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.
Yoshikawa
F
F
Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Iwasaki
H
H
Michikawa
T
T
Furuichi
T
T
Mikoshiba
K
K
D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D015544:Inositol 1,4,5-Trisphosphate; D005982:Glutathione Transferase; D014357:Trypsin