Determination of the Critical Region of KRAS-induced Actin-Interacting Protein for the Interaction With Inositol 1,4,5-trisphosphate Receptor

Biochem Biophys Res Commun. 2011 May 6;408(2):282-6. doi: 10.1016/j.bbrc.2011.04.016. Epub 2011 Apr 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Inositol 1,4,5-Trisphosphate Receptors / chemistry
  • Inositol 1,4,5-Trisphosphate Receptors / genetics
  • Inositol 1,4,5-Trisphosphate Receptors / metabolism*
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism*
  • Molecular Sequence Data
  • Phenylalanine / chemistry
  • Phenylalanine / genetics

Substances

  • ITPR3 protein, human
  • Inositol 1,4,5-Trisphosphate Receptors
  • KRAP protein, human
  • Membrane Proteins
  • Microfilament Proteins
  • Phenylalanine