Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

Area of Science:

• Cell Biology
• Molecular Biology
• Integrin Signaling

Background:

• The urokinase receptor (uPAR) mediates cellular migration via pericellular proteolysis, integrin regulation, and cell signaling upon urokinase (uPA) binding.
• uPAR was recently identified as a ligand for the beta2 integrin CD11b/CD18 (Mac-1), but its interactions with beta1 integrins were less defined.

Purpose of the Study:

• To identify the specific beta1 integrin partner involved in uPAR/uPA signaling.
• To elucidate the role of this integrin in uPAR-mediated cell adhesion and signaling pathways.

Main Methods:

• Co-complexing analysis of uPAR with alpha3beta1 integrin in transfected cells.
• Surface plasmon resonance to quantify soluble uPAR binding to recombinant alpha3beta1.
• Inhibition studies using antibodies and peptides targeting alpha3beta1.
• Cellular adhesion, spreading, and focal adhesion kinase phosphorylation assays.
• Analysis of uPA signaling in alpha3-deficient epithelial cells.

Main Results:

• uPAR predominantly complexes with alpha3beta1 integrin (>90%) in uPAR-transfected cells.
• uPA-dependent binding of soluble uPAR to recombinant alpha3beta1 was observed (K(d) < 20 nM), inhibited by an alpha3beta1-specific peptide.
• uPAR and alpha3beta1 colocalized in MDA-MB-231 cells; uPA enhanced cell spreading and FAK phosphorylation on fibronectin and collagen in an alpha3beta1-dependent manner.
• Studies in alpha3-deficient mice confirmed the critical role of alpha3beta1 in uPA signaling.

Conclusions:

• uPAR preferentially forms complexes with alpha3beta1 integrin, mediating both direct (vitronectin) and indirect (fibronectin, collagen) cell adhesion.
• The signaling pathways involving alpha3beta1 and other beta1 integrins are dependent on heterotrimeric G proteins.
• This interaction highlights a novel mechanism for uPAR in regulating cell adhesion and migration through alpha3beta1 integrin.