Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity - Generation of a new integrin inhibitor

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alpha v beta 3. Vitronectin (VN) induces cell migration by binding to alpha v beta 3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alpha v beta 3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alpha v beta 3 and activates alpha v beta 3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alpha v beta 3 and uPAR-alpha 5 beta 1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alpha v beta 3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.