To define the structural determinants for inositol 1,4,5-trisphosphate (IP 3 ) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R1), we developed a means of expressing the N-terminal 734 amino acids of IP 3 R1 (T734), which contain the IP 3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP 3 as the native IP 3 R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP 3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224 -579) exhibited specific binding activity. Taken together, residues 226 -578 were sufficient and close enough to the minimum region for the specific IP 3 binding, and thus formed an IP 3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP 3 R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP 3 .Many cellular responses to hormones, neurotransmitters, growth factors, etc. are mediated by the intracellular second messenger inositol 1,4,5-trisphosphate (IP 3 or (1,4,5)IP 3 ) 1 (1).IP 3 releases Ca 2ϩ from intracellular stores by binding to the IP 3 receptor (IP 3 R) (2), which is a tetrameric IP 3 -gated Ca 2ϩ release channel (3-5). There are at least three types of IP 3 R derived from distinct genes in mammals (6 -12). Structural and functional studies on type I IP 3 R (IP 3 R1) (2749 amino acids, 313 kDa) have revealed that it is structurally divided into three parts: a large N-terminal cytoplasmic arm (83% of the receptor molecule); a putative six membrane-spanning domains clustered near the C terminus, which are thought to constitute an ion channel by forming a tetramer; and a short C-terminal cytoplasmic tail (13,14).The binding of IP 3 to this receptor purified from mouse cerebella is stoichiometric (K d ϭ ϳ100 nM, Hill coefficient ϭ ϳ1.0) (2, 15). To localize the IP 3 binding site, deletion mutagenesis studies showed that IP 3 R1 binds IP 3 within the N-terminal 650 amino acids independently of the tetramer formation (16,17). Newton et al. (18) have reported that the N-terminal 576 amino acids...

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