Mutational analysis of the ligand binding site of the inositol 1,4,5- trisphosphate receptor

Fumio Yoshikawa, Mitsuhiro Morita, Toshiaki Monkawa, Takayuki Michikawa, Teiichi Furuichi, Katsuhiko Mikoshiba

Research output: Contribution to journalArticle

179 Citations (Scopus)

Abstract

To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amine acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 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 amine 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 IP3 binding, and thus formed an IP3 binding 'core.' Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amine acids of T734. We showed that single amine acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R 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 IP3.

Original languageEnglish
Pages (from-to)18277-18284
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number30
DOIs
Publication statusPublished - 1996

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Inositol 1,4,5-Trisphosphate Receptors
Amines
Binding Sites
Ligands
Mutagenesis
Acids
Escherichia coli
Inositol 1,4,5-Trisphosphate
Inositol Phosphates
Site-Directed Mutagenesis
Cerebellum
Substitution reactions
Phosphates
Amino Acids
Peptides
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutational analysis of the ligand binding site of the inositol 1,4,5- trisphosphate receptor. / Yoshikawa, Fumio; Morita, Mitsuhiro; Monkawa, Toshiaki; Michikawa, Takayuki; Furuichi, Teiichi; Mikoshiba, Katsuhiko.

In: Journal of Biological Chemistry, Vol. 271, No. 30, 1996, p. 18277-18284.

Research output: Contribution to journalArticle

Yoshikawa, Fumio ; Morita, Mitsuhiro ; Monkawa, Toshiaki ; Michikawa, Takayuki ; Furuichi, Teiichi ; Mikoshiba, Katsuhiko. / Mutational analysis of the ligand binding site of the inositol 1,4,5- trisphosphate receptor. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 30. pp. 18277-18284.
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