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

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 (IP(3)R1) to identify IP(3)R1-associated proteins. In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein. Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed. Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1. Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1. Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647. CARP inhibits IP3 binding to IP(3)R1 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 coexpression of CARP with IP3R in Purkinje cells and its inhibitory effects on IP3 binding.