Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1

Abstract

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular IP3-gated Ca2+ channel that is located on intracellular Ca2+ stores and modulates Ca2+ signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP3R type 1 (IP3R1) to identify IP3R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP3R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP3R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP3R1. Biochemical analysis also showed that CARP is co-precipitated with IP3R1. Using deletion mutagenesis, we established that amino acids 45–291 of CARP are essential for its association with IP3R1, and that the CARP-binding site is located within the modulatory domain of IP3R1 amino acids 1387–1647. CARP inhibits IP3 binding to IP3R1 by reducing the affinity of the receptor for IP3. As reported previously, sensitivity to IP3 for IP3-induced Ca2+ release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP3R in Purkinje cells and its inhibitory effects on IP3 binding.