Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors

Three isoforms of the inositol 1,4,5-trisphosphate ( IP3) receptor ( IP3R), IP(3)R1, IP(3)R2, and IP(3)R3, have different IP3-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP3R types exhibited K-d values of 49.5 +/- 10.5, 14.0 +/- 3.5, and 163.0 +/- 44.4 nM, which are close to the intrinsic IP3-binding affinity previously estimated from the analysis of full-length IP(3)Rs. In contrast, residues 224-604 of IP(3)R1 and IP(3)R2 and residues 225-604 of IP(3)R3, which contain the IP3-binding core domain but not the suppressor domain, displayed an almost identical IP3-binding affinity with a K-d value of similar to 2 nM. Addition of 100-fold excess of the suppressor domain did not alter the IP3-binding affinity of the IP3-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP3-binding core domain from different isoforms exhibited IP3-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP3-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP3-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP3-binding affinity of IP(3)Rs is specifically tuned through the intramolecular attenuation of IP3-binding affinity of the IP3-binding core domain by the aminoterminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP3R isoforms originates from at least the structural difference identified on the suppressor domain.