Coupling of cellular adhesiveness with proteolytic cascades is an increasingly recognized paradigm for coordinating focal attachment and detachment important to cell migration (Werb, 1997
). The uPAR is a prototypical example of this strategy (Chapman, 1997
). By localizing with integrins and binding urokinase, uPAR focuses plasmin activation at or near sites of focal contact between the cell surface and extracellular matrix proteins (Blasi et al., 1987
). Plasmin activates cascades involving both matrix metalloproteases and growth factors in the pericellular milieu (Carmeliet et al., 1997
). Prior studies have also shown that the complexes uPAR forms with integrins are important to binding and adhesion of hematopoietic cells to matrix vitronectin, plasmin cleavage of vitronectin reversing this attachment (Wei et al., 1996
; Waltz et al., 1997
). Results reported here further develop this paradigm by elucidating the specificity of interaction between uPAR and β1 integrins. Our results indicate that uPAR preferentially interacts with α3β1 and that this interaction has two important functional consequences: 1) uPAR/α3β1 complexes enable a pathway of cellular adhesion to Vn, especially in cells with little or no αvβ3; and 2) these complexes initiate a signaling pathway promoting the function of α5β1 and α2β1. Both pathways of signaling and enhanced adhesion are activated by concurrent binding of urokinase to uPAR. The pathways are nonetheless distinct because pertussis toxin only blocks the cross talk between α3β1/uPAR and other β1 integrins and not α3β1/uPAR-dependent Vn adhesion. The observation that urokinase signals through uPAR/α3β1 complex formation is consistent with a recent report that urokinase induces metalloproteinases in oral keratinocytes through an α3β1-dependent mechanism (Ghosh et al., 2000
). Thus, the intricate connections between expression of proteases and function of the adhesive machinery of cells is epitomized by the reorganization of membrane partners induced by uPAR expression and its association with α3β1.
Our current findings may help clarify previously reported, apparently contradictory observations regarding the influence of uPAR on the function of the Fn receptor α5β1. In 293 cells, high levels of uPAR expression impair the function of Fn receptors (Wei et al., 1996
). Yet our data (Figure ) indicate that the majority uPAR in these cells is associated with α3β1 and not the Fn receptor. This finding suggests that the inhibition of Fn receptor function by uPAR is probably indirect. Because caveolin-1 is important to β1 integrin signaling and preferentially associates with uPAR/integrin complexes, and because 293 cells express relatively low levels of caveolin-1, overexpression of uPAR may enrich α3β1 complexes with caveolin and at the same time deplete Fn receptors of caveolin. This may explain why impaired Fn receptor function in uPAR-transfected 293 cells is reversed by overexpression of caveolin-1 (Wei et al., 1999
). In contrast, physiological levels of uPAR expression in most cells appear to promote rather than impair Fn receptor function. Our data suggest that this operates, at least in part, through signals derived from uPAR/α3β1 complexes. Although Fn receptors do not require such signals for adhesion, the presence of these signals accelerates FAK phosphorylation and cell spreading on Fn, and therefore may promote Fn receptor-dependent cell migration. We have previously reported that peptides that disrupt uPAR/integrin association impairs smooth muscle cell migration on Fn (Wei et al., 1999
). We postulate that this may explain the recently observed requirement for uPAR expression in Fn receptor-dependent tumor invasion (Aguirre Ghiso et al., 1999
). This pathway may also underlie the requirement of uPAR for αvβ5-dependent migration of pancreatic carcinoma cells on vitronectin even though uPAR was not required for vitronectin adhesion of these cells (Yebra et al., 1996
A series of recent studies by Blasi and colleagues have defined a pathway of urokinase- and uPAR-mediated chemotaxis (Fazioli et al., 1997
; Degryse et al., 1999
). Urokinase stimulates chemotaxis of uPAR-bearing cells in a pathway involving Src kinase activation and sensitive to heterotrimeric G protein inactivation with pertussis toxin. The requirements for FAK and Src kinase activation for this migration favor integrin activation as a critical feature of urokinase-dependent chemotaxis. Data reported here may shed light on these observations. We find urokinase, by promoting uPAR/α3β1 interactions, promotes FAK activation and spreading of MDA-MB-231 cells on either fibronectin or collagen in a G protein-dependent manner (Figures and ). This signaling is blocked by peptides that dissociate uPAR and Gαi-3 from α3β1, increasing the possibility that urokinase is chemotactic for cells because urokinase enables ligand-dependent G protein activation through an integrin. It remains to be determined how conformational changes in uPAR or α3β1 induced by urokinase could mediate Gα or Gβγ activation. Although the mechanism is not defined, our observations are conceptually similar to recent reports that the integrin-associated protein CD47 promotes association of αvβ3 with heterotrimeric G proteins and that this is important to spreading mediated by this integrin (Frazier et al., 1999
; Green et al., 1999
). The finding of two distinct examples of coupling of integrins to heterotrimeric G proteins by integrin-associated proteins suggest this may be a common adaptive mechanism of cells to link matrix attachment to cell migration.
Prior studies have indicated that in addition to binding laminin-5, the integrin α3β1 regulates the function of other β1 integrins (DiPersio et al., 1995
; Fukushima et al., 1998
; Hodivala-Dilke et al., 1998
). Antibodies (P1B5) to α3β1 that block laminin-5 attachment promote spreading and migration of cells on Col and/or Fn (Kubota et al., 1997
; Lichtner et al., 1998
), consistent with findings reported here (Figure A). Furthermore, epithelial cells from mice deficient in α3 show altered organization of integrin focal contacts and enhanced spreading on Fn, suggesting an inhibitory role for α3β1 on Fn and Col integrin receptors (Lichtner et al., 1998
). Our finding that urokinase, mimicking P1B5, evokes α3β1-dependent signals promoting activation of several β1 integrins indicates uPA-dependent association of uPAR with α3β1 attenuates and even reverses the dominant negative function of α3β1 on other β1 integrins. This observation raises the possibility that prior observations of “integrin activation” by soluble uPAR may operate through its association with α3β1 (Aguirre Ghiso et al., 1999
). In addition, our observations may also shed some light on the possible molecular basis for such cross talk. The association of α3β1 with uPAR appears to be required for coprecipitation of Gα and Src family kinases with this integrin. Such complexes may complement the binding of the same integrin to CD151, a tetraspan family member that associates specifically with α3β1 and that has been recently linked to signaling and migration of cells via this integrin (Yauch et al., 1998
; Berditchevski and Odinstova, 1999
). Antibodies to CD151 coprecipitate uPAR in uPAR-transfected 293 cells. However, peptides α325 and M25, which disrupt uPAR/integrin interactions, have no effect on α3β1/CD151 complexes (Wei and Hemler, unpublished observations), consistent with the mapping of the interaction site between CD151 and α3β1 to the membrane proximal region of the α chain and the mapping of the uPAR/integrin interaction site to the β-propeller (Simon et al., 2000
; Yauch et al., 2000
). We postulate that multimeric complexes involving CD151, uPA/uPAR, and α3β1 have distinct signaling capacity promoting integrin signaling and migration on multiple matrix ligands for β1 integrins. How these complexes organize and whether other membrane adaptor proteins contribute importantly to their signaling function remains to be determined. It is important to reiterate that the discovery that uPAR associates preferentially with α3β1 is based on sequence homology with a previously defined integrin interaction site on CD11b/CD18 (Mac-1). The α6 amino acid sequence in the same region is also quite homologous and we show here that a α6 peptide based on this sequence also disrupts uPAR/integrin coprecipitation and uPAR-dependent adhesion, whereas peptides of the identical region of α5 (Figure A) or αv were inactive. The possible functional significance of uPAR/α6β1 complexes in cells expressing both of these receptors remains to be defined.
Finally, our observations that uPAR associates preferentially with integrin α chains mediating laminin-5 binding may provide an explanation for prior findings that uPAR colocalizes with the distribution of laminin-5 in vivo at sites of tumor cell invasion (Pike et al., 1995
). Laminin-5 is a major basement membrane matrix protein that is breeched during the invasion of metastatic cells into or out of blood vessels. The finding that uPAR associates preferentially with the laminin-5 binding β1 integrins supports the hypothesis that invasive tumor cells have exploited the advantage of coordinate signaling of integrins, proteases, and protease receptors embodied by uPAR/integrin interactions to promote invasion and metastasis. This is also supported by studies correlating uPAR expression with metastatic capacity and poor prognosis of breast cancer patients (Solberg et al., 1994
). If so, our studies identifying a critical site for interaction between uPAR and laminin-5 binding integrins may be a site for intervention in the invasive process.