Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor

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 (IP3R) and is critical for the proper subcellular localization and function of IP3R. However, the molecular mechanisms underlying the regulation of IP3R by KRAP remain elusive. In this report, to determine the critical region of KRAP protein for the regulation of IP3R, we generate several mutants of KRAP and examine the association with IP3R 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 IP3R, indicating that the 19-length amino-acid residues (200–218) are essential for the association with IP3R. 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 IP3R, 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 IP3R, in which exogenous expression of full-length of KRAP properly restores the subcellular localization of IP3R, 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–IP3R complex. All these results provide insight into the understandings for the molecular mechanisms underlying the regulation of IP3R, and would reveal a potent strategy for the drug development targeting on IP3R.

► We determined the critical region of KRAP for the association with IP3R.
► Amino-terminal region of KRAP is sufficient for the association per se with IP3R.
► Two consecutive phenylalanines in KRAP are critical for the interaction with IP3R.
► KRAP is required for the proper subcellular localization of IP3R in HeLa cells.
► Carboxyl-terminal region of KRAP is also required for the localization of IP3R.