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 fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. 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 of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.