Paired Ig-like type 2 receptors ( PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory ( PILR alpha) and activating ( PILR beta) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance ( SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILR alpha can bind to CD99 with low affinity ( K-d similar to 2.2 mu M), but activating PILR beta binds with similar to 40 times lower affinity ( K-d similar to 85 mu M). In addition to our previous mutagenesis study ( Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. ( 2008) J. Immunol. 180, 1686 - 1693), the SPR analysis showed that PILR alpha can bind to each Ala mutant of the two CD99 O-glycosylated sites ( Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILR alpha binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILR beta can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILR alpha and PILR beta. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILR alpha-CD99 interaction is enthalpically driven with a large entropy loss (-T Delta S = 8.9 kcal.mol(-1)), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.