Construction and expression of an enzymatically active form of PECAM-1 containing the phosphatase domain of the protein tyrosine phosphatase, SHP-2

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a 130-kDa transmembrane glycoprotein that is expressed on the surfaces of platelets, endothelial cells, and certain leukocyte subsets. The extracellular region of PECAM-1 contains six immunoglobulin homology domains, two of which (doma ins 1 and 2) mediate PECAM-1 hemophilic interactions. Recent evidence sugge sts that a major function of the extracellular region of PECAM-1 is to dete rmine its localization within the plane of the plasma membrane. The cytopla smic domain of PECAM-1 contains an immunoreceptor tyrosine-based inhibitory motif that, upon tyrosine phosphorylation, supports recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase, SHP-2, However, neither the targets of this PECAM-1/SHP-2 complex nor the significance of localizing SHP-2 to the borders of opposing PECAM-1-expressing cells is yet known. As a first step in addressing these issues, we designed a cDNA enco ding a chimeric protein composed of the PECAM-1 extracellular domain fused to the phosphatase domain of SHP-2, which we call PECAM-1/SHP-2. When immun opurified from stably transfected HEK293 cell lines expressing this recombi nant protein, PECAM-1/PhD2 was found to possess constitutive enzymatic acti vity and appropriate border localization. This constitutively active chimer ic protein will be useful in future studies designed to define the componen ts of signal transduction pathways modulated by PECAM-1/SHP-2 signaling com plexes. (C) 2001 Academic Press.