(Received for publication, November 27, 1995; and in revised form, January 2, 1996)
From the
Cellular disintegrins are a family of membrane-anchored proteins with structural homology to snake venom metalloproteases and disintegrins. We report here the cDNA cloning and initial biochemical characterization of the first cellular disintegrin protein with an RGD sequence in its disintegrin domain, which we propose to name metargidin (for metalloprotease-RGD-disintegrin protein). The domain organization of metargidin is identical with that of previously reported members of the cellular disintegrin family, comprising (i) a pro- and a metalloprotease domain including a zinc-binding consensus motif, (ii) a disintegrin domain containing the RGD motif, (iii) a cysteine-rich domain, (iv) an epidermal growth factor-like domain, (v) a hydrophobic transmembrane domain, and (vi) a cytoplasmic tail with proline-rich sequences that could act as potential SH3 ligands. Antibodies raised against the cytoplasmic tail of metargidin recognize a glycoprotein of 110 kDa in MDA-MB-468 mammary epithelial carcinoma cells, which can be cell surface-biotinylated, indicating its localization in the plasma membrane. A second protein of 56 kDa co-immunoprecipitates with metargidin, suggesting that it is part of a protein complex. These features are consistent with a model in which metargidin is an integrin ligand which, as a transmembrane protein, might function in cell-cell adhesion and/or signaling.
Cellular disintegrins are a family of proteins that are defined
by their sequence similarity to snake venom integrin ligands and
metalloproteases. Disintegrins were first described as short soluble
protein components of Viperidae snake venoms that efficiently
interfere with platelet aggregation by binding to the platelet integrin
via an RGD
sequence(1, 2, 3) . In recent years, a family
of membrane-anchored proteins containing a metalloprotease, a
disintegrin, a cysteine-rich, and an epidermal growth factor-like
domain followed by a transmembrane domain and a cytoplasmic tail has
been
discovered(4, 5, 6, 7, 8) .
The best-characterized cellular disintegrins are the
and
subunit of the heterodimeric sperm-surface protein fertilin (formerly
termed PH-30)(4, 9) . Fertilin has been implicated in
gamete membrane binding and fusion(10, 11) , likely
involving binding to the oocyte integrin
in mouse(12) . A recent study has demonstrated a
potential role of another member of this protein family, meltrin
,
in myoblast fusion(13) . Based on the properties of snake venom
disintegrins, as well as of fertilin and meltrin
, it is likely
that other members of the membrane-anchored cellular disintegrin
protein family might also play a role in cell-cell interactions. We
report here the identification of the first cellular disintegrin
protein to display an RGDC motif in its putative integrin-binding site
and discuss the possible implications of this finding.
Figure 1:
Deduced protein
sequence of metargidin and alignment of the disintegrin domains of
metargidin and other related cellular and snake venom disintegrins. The
complete deduced protein sequence of metargidin is shown in A.
The predicted signal sequence cleavage site is marked by an arrowhead, and the putative protein domains of metargidin are
labeled (see Refs. 7 and 20) for alignment of other cellular and snake
venom disintegrin proteins). The metalloprotease domain includes the
zinc-binding consensus sequence HEXXHXLGXXHD (boxed, hatched line)(19) . Five potential N-linked glycosylation sites are marked with an asterisk, and two potential proline-rich cytoplasmic SH3
ligand domains, PPPPRKP and
RPAPPPP(37, 38, 39) , are boxed. B shows an alignment of the disintegrin domain of metargidin
with the disintegrin domains of the RGD-type snake venom disintegrins
bitistatin (43) and kistrin(44) , with the non-RGD-type
snake venom disintegrin HR1B(45) , and with the cellular
disintegrins guinea pig fertilin (4) and human
MDC9(15) . Conserved cysteine residues are shaded, and
the known integrin-binding motif of kistrin and bitistatin and the
predicted integrin-binding motif of metargidin, HR1B, fertilin
,
and MDC9 are boxed(5) .
In order to identify novel cellular disintegrins, cDNA tags were generated by PCR on reverse-transcribed cDNA from the human mammary epithelial cell line MDA-MB-468 using degenerate primers. With this approach, a cDNA tag of a disintegrin protein harboring an RGD sequence was isolated. To isolate the full-length cDNA, we constructed a cDNA library from MDA-MB-468 cells, which was screened with the labeled cDNA tag. Sequencing of the clone with the longest insert thus isolated revealed a cDNA of 2740 base pairs, with an open reading frame encoding an 814-amino acid protein (Fig. 1A). This protein was named metargidin (metalloprotease-RGD-disintegrin; for reference purposes the metargidin cDNA was named ADAM 15, see also (8) ). Northern blot analysis revealed the metargidin mRNA to be close to 3 kilobases in length and to be present in all human tissues examined (Fig. 2).
Figure 2: Northern blot analysis of metargidin expression in human tissues. A Northern blot of RNA extracted from different human tissues (Clontech, 2 µg of poly(A)-selected mRNA per lane) was probed with a metargidin cDNA probe under high stringency conditions as described under ``Materials and Methods.'' The source of mRNA is indicated for each lane.
Excluding the putative signal peptide (Fig. 1A), the mature metargidin protein has a predicted molecular mass of 85 kDa, and its extracellular moiety contains 5 potential sites of N-linked glycosylation. The deduced metargidin protein sequence includes all the domains previously found in cellular disintegrin proteins (see above and Fig. 1A). The metalloprotease domain of metargidin has the HEXXHXLGXXHD zinc-binding motif, which suggests metalloprotease activity(4, 8, 19, 20) . The putative boundary between the prodomain and the metalloprotease domain has four consecutive arginine residues, creating a potential cleavage site for serine proteases such as the proprotein convertase furin(21, 22, 23) . As is the case for other cellular disintegrin-metalloprotease domains that contain a zinc-binding consensus sequence, the prodomain contains an odd number of cysteine residues, one of which may regulate the activity of the metalloprotease through a cysteine switch mechanism (24) .
Metargidin is the first cellular disintegrin to contain the RGD
integrin ligand consensus motif in a position analogous to that found
in snake venom disintegrins (see alignment in Fig. 1B).
The RGD sequence is followed by an additional cysteine residue which so
far has only been found in non-RGD-type snake venom disintegrins and
cellular disintegrins, but not in RGD-type snake venom disintegrins.
The structure of several snake venom disintegrins has been solved by
NMR, revealing that the RGD sequence is present in a flexible hairpin
loop(25, 26) . An RGD-containing loop structure has
also been found in the otherwise unrelated Elapidae snake
venom toxin dendroaspin(27, 28) , in the leech
anticoagulant decorsin(29) , and in the fibronectin type III
repeat(30, 31) , and thus appears to be a preferred
context for RGD motifs showing high affinity binding to an integrin. It
is conceivable that the additional cysteine residue in the metargidin
disintegrin domain may change its structure such that the RGDC sequence
becomes cryptic. However, if the RGDC sequence is accessible to
integrin binding, the cysteine residue may contribute to the
specificity for binding to a certain integrin, since the amino acid
residues following the RGD sequence are known to confer some
specificity for different
integrins(32, 33, 34) . Data from phage
display studies have shown that RGD sequences in the context of
disulfide-bonded cysteine residues are high affinity integrin ligands (34, 35, 36) , and an RGDC sequence in a
cyclic peptide with eight amino acid residues
(CXC) has high affinity for the platelet integrin
(35) . If metargidin is
indeed capable of interacting with integrins through its RGD sequence,
it would represent a new type of membrane-anchored integrin ligand
containing an RGD sequence.
Two cellular disintegrin proteins,
fertilin (4) and meltrin
(13) , are thought
to be involved in membrane fusion. In both proteins, the cysteine-rich
domain displays a short hydrophobic stretch that has been suggested to
function as a fusion peptide(4, 13) . Comparison of a
hydrophobicity plot of metargidin with fertilin
and meltrin
shows that metargidin lacks a similarly hydrophobic region in the
cysteine-rich domain (data not shown). Metargidin is therefore not
likely to play a direct role in membrane fusion.
Two short
proline-rich sequences present in the cytoplasmic domain of metargidin
(PPPPRKP and RPAPPPP, see Fig. 1A) show similarity to
Src homology 3 (SH3) ligand
domains(37, 38, 39) . If active as SH3
ligands, these motifs in metargidin could mediate intracellular
signaling or cytoskeletal attachment via proteins containing SH3
domains(40, 41) . Similar proline-rich sequences are
also found in the cytoplasmic tail of some other cellular disintegrins,
such as MDC9 (15) , MS2(42) , and meltrin
(13) , and the putative proline-rich cytoplasmic SH3
ligand domains of MDC9 have been shown to interact with the SH3 domain
of Src in a blot overlay assay(15) .
To analyze the domain
organization and potential processing of metargidin in MDA-MB-468
cells, antibodies were raised against a fusion protein between the
metargidin cytoplasmic tail domain and GST. For use in Western blot
analysis and immunoprecipitation, purified IgG were depleted of
antibodies reactive to GST (referred to as anti-metargidin IgG), or
depleted of GST-cytoplasmic tail-specific antibodies (referred to as
control IgG, see ``Materials and Methods''). MDA-MB-468 cell
extracts enriched for glycoproteins through precipitation on
concanavalin A were analyzed by immunoblotting with anti-metargidin
IgG. A band of 100 kDa was recognized under nonreducing conditions (Fig. 3A, lane 1), and a band of 110 kDa under
reducing conditions (Fig. 3A, lane 2). The
increase in apparent molecular mass upon reduction is consistent with
the behavior of a cysteine-rich cellular disintegrin protein. When
identical samples were probed with control IgG, three weaker
nonspecific bands of 63, 73, and 90 kDa were visible under nonreducing (Fig. 3A, lane 5) or reducing conditions (Fig. 3A, lane 6). After removal of N-linked carbohydrate residues with Endo H (Fig. 3A, lane 3), reduced metargidin
has an apparent molecular mass of 95 kDa, whereas mock Endo
H
-treated metargidin has an apparent molecular mass of 110
kDa (Fig. 3A, lane 4). The apparent molecular
mass of 95 kDa for Endo H
-treated metargidin is close to
its predicted molecular mass of 85 kDa.
Figure 3:
Western blot analysis and
immunoprecipitation of cell surface-biotinylated metargidin. In A, MDA-MB-468 extracts were incubated with concanavalin A, and
bound glycoproteins were eluted in sample loading buffer, separated on
a 10% SDS-polyacrylamide gel, and transferred to nitrocellulose
membranes. Samples in lanes 2-4 and 6 were
reduced and alkylated prior to electrophoresis. No DTT was added to
samples in lanes 1 and 5. The sample in lane 3 was deglycosylated with Endo H, whereas the sample in lane 4 was mock-deglycosylated. Lanes 1-4 were
probed with anti-metargidin IgG, and lanes 5 and 6 with control IgG, both prepared as described under
``Materials and Methods.'' B, lanes 1-3 show the results of cell surface biotinylation and
immunoprecipitation of metargidin. Extracts of cell
surface-biotinylated MDA-MB-468 cells were immunoprecipitated with
anti-metargidin IgG (lane 1), control IgG (lane 2),
or preimmune IgG (lane 3). Precipitated proteins were reduced
with 10 mM DTT prior to electrophoresis on 7.5%
SDS-poly-acrylamide gels, transferred to nitrocellulose and probed with
horseradish peroxidase-coupled
streptavidin.
The size of metargidin on
MDA-468-cells suggests that both the disintegrin and metalloprotease
domains occur in a membrane-anchored form. The presence of both an
integrin ligand domain and a metalloprotease domain in the metargidin
protein suggests the possibility that both domains are active
simultaneously. Alternatively, the RGD sequence may only become exposed
after proteolytic processing, and the metalloprotease and disintegrin
domains may thus function sequentially. The sperm protein fertilin
is an example of a membrane-anchored cellular disintegrin that is
processed at the N terminus of the disintegrin domain during sperm
maturation, and processing correlates with the acquisition of
fertilization competence(4, 9) . Furthermore,
expression of a variant of the mouse cellular disintegrin meltrin
lacking its metalloprotease domain in C2C12 myoblasts resulted in
increased myoblast fusion, whereas expression of the full-length
meltrin
led to decreased fusion(13) , indicating that
certain cellular disintegrins may need to be processed in order to
become active. Experiments are currently in progress to determine
whether the RGD sequence within the metargidin-disintegrin domain
indeed functions as an integrin ligand, and, if so, whether the
prodomain or the metalloprotease domain needs to be removed for the RGD
to become available for binding.
To determine whether metargidin, like fertilin, is also part of a protein complex on the cell surface, MDA-MB-468 cells were labeled with the water-soluble biotinylation reagent NHS-LC-biotin. Labeled extracts were immunoprecipitated with anti-metargidin IgG or with control IgG. Anti-metargidin IgG immunoprecipitated a protein with an apparent molecular mass of 110 kDa under reducing conditions (Fig. 3B, lane 1) and 100 kDa under nonreducing conditions (not shown). This protein was not immunoprecipitated by control IgG (Fig. 3B, lane 2), or by preimmune IgG (Fig. 3B, lane 3). These results indicate that metargidin is present on the plasma membrane of MDA-MB-468 cells. In addition to the band of 110 kDa, a second band of 56 kDa was also immunoprecipitated by anti-metargidin IgG, but not by control antibodies. The 56-kDa protein also underwent a shift in apparent molecular mass upon reduction (not shown), indicating the existence of extracellular disulfide bonds. In a separate Western blot analysis using anti-metargidin IgG, the 56-kDa protein was not recognized (not shown), suggesting that it is distinct from metargidin. Since fertilin is a heterodimer consisting of two cellular disintegrin proteins, it will be interesting to determine whether the 56-kDa protein co-precipitating with metargidin is also a cellular disintegrin.
In conclusion, the presence of the RGD sequence in metargidin strongly suggests that metargidin could act as a membrane-anchored integrin ligand containing the RGD motif. This finding raises the possibility that, in addition to their role in cell matrix interactions, RGD-binding integrins might also mediate direct cell-cell interactions. Since metargidin contains potential cytoplasmic SH3 ligand domains, it is possible that it not only functions as a membrane-anchored integrin ligand, but also as a counter-receptor for integrins or other proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41767[GenBank].