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J. Biol. Chem., Vol. 282, Issue 17, 12755-12764, April 27, 2007

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Molecular Basis of the Isoform-specific Ligand-binding Affinity of Inositol 1,4,5-Trisphosphate Receptors^*

Miwako Iwai, Takayuki Michikawa^¶1, Ivan Bosanac^||, Mitsuhiko Ikura^||, and Katsuhiko Mikoshiba^¶2

From the Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, the Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan, the ^¶Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan, and the ^||Division of Signaling Biology, Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada

Three isoforms of the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), IP₃R1, IP₃R2, and IP₃R3, 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 IP₃Rs. In contrast, residues 224–604 of IP₃R1 and IP₃R2 and residues 225–604 of IP₃R3, 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 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 IP₃Rs is specifically tuned through the intramolecular attenuation of IP₃-binding affinity of the IP₃-binding core domain by the amino-terminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP₃R isoforms originates from at least the structural difference identified on the suppressor domain.

Received for publication, October 18, 2006 , and in revised form, February 1, 2007.

^* This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants 17017013 and 17570128 to T. M. and Grant 15100006 to K. M.), by the Heart and Stroke Foundation of Canada (to M. I.), and by the Moritani Scholarship Foundation (to T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3, Figs. S1–S3, and an additional reference.

¹ To whom correspondence may be addressed. Tel.: 81-3-5449-5317; Fax: 81-3-5449-5420; E-mail: takamich{at}ims.u-tokyo.ac.jp. ² To whom correspondence may be addressed. Tel.: 81-3-5449-5316; Fax: 81-3-5449-5420; E-mail: mikosiba{at}ims.u-tokyo.ac.jp.

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