/DDBJ under accession number X93328. B, diagramatic representation of the F4/80 protein structure. EGF-like domains (triangles), N-linked glycosylation sites (lollipops), and the seven transmembrane segments are shown.
Figure 2: Northern blot analysis of F4/80 mRNA expression in mouse cell lines. 15 µg of total RNA from each cell line was electrophoresed through a 1.2% denaturing agarose gel, transferred to a Genescreen Plus membrane and probed with a 757-bp fragment from pF4/80(12.2) (top panel). Ethidium bromide staining of the gel (bottom panel) demonstrates equal loading of the samples. The relative positions of RNA markers are shown.
Figure 3:
Fluorescence-activated cell sorting
analysis of pcDNA3/F4/80 transfected CHO-K1 cells, stained with F4/80 (c) and 5C1 (d) shown as solid lines.
CAMPATH-9 and MRC-OX20 control staining are shown as dashed
lines. Cells transfected with a control pcDNA3/-galactosidase
construct are shown in panels a and b.
The primary amino acid sequence of F4/80 demonstrates a
high degree of homology to members of two independent protein
superfamilies. First, the NH-terminal region of the protein
contains seven tandem EGF-like domains(13) . These repeats of
approximately 50 amino acids are characterized by the spatial
arrangement of six cysteine residues that form three disulfide bonds
within each domain, thereby generating a tightly folded structure. A
search of the SwissProt and NBRF-PIR data bases with the F4/80 sequence
identified a high degree of homology to a number of proteins containing
EGF repeats, with the highest scores between F4/80 and connective
tissue components such as fibrillin 1 (14) and fibulin
2(15) . As well as the six invariant cysteines, five of the
EGF-like domains contain consensus motifs implicated in Ca
binding(16) , which may play a role in stabilizing the
conformation required for ligand interaction. Preliminary analysis of
the mouse F4/80 gene demonstrates that each separate EGF-like
domain is encoded by a single exon as described for the genomic
organization of other EGF superfamily members. (
)A role for
EGF-like domains in numerous protein-protein interactions has been
proposed, such as the critical requirement of two EGF repeats in the
neurogenic Drosophila protein Notch for its
interaction with the Delta and Serrate proteins(17) .
The second region of homology identified
between F4/80 and members of a protein superfamily is located at the
COOH-terminal region of the F4/80 protein. A hydropathy profile of the
F4/80 sequence (18) demonstrated an abundance of hydrophobic
residues within a region of approximately 250 COOH-terminal amino acids (Fig. 4), suggesting that the molecule may span the cell
membrane a number of times. Protein data base searches identified
significant homology scores between F4/80 and members of the Tm7
hormone receptor family, including the receptors for peptidic hormones
such as parathyroid hormone, calcitonin, vasoactive intestinal peptide,
glucagon, and secretin(19) . This recently described receptor
family shares a common overall topology with an extracellular NH terminus, an intracellular COOH terminus, and a central region
consisting of seven transmembrane segments, which results in three
external loops and three internal loops. Characteristic residues were
found to be conserved between F4/80 and the members of this particular
subset of Tm7 receptors in the transmembrane segments (with the
exception of Tm6), the three intracellular loops and the cytoplasmic
tail immediately following Tm7. In addition, F4/80, in common with the
other members of the family, contains a cysteine residue in each of the
first and second extracellular loops. The formation of a disulfide
bridge between these cysteines is believed to be crucial to the overall
tertiary structure of the receptors. The Tm7 receptors interact with
heterotrimeric
-
-
G-proteins, on the cytosolic surface
of the membrane, which are involved in signal transduction following
ligand binding to the extracellular loops of the Tm7
molecule(20) . The F4/80 ligand, and function, remains unknown,
and its identification will determine whether F4/80 serves as a
receptor for a hormone involved in macrophage differentiation and
function. The presence of seven EGF-like repeats in the
NH
-terminal region of F4/80 is an unusual divergence for
Tm7 molecules which, with a limited number of exceptions (20, 21) , have relatively short NH
termini with no defined protein superfamily domains. We suggest
that F4/80 possibly interacts with two separate ligands, via the
EGF-like domains and an extracellular portion of the Tm7 multispan
region, respectively. This notion is strengthened by the presence of an
Arg-Gly-Asp motif (Fig. 1A; amino acids 506-508),
often found in matrix proteins with multiple EGF repeats, which could
play a role in cell adhesion following recognition by an integrin
molecule(22) . Based upon the structural elements and
macrophage-restricted expression pattern of the molecule, we propose
that F4/80 is involved in macrophage adhesion within tissues combined
with receptor signaling following its interaction with a peptide
ligand, possibly resulting in the activation of adenylate cyclase and
increased intracellular cAMP levels(19) . This receptor
activity may therefore influence macrophage responses within a defined
tissue microenvironment.
Figure 4: Hydropathy plot of the 931 amino acid F4/80 sequence. The Kyte-Doolittle algorithm and hydropathy values were applied(18) . The seven predicted transmembrane spanning regions are numbered.
A recent report has described the cloning of a cell surface molecule (designated EMR1) from a human neuroectodermal cDNA library, which shows an extremely high degree of similarity to the F4/80 sequence described here(23) . This sequence shows 68% overall identity to F4/80 and contains six EGF-repeats and seven postulated transmembrane segments. Reverse transcriptase PCR analysis suggests that expression of the EMR1 molecule is not as tightly regulated as F4/80 is in mice, although increased levels of EMR1 transcripts appear to be expressed in peripheral blood mononuclear cells. The wide distribution of EMR1 expression, in comparison with the restricted pattern of F4/80 expression, is intriguing. The development of mAbs directed to the human protein will aid greatly in defining the localization of cells expressing this molecule, as will in situ hybridization studies on normal human tissue sections. The deduced structure of the F4/80 and EMR1 proteins suggests a role in the cellular response to an undefined hormone or an interaction, possibly through the EGF-like repeats, with an alternative protein ligand. The identification of the F4/80 ligand(s) will help to elucidate the function of this specialized molecule in macrophage physiology.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X93328[GenBank].