Characterization of binding activity between nuclear factor of activated T cells and calcineurin by amplified luminescent proximity homogeneous assay

For quantitative evaluation of the relationship between biological binding partners, including protein-protein interactions, a novel analyzing system, amplified luminescent proximity homogeneous assay (ALPHA), has been developed. We here employed ALPHA for accurate assessment of the binding properties between nuclear factor of activated T cells 1(NFAT1) and calcineurin (CN), which is essential for Ca2+-dependent regulation of immune responses. A recombinant protein of the Ca2+ regulatory domain (CRD) of NFAT1 was prepared and its binding activity with biotinylated CN was determined by ALPHA (K-d=0.20 mu M). The contribution of each CN-binding component involved in the CRD of NFAT1 to CN/NFAT1 binding was next examined by competitive assay. Not only the whole CRD but also the N- and C-terminal CN-binding regions (CNBR1 and CNBR2, respectively) dose-dependently blocked CN/NFAT1 binding and their potency dose-dependently blocked CN/NFAT1 binding and their potency was CRD >> CNBR2 >= CNBR1. CN/NFAT1-binding properties were further characterized using short inhibitory peptides derived from NFAT1-CNBR1 as well as NAFT4-CNBR2. In conclusion, ALPHA is a useful system to analyse biological signaling cascades, due to its capability of quantitative evaluation of protein-protein interactions. (c) 2006 Elsevier B.V. All rights reserved.