Cooperative formation of the ligand-binding site of the inositol 1,4,5-trisphosphate receptor by two separable domains

Limited trypsin digestion of mouse cerebellar membrane fractions leads to f ragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furu ichi, T., and Mikoshiba, K, (1999) J, Biol. Chem. 274, 316-327), Here we re port that trypsin-fragmented mouse IP(3)R1 (mIP(3)R1) retains significant i nositol 1,4,5-trisphosphate (IP,) binding activity that is comparable to th e intact receptor in affinity, capacity, and specificity. This is despite t he fact that the IP,binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fra gments at the Arg-343 and/or Arg-345, around the center of the core. Furthe rmore, we have examined whether binding activity could be complemented in v itro by mixing two distinct glutathione S-transferase (GST) fusion proteins , which were respectively composed of residues 1-343 and 341-604, almost co rresponding to two split binding components, and separately expressed in Es cherichia coil. The GST-fused residues 1-343 (GN) showed no binding affinit y for IF,, whereas the GST-fused residues 341-604 (GC) displayed weak but d efinite activity with an affinity >100-fold lower than that of the native r eceptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared, We suggest that the IP3-binding pocket consists o f two non-covalently but tightly associated structural domains each of whic h has a discrete function: the C-terminal domain alone has low affinity for IF,, whereas the N-terminal one alone is incapable of binding but is capab le of potentiating binding affinity.