(Received for publication, June 5, 1995; and in revised form, July 26, 1995)
From the
Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Analysis of PECAM-1 expression in the developing mouse embryo has revealed the presence of multiple isoforms of murine PECAM-1 (muPECAM-1) that appeared to result from the alternative splicing of exons encoding cytoplasmic domain sequences (exons 10-16) (Baldwin, H. S., Shen, H. M., Yan, H., DeLisser, H. M., Chung, A., Mickanin, C., Trask, T., Kirschbaum, N. E. Newman, P. J., Albelda, S., and Buck, C. A.(1994) Development 120, 2539-2553). To investigate the functional consequences of alternatively spliced muPECAM-1 cytoplasmic domains, L-cells were transfected with cDNA for each variant and their ability to promote cell aggregation was compared. In this assay, full-length muPECAM-1 and all three isoforms containing exon 14 behaved like human PECAM-1 in that they mediated calcium- and heparin-dependent heterophilic aggregation. In contrast, three muPECAM-1 variants, all missing exon 14, mediated calcium- and heparin-independent homophilic aggregation. Exon 14 thus appears to modulate the ligand and adhesive interactions of the extracellular domain of PECAM-1. These findings suggest that alternative splicing may represent a mode of regulating the adhesive function of PECAM-1 in vivo and provides direct evidence that alternative splicing involving the cytoplasmic domain affects the ligand specificity and binding properties of a cell adhesion receptor.
PECAM-1 ()(CD31) has the distinctive feature of being
found on platelets, leukocytes, and endothelial cells (reviewed in
DeLisser et al. (1994b)). Recent observations have implicated
PECAM-1 in a number of important processes. PECAM-1 facilitates the
diapedesis of leukocytes both in vitro and in vivo (Muller et al., 1993; Vaporciyan et al., 1993;
Bogen et al., 1994), acts as a trigger for up-regulating
integrins on leukocytes (Tanaka et al., 1992; Piali et
al., 1993; Berman and Muller, 1995; Leavesley et al.,
1994), and thus appears to play a role in cell-cell interactions during
an inflammatory response. It is also one of the first cell surface
molecules to be expressed by endothelial and endocardial cells during
embryonic development, suggesting that it may be involved in the
establishment of the early cardiovascular system (Baldwin et
al., 1994).
PECAM-1 is organized into an extracellular amino-terminal domain containing 6 immunoglobulin (Ig)-like repeats, a short hydrophobic transmembrane domain, and long cytoplasmic tail (Newman et al., 1990). The gene for human PECAM-1 (huPECAM-1) has recently been characterized (Kirschbaum et al., 1994) and is composed of 16 exons separated by introns ranging in size from 86 to more than 12,000 base pairs in length. Each of the six extracellular Ig homology domains is encoded by a single exon (exons 3-8). The transmembrane region is encoded by one exon (exon 9), while the cytoplasmic tail is encoded by a series of six short exons (exons 10-16). This multi-exon structure of the cytoplasmic domain is quite unusual for Ig superfamily members. A number of cytoplasmic domain variants of PECAM-1, presumably arising from alternative splicing, have been identified in huPECAM-1 (Goldberger et al., 1994; Kirschbaum et al., 1994) and in murine PECAM-1 (muPECAM-1) (Baldwin et al., 1994). Analysis of PECAM-1 expression in the developing mouse embryo documented the presence of at least six isoforms of muPECAM-1 that appeared to result from the alternative splicing of the exons encoding the cytoplasmic domain (Baldwin et al., 1994).
Although the role of these multiple isoforms of PECAM-1 is currently unknown, studies examining the binding characteristics of huPECAM-1 suggest that alterations in the cytoplasmic domain have important functional implications. Previous experiments showed that full-length huPECAM-1 promoted heterophilic aggregation in transfected L-cells in a divalent cation-dependent, heparin-sensitive manner (Albelda et al., 1991; Muller et al., 1992; DeLisser et al., 1993). In contrast, mutants of huPECAM-1 with partially truncated cytoplasmic domains mediated aggregation that was quite different in that it was homophilic, divalent cation-independent, and heparin-insensitive (DeLisser et al., 1994a). These findings raised the possibility that naturally occurring alternatively spliced forms of PECAM-1 might also function differently (Baldwin et al., 1994).
The purpose of this study was to analyze the adhesive properties of each of the muPECAM-1 isoforms detected in early mouse embryos (Baldwin et al., 1994) and, if possible, identify specific regions of the cytoplasmic domain that determined the binding characteristics of the molecule. To accomplish this, L-cells were transfected with the cDNA for each variant and the functional properties of each isoform analyzed using the L-cell aggregation assay. These experiments documented that consistent changes in the aggregation properties of the cells were correlated with specific isoforms of muPECAM-1. Full-length muPECAM-1 and all isoforms containing peptide sequences encoded in cytoplasmic exon 14 mediated heterophilic, calcium-dependent, heparin-sensitive aggregation. In contrast, all isoforms missing this peptide sequence in their cytoplasmic domains demonstrated homophilic aggregation that was calcium-independent and heparin-insensitive. These findings provide direct evidence for the hypothesis that naturally occurring alternatively spliced PECAM-1 isoforms have different ligand specificity and suggest that alternative splicing may be a method of regulating the adhesive function of PECAM-1 in vivo. In addition, these results pinpoint exon 14 as a key region of the cytoplasmic domain that determines the ligand and adhesive interactions of PECAM-1.
Figure 1:
Comparison
of nucleotide sequences of muPECAM-1 isoforms and huPECAM-1. The
nucleotide sequences encoding the cytoplasmic domains of the seven
muPECAM-1 (Mu) isoforms are compared to the sequence for
huPECAM-1 (Hu) as previously reported by Newman et
al.(1990). Identical residues are indicated by hyphens(-), while the solid bars designate
deleted nucleotide sequences. A space appears between
predicted exon sequences (Kirschbaum et al., 1994). Note the
change in the reading frame of exon 16 and the resulting truncation of
the cytoplasmic domain of muPECAM-115, muPECAM-1
12,15,
muPECAM-1
14,15, and
muPECAM1
12,14,15.
Figure 2:
Surface expression of muPECAM-1 isoforms
expressed in L-cells. Cell surface expression of L-cells transfected
with muPECAM-1 (A), muPECAM-112 (B),
muPECAM-1
14 (C), muPECAM-1
15 (D),
muPECAM-1
12,15 (E), muPECAM-1
14,15 (F), and
muPECAM-1
12,14,15 (G) constructs were assessed by
fluorescence activated cell sorting using the anti-muPECAM-1 monoclonal
antibody, mAb 390. For each L-cell transfectant >90% of the cells
were positive. The antibody did not bind to control cells transfected
with vector alone (H).
Figure 3:
Immunoprecipitation of muPECAM-1 isoforms
expressed in L-cell. To confirm the molecular mass of each isoform,
surface biotinylated protein extracts from L-cells expressing each
variant were immunoprecipitated with mAb 390. FL designates
(full-length) muPECAM-1; 15, muPECAM-1
15;
12, muPECAM-1
12, etc. To facilitate comparison,
constructs were ordered on the gel based on the number of amino acids
in their cytoplsmic domain. The molecular mass of the different
muPECAM-1 constructs varied from
110 to 130
kDa.
Figure 4:
Aggregation of muPECAM-1 isoforms
expressed in L-cells. The aggregation assay was performed as described
under ``Experimental Procedures.'' L-cell transfectants
expressing each isoform were allowed to aggregate in the absence or
presence of 1 mM calcium. muPECAM-1, muPECAM-112,
muPECAM-1
15, and muPECAM-1
12,15 (Group 1) demonstrated
calcium-dependent aggregation (A) while muPECAM-1
14,
muPECAM-1
14,15, and muPECAM-1
12,14,15 (Group 2) displayed
calcium-independent aggregation (B). Heparin (50 µg/ml)
blocked the aggregation of Group 1 (A) but did not
significantly inhibit the aggregation of Group 2 (B) even at
heparin concentrations of 500 µg/ml (data not shown). For Group 2,
similar results were obtained for experiments performed in the absence
of calcium. The data presented are representative of at least three
experiments done in duplicate or triplicate and of at least two
independent clones. Standard deviation is
shown.
Figure 5:
Effect of anti-muPECAM-1 antibodies on the
aggregation of muPECAM-1 isoforms expressed in L-cells. Three
monoclonal antibodies (mAb 390, EA-3, and Mec 13.3; 50 µg/ml) were
studied for their effect on the aggregation process in the presence of
calcium. All three antibodies inhibited aggregation mediated by
muPECAM-1, muPECAM-112, muPECAM-1
15, and muPECAM-1
12,15 (A) while for muPECAM-1
14, muPECAM-1
14,15,
muPECAM-1
12,14,15 EA-3 and Mec 13.3 but not 390 were blocking (B). The data presented are representative of at least two
experiments done in duplicate or triplicate. For muPECAM-1
14,
muPECAM-1
14,15, and muPECAM-1
12,14,15 similar results were
obtained for experiments performed in the absence of calcium. Standard
deviation is shown.
Figure 6:
Mixed aggregation studies. Mixed
aggregation assays were performed in which equal numbers of
non-transfected and transfected L-cells were mixed together after
fluorescent labeling of one of the cell lines. After incubation, the
number of labeled cells within each 5 cell aggregate was counted. The
data is representative of at least three experiments. A, when
muPECAM-1, muPECAM-112, muPECAM-1
15, and muPECAM-1
12,15
L-cell transfectants (Group 1) were mixed with non-transfected L-cells
the majority of aggregates were made up of mixtures of transfected and
non-transfected cells. A ``normal'' distribution is noted,
indicative of a heterophilic interaction (see DeLisser et al.,
1993). B, similar mixed aggregation experiments with
muPECAM-1
14, muPECAM-1
14,15, and muPECAM-1
12,14,15
(Group 2) yielded aggregates that were composed primarily of
transfected cells. The frequency distribution is shifted toward the
right, reflecting a homophilic interaction.
Figure 7:
Phosphorylation of muPECAM-1 isoforms.
Transfected L-cells were labeled in parallel with
[S]methionine (A) or
[
P]orthophosphate (B), extracted with
non-ionic detergents in the presence of vanadate and immunoprecipitated
with mAb 390. FL designates (full-length) muPECAM-1,
15, muPECAM-1
15,
12, muPECAM-1
12,
etc. Full-length muPECAM-1 and muPECAM-1
14 demonstrated high
levels of constitutive phosphorylation while the deletion of exon 12
and/or 15 was accompanied by reduced incorporation of
P
label. Numbers on the right represent molecular size
markers in kilodaltons.
Figure 8:
The effect of deglycosylation on
muPECAM-1-dependent aggregation. A, to confirm the activity of
these deglycosylating compounds, extracts of full-length muPECAM-1 (FL) and muPECAM-114 (
14) L-cell
transfectants, treated with each agent, were immunoprecipitated with
mAb 390. Increased mobility was noted for both isoforms following
deglycosylation by each agent. B, deoxymannojirimycin (dMM), swainsonine, and neuraminidase were studied for their
effect on the aggregation process in the presence of calcium (1
mM). All three compounds were found to inhibit aggregation
mediated by muPECAM-1 but not that of muPECAM-1
14. The data
presented are representative of at least two experiments done in
duplicate or triplicate. Standard deviation is
shown.
To determine the functional consequences of alternative
splicing of muPECAM-1, the behavior of L-cell transfectants expressing
various isoforms was compared in an established adhesion assay (Albelda et al., 1991; Muller et al., 1992; DeLisser et
al., 1993). Full-length muPECAM-1 and the three isoforms
containing exon 14 (muPECAM-112, muPECAM-1
15, and
muPECAM-1
12,15) mediated calcium-dependent, heparin-sensitive,
heterophilic aggregation that was inhibited by the anti-murine PECAM-1
monoclonal antibody, mAb 390, and was sensitive to deglycosylation. In
contrast, all three of the muPECAM-1 variants missing exon 14
(muPECAM-1
14, muPECAM-1
14,15, and muPECAM-1
12,14,15)
mediated homophilic aggregation that was calcium-independent,
heparin-insensitive and not inhibited by mAb 390 or affected by
deglycosylation (see Table 2). These findings are consistent with
experiments showing that deletion of regions of the cytoplasmic domain
of huPECAM-1 containing exon 14 resulted in a switch in the aggregation
properties of transfected cells (DeLisser et al., 1994a) and
give a possible biological context to these observations.
These results provide further evidence that naturally occurring isoforms produced by alternative splicing lead to functionally different cytoplasmic domain variants of muPECAM-1. Given its wide distribution among vascular-associated cells and its putative roles in inflammation (Tanaka et al., 1992; Muller et al., 1993; Piali et al., 1993; Vaporciyan et al., 1993; Bogen et al., 1994; Berman and Muller, 1995), hematopoietic development (Leavesley et al., 1994), angiogenesis (Albelda et al., 1990), and vascular development (Baldwin et al., 1994), changes in the cytoplasmic domain of PECAM-1 generated by alternative splicing may play a role in modulating the ligand interactions of PECAM-1 and thus allow for a diversity of function.
A new finding in this study was that heterophilic aggregation mediated by PECAM-1 was sensitive to deglycosylation, while homophilic aggregation was unaffected by removal of carbohydrate groups. In this regard, the heterophilic binding of PECAM-1 resembles a number of other Ig superfamily members such as NCAM (Walsh et al., 1989; Doherty et al., 1992), L1 (Kadmon et al., 1990; Horstkorte et al., 1993), myelin Po protein (Filbin and Tennekoon, 1993), and ICAM-1 (Diamond et al., 1991).
Our results, however, differ from those reported by Xie and Muller (1993) who described a full-length muPECAM-1 that mediated calcium-independent, homophilic aggregation. The reasons for these differences are unclear, although they may be partially explained by somewhat different assay conditions in that a greater concentration of cells, higher divalent cation concentrations, and longer incubation time were used. Since muPECAM-1 and huPECAM-1 have a high degree of homology and resemble each other in all other respects studied, it seems reasonable that the basic aggregation characteristics of each form would also be similar.
A key question is how small changes in cytoplasmic domain can dramatically affect the binding characteristics of PECAM-1. The localization of exon 14, a small exon composed of 18 amino acids (LGTRATETVYSEIRKVDP), as the key region may be especially useful. A portion of this exon is very highly conserved between mouse and human PECAM-1. Interestingly, the 10-amino acid region, TETVYSEIRK, is almost identical to the cytoplasmic terminus (TETVYSEVKK) of the related Ig-superfamily member biliary glycoprotein (Rojas et al., 1990). Like PECAM-1 biliary glycoprotein mediates calcium-dependent adhesion. Loss of exon 14 could result in a number of possible effects including changes in phosphorylation or glycosylation, alterations in associations with cytoplasmic partners, and direct conformational changes.
Since there are 5 amino acids in exon 14 that could potentially serve as phosphorylation sites, one potential mechanism responsible for the changes in function resulting from loss of exon 14 could involve loss of a key phosphorylation site. PECAM-1 is constitutively phosphorylated and there is evidence to suggest that cellular activation is accompanied by changes in phosphorylation at serine and threonine residues (Newman et al. 1992; Zehnder et al., 1992). Our data (Fig. 8), however, suggest that changes in the overall level of phosphorylation do not correlate with the deletion of exon 14. However, these data do not rule out the possibility that the absence of one or more of the specific phosphorable residues in exon 14 may be important.
Since
glycosylation of the extracellular domain has been shown to play a
crucial role in regulating the binding properties of a number of Ig
superfamily members, now including PECAM-1, it is possible that loss of
exon 14 could alter post-translational processing and thus lead to a
change in adhesive function of PECAM-1. Although there may be changes
in specific carbohydrate residues induced by loss of exon 14, we have
been unable to observe any gross changes in the overall level of
glycosylation, as determined by the change in molecular size after
deglycosylation. ()
Another possibility, currently being
examined, is that association of the cytoplasmic domain of PECAM-1 with
another membrane or cytoplasmic protein may be critical for its
function. Exon 14 may be important in regulating this association.
Preliminary experiments using differential detergent extractions have
not revealed differences in the ability of the various isoforms to
associate with the actin cytoskeleton. ()However, this
PECAM-1-associated molecule may be another PECAM-1 molecule, a molecule
with enzymatic activity (such as the association of the cytoplasmic
domain of CD4 with the tyrosine kinase p56
(Turner et
al., 1990) or a novel protein.
Finally, it is theoretically possible that loss of exon 14 might directly lead to a change in the overall conformation of the molecule. A proline residue is the terminal amino acid of exon 14. Loss of this amino acid may induce an important conformational change that alters the ability of the cytoplasmic domain to fold normally. Clearly, this change must be rather subtle, since all of the mAbs tested bind equally well to all isoforms.
Regardless of the mechanism(s) involved in regulating PECAM-1 ligand interactions, the implications of these findings extend beyond their significance to the structure-function relationships of PECAM-1. First, it is possible that the sequences found within exon 14 may have importance in the function of other Ig superfamily members (Rojas et al., 1990). Second, our findings indicate that observations made with artificial mutations of huPECAM-1 (DeLisser et al., 1994a) are likely to have physiological relevance and thus advance the emerging concept of the cytoplasmic domain as a regulator of not only the strength but the mechanism of adhesion. Finally, these data also suggest that alternative splicing of cytoplasmic domain regions may represent a novel way a cell can alter its interactions with the environment during development, inflammation, and wound healing.