Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1.
The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling. Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R 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 IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3). As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues. This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.
Hirota
Junji
J
Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Japan. hirotaj@mail.rockefeller.edu
Ando
Hideaki
H
Hamada
Kozo
K
Mikoshiba
Katsuhiko
K
D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015723:Gene Library; D007124:Immunoenzyme Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011689:Purkinje Cells / Q000378:metabolism; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques
D015220:Calcium Channels; D017931:DNA Primers; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009419:Nerve Tissue Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002256:Carbonic Anhydrases