A region in urokinase plasminogen receptor domain III controlling a functional association with α5β1 integrin and tumor growth

Highly expressed urokinase plasminogen activator receptor (uPAR) can interact with alpha 5 beta 1 integrin leading to persistent ERK activation and tumorigenicity. Disrupting this interaction reduces ERK activity, forcing cancer cells into dormancy. We identified a site in uPAR domain III that is indispensable for these effects. A 9-mer peptide derived from a sequence in domain III ( residues 240-248) binds purified alpha 5 beta 1 integrin. Substituting a single amino acid ( S245A) in this peptide, or in full-length soluble uPAR, impairs binding of the purified integrin. In the recently solved crystal structure of uPAR the Ser-245 is confined to the large external surface of the receptor, a location that is well separated from the central urokinase plasminogen binding cavity. The impact of this site on alpha 5 beta 1 integrin-dependent cell functions was examined by comparing cells induced to express uPAR(wt) or the uPAR(S245A) mutant. Transfecting uPARwt into cells with low endogenous levels of uPAR, inactive integrin, lowERK activity, and a dormant phenotype in vivo restores these functions and reinstates growth in vivo. In contrast, transfection of the same cells with uPAR(S245A) elicits only very small changes. Incubation of highly malignant cells with the wild-type, but not the S245A mutant peptide, disrupts the uPAR integrin interaction leading to down-regulation of ERK activity. The relevance of this binding site, and of the lateral uPAR-alpha 5 beta 1 integrin interaction, to ERK pathway activation and tumor growth implicates it as a possible specific target for cancer therapy.