Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor - Structural and functional coupling of cleaved ligand binding and channel domains

The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel (calculated molecular mass = similar to 313 kDa/subunit). We studied struc tural and functional coupling in this protein complex by limited (controlle d) trypsinization of membrane fractions from mouse cerebellum, the predomin ant site for IP(3)R1. Mouse IP(3)R1 (mIP(3)R1) was trypsinized into five ma jor fragments (I-V) that were positioned on the entire mIP(3)R1 sequence by immune-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I-TV were derived from the N-terminal cytopl asmic region where the IP3-binding region extended over two fragments I (40 /37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the m embrane-spanning channel region. All five fragments were pelleted by centri fugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggest ed that the native mIP(3)R1-channel is an assembly of four subunits, each o f which is constituted by non-covalent interactions of five major, well fol ded structural components I-V that are not susceptible to attack by mild tr ypsinolysis. Ca2+ release experiments further revealed that even the comple tely fragmented mIP(3)R1 retained significant IP3-induced Ca2+ release acti vity. These data suggest that structural coupling among five split componen ts conducts functional coupling for IP3-induced Ca2+ release, despite the l oss of peptide linkages. We propose structural-functional coupling in the m IP(3)R1, that is neighboring coupling between components I and II for IP3 b inding and long-distant coupling between the IP3 binding region and the cha nnel region (component V) beyond trypsinized gaps for ligand gating.