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 (Kd = 0.20 microM). 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 was CRD >> CNBR2 > or = CNBR1. CN/NFAT1-binding properties were further characterized using short inhibitory peptides derived from NFAT1-CNBR1 as well as NFAT4-CNBR2. In conclusion, ALPHA is a useful system to analyze biological signaling cascades, due to its capability of quantitative evaluation of protein-protein interactions.
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