title: Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor.
abstract: KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous-actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization and function of IP(3)R. However, the molecular mechanisms underlying the regulation of IP(3)R by KRAP remain elusive. In this report, to determine the critical region of KRAP protein for the regulation of IP(3)R, we generate several mutants of KRAP and examine the association with IP(3)R using coimmunoprecipitation and confocal imaging assays. Coimmunoprecipitations using the deletion mutants reveal that amino-acid residues 1-218 but not 1-199 of KRAP interact with IP(3)R, indicating that the 19-length amino-acid residues (200-218) are essential for the association with IP(3)R. This critical region is highly conserved between human and mouse KRAP. Within the critical region, substitutions of two phenylalanine residues (Phe202/Phe203) in mouse KRAP to alanines result in failure of the association with IP(3)R, suggesting that the two consecutive phenylalanine residues are indispensable for the association. Moreover, the KRAP-knockdown stable HeLa cells exhibit the inappropriate subcellular localization of IP(3)R, in which exogenous expression of full-length of KRAP properly restores the subcellular localization of IP(3)R, but not the 1-218 or 1-236 mutant, indicating that the residual carboxyl-terminal region is also required for the proper subcellular localization of KRAP-IP(3)R complex. All these results provide insight into the understandings for the molecular mechanisms underlying the regulation of IP(3)R, and would reveal a potent strategy for the drug development targeting on IP(3)R.
authors.affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Machida
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Tsunoda
authors.forename: Keiko
authors.initials: K
authors.lastname: Doi
authors.forename: Takeharu
authors.initials: T
authors.lastname: Ota
authors.forename: Masahide
authors.initials: M
authors.lastname: Kuroki
authors.forename: Senji
authors.initials: S
authors.lastname: Shirasawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics
chemical_list: C506929:ITPR3 protein, human; C482653:ITPRID2 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010649:Phenylalanine