Functional characterization of αMβ2-uPar interaction

Abstract

The cell adhesion molecule integrin αMβ2, one of the β2 integrins, associates with the urokinase-type plasminogen activator receptor (uPAR) on monocytes and neutrophils. The GPI-linked uPAR also associates with members of the βl and β3 integrins, and it modulates the ligand-binding and migration functions of these integrins. In the first part of this study, we show that co-expressing uPAR with αMβ2 in HEK-293 transfectants down-regulates the ligand-binding capacity of αMβ2 to denatured proteins, fibrinogen, and the intercellular adhesion molecule 1 (ICAM-l). Migration of transfectants on fibrinogen mediated by αMβ2 was reduced in the presence of uPAR. In addition, the constitutive ligand-binding property of an αMβ2 mutant was attenuated by its association with uPAR. Co-immunoprecipitation analyses using a panel of αMβ2-specific mAbs suggest shielding of the ligand-recognition site of αMβ2 by uPAR. Although devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules integrin α5β1, and elicits cytoplasmic signaling. The separation or re-orientation of integrin transmembrane domains (TMs) and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In the second part of this study, we used reporter mAbs and FRET analyses to show conformational changes in the αMβ2 headpiece and re-orientation of its TMs when αMβ2 interacts with uPAR.