Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Abstract

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.

Figure 1

Construction of PECAM-1/FKBP chimeric proteins. (A) Schematic diagram detailing the production of PECAM-1/FKBP-1 and PECAM-1/FKBP-2. cDNAs encoding PECAM-1 and FKBP were used as templates for standard overlap PCR to produce the resulting chimeric proteins, which contain either one or two FKBP dimerization domains. (B) Controlled dimerization and oligomerization of PECAM-1. PECAM-1/FKBP-1, containing one FKBP domain, forms self-limiting dimers upon exposure to the cell permeable synthetic dimerizer AP1510, whereas PECAM-1/FKBP-2 is capable of forming oligomers.

Figure 6

Model of PECAM-1/IgG binding to monomers, dimers, and oligomers of PECAM-1 on the cell surface. Note that the number of PECAM-1/IgG molecules bound is limited by the availability of free Ig domain 1. The predicted stoichiometry of PECAM-1/IgG to cell surface PECAM-1 correlates with the flow cytometric data shown in Fig. 5i.e., cell surface PECAM-1 dimers maximally support trans homophilic adhesion, whereas PECAM-1 oligomers are involved in cis interactions.

Figure 2

Chemical cross-linking of cell surface PECAM-1 results in formation of stable high molecular weight complexes. (A) HEL cells were treated with chemical cross-linking agents BS³ or DTSSP, and detergent lysates were analyzed by SDS-PAGE using the anti–PECAM-1 mAb, PECAM-1.3. The prominent 130-kD doublet observed in untreated cells corresponds to variously glycosylated monomeric PECAM-1. Note the high molecular weight forms corresponding to PECAM-1 dimers and oligomers. (B) Immunoblot analysis of PECAM-1–transfected HEK-293 lysates that had been exposed to either BS³ (noncleavable) or DTSSP (cleavable). Note that the high molecular weight forms of PECAM-1 present under nonreducing conditions after cross-linking with DTSSP all revert upon reduction to a single band with a mobility identical to monomeric PECAM-1, suggesting the presence of homodimers and homooligomers.

Figure 3

PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2 expression on 293 cell surface. (A) Flow cytometric analysis of cell surface expression. Note that untransfected 293 cells do not express PECAM-1, whereas the transfectants express nearly equivalent level of PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2, respectively. (B) PECAM-1.3 immunoblot analysis of untransfected 293 cells, or 293 cells transfected with wild-type PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2. Identical blots were probed with either PECAM-1.3 (left) or anti-FKBP (right). Note that addition of FKBP domains decreases the mobility of PECAM-1 (left), and also confers immunoreactivity to anti-FKBP-12 antibodies (right).

Figure 3

PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2 expression on 293 cell surface. (A) Flow cytometric analysis of cell surface expression. Note that untransfected 293 cells do not express PECAM-1, whereas the transfectants express nearly equivalent level of PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2, respectively. (B) PECAM-1.3 immunoblot analysis of untransfected 293 cells, or 293 cells transfected with wild-type PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2. Identical blots were probed with either PECAM-1.3 (left) or anti-FKBP (right). Note that addition of FKBP domains decreases the mobility of PECAM-1 (left), and also confers immunoreactivity to anti-FKBP-12 antibodies (right).

Figure 4

AP1510-induced dimerization and oligomerization of PECAM-1. (A) 293 cells stably expressing PECAM-1/FKBP-2 were treated with AP1510, and the distribution PECAM-1 visualized with Alexa 488–conjugated mAb PECAM-1.3. Whereas PECAM-1 oligomers are readily observed, changes in cell surface distribution of PECAM-1/FKBP-1, upon treatment with AP1510, were not apparent (not shown), probably due to the inability of light microscopy to visualize simple dimers of the receptor. Bar, 4 μM. (B) 293 cells stably expressing wild-type PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2 were treated with the chemical cross-linking agent DTSSP in the presence or absence of AP1510, and subjected to immunoblot analysis using the anti–PECAM-1 mAb, PECAM-1.3. Note that nearly all the cell surface PECAM-1/FKBP-1 is forced into dimers by AP1510, whereas the majority of PECAM-1/FKBP-2 exists in high molecular weight oligomers after AP1510 treatment.

Figure 4

AP1510-induced dimerization and oligomerization of PECAM-1. (A) 293 cells stably expressing PECAM-1/FKBP-2 were treated with AP1510, and the distribution PECAM-1 visualized with Alexa 488–conjugated mAb PECAM-1.3. Whereas PECAM-1 oligomers are readily observed, changes in cell surface distribution of PECAM-1/FKBP-1, upon treatment with AP1510, were not apparent (not shown), probably due to the inability of light microscopy to visualize simple dimers of the receptor. Bar, 4 μM. (B) 293 cells stably expressing wild-type PECAM-1, PECAM-1/FKBP-1, and PECAM-1/FKBP-2 were treated with the chemical cross-linking agent DTSSP in the presence or absence of AP1510, and subjected to immunoblot analysis using the anti–PECAM-1 mAb, PECAM-1.3. Note that nearly all the cell surface PECAM-1/FKBP-1 is forced into dimers by AP1510, whereas the majority of PECAM-1/FKBP-2 exists in high molecular weight oligomers after AP1510 treatment.

Figure 5

Effect of PECAM-1 dimerization and oligomerization on PECAM-1 homophilic adhesion. (A) PECAM-1/IgG was added to the indicated transfected 293 cell lines in either the absence (top) and presence (lower) of AP1510. After incubation with FITC-labeled mouse anti–human Fc, binding was quantified by flow cytometry. At 1 μM, AP1510 more than doubled the amount of PECAM-1/IgG that became associated with PECAM-1/FKBP-1–transfected cells, an effect that was dose dependent (B) and reversible at high concentrations of the drug, probably due to saturation of available FKBP sites and the resulting formation of PECAM-1 dimers and monomers. AP1510 had no effect on PECAM-1/IgG binding to cells expressing wild-type PECAM-1, as expected, however, oligomerization of PECAM-1/FKBP-2 results in decreased homophilic binding capacity, suggesting that PECAM-1/PECAM-1 cis interactions occur at the expense of its ability to support trans homophilic interactions.

Figure 5

Effect of PECAM-1 dimerization and oligomerization on PECAM-1 homophilic adhesion. (A) PECAM-1/IgG was added to the indicated transfected 293 cell lines in either the absence (top) and presence (lower) of AP1510. After incubation with FITC-labeled mouse anti–human Fc, binding was quantified by flow cytometry. At 1 μM, AP1510 more than doubled the amount of PECAM-1/IgG that became associated with PECAM-1/FKBP-1–transfected cells, an effect that was dose dependent (B) and reversible at high concentrations of the drug, probably due to saturation of available FKBP sites and the resulting formation of PECAM-1 dimers and monomers. AP1510 had no effect on PECAM-1/IgG binding to cells expressing wild-type PECAM-1, as expected, however, oligomerization of PECAM-1/FKBP-2 results in decreased homophilic binding capacity, suggesting that PECAM-1/PECAM-1 cis interactions occur at the expense of its ability to support trans homophilic interactions.

Figure 7

PECAM-1 oligomerization is required to effect integrin activation. (A) 2 × 10⁵ cells in 200 μl of serum-free cell culture media were added to microtiter wells containing immobilized fibronectin or BSA in the presence or absence of 1 μM AP1510, and cell adhesion was visualized using light microscopy (left) or quantitated using a fluorescent plate reader (right). Note that oligomerization of PECAM-1 conferred increased adhesion to fibronectin only for PECAM-1/FKBP-2–transfected cells. (B) The degree of PECAM-1 oligomerization correlated in a dose-dependent manner with cell adhesion to fibronectin, with optimal adhesion occurring at 1 μM AP1510. At higher doses of AP1510, the drug is likely to saturate available FKBP sites, leading to the formation of dimers and monomers.

Figure 7

PECAM-1 oligomerization is required to effect integrin activation. (A) 2 × 10⁵ cells in 200 μl of serum-free cell culture media were added to microtiter wells containing immobilized fibronectin or BSA in the presence or absence of 1 μM AP1510, and cell adhesion was visualized using light microscopy (left) or quantitated using a fluorescent plate reader (right). Note that oligomerization of PECAM-1 conferred increased adhesion to fibronectin only for PECAM-1/FKBP-2–transfected cells. (B) The degree of PECAM-1 oligomerization correlated in a dose-dependent manner with cell adhesion to fibronectin, with optimal adhesion occurring at 1 μM AP1510. At higher doses of AP1510, the drug is likely to saturate available FKBP sites, leading to the formation of dimers and monomers.

Figure 8

PECAM-1 is a specific integrin activator. (A) Calcein AM–labeled 293 cells expressing PECAM-1/FKBP-2 were added to microtiter wells containing immobilized poly-l-lysine or fibronectin in the presence or absence of AP1510. Note that the increase in cell adhesion induced by oligomerization of PECAM-1 is specific for integrin substrates, and is mediated by the integrin α₅β₁, as antibodies to either of these two integrin subunits reduced cell adhesion to near background levels (B). The AP1510-induced increase in cell adhesion appears to operate via either affinity or avidity regulation, as oligomerization of PECAM-1 does not increase the level of cell surface expression of integrin α₅β₁ (C).

Figure 8

PECAM-1 is a specific integrin activator. (A) Calcein AM–labeled 293 cells expressing PECAM-1/FKBP-2 were added to microtiter wells containing immobilized poly-l-lysine or fibronectin in the presence or absence of AP1510. Note that the increase in cell adhesion induced by oligomerization of PECAM-1 is specific for integrin substrates, and is mediated by the integrin α₅β₁, as antibodies to either of these two integrin subunits reduced cell adhesion to near background levels (B). The AP1510-induced increase in cell adhesion appears to operate via either affinity or avidity regulation, as oligomerization of PECAM-1 does not increase the level of cell surface expression of integrin α₅β₁ (C).