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 (domains 1 and 2) mediate PECAM-1 homophilic interactions. Recent evidence suggests that a major function of the extracellular region of PECAM-1 is to determine its localization within the plane of the plasma membrane. The cytoplasmic 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 encoding a chimeric protein composed of the PECAM-1 extracellular domain fused to the phosphatase domain of SHP-2, which we call PECAM-1/PhD2. When immunopurified from stably transfected HEK293 cell lines expressing this recombinant protein, PECAM-1/PhD2 was found to possess constitutive enzymatic activity and appropriate border localization. This constitutively active chimeric protein will be useful in future studies designed to define the components of signal transduction pathways modulated by PECAM-1/SHP-2 signaling complexes.
Noda
M
M
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI 53201, USA.
Paddock
C
C
Newman
P J
PJ
D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011993:Recombinant Fusion Proteins; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases