The GPI-linked uPAR (urokinase-type plasminogen activator receptor) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. 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 alpha5beta1, 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 this investigation, we used reporter mAbs and FRET analyses to show conformational changes in the alphaMbeta2 headpiece and re-orientation of its TMs when alphaMbeta2 interacts with uPAR.