Possible binding sites for inositol 1,4,5-trisphosphate in canine tracheal smooth muscle cells and rat liver cells.

To search for binding site of inositol 1,4,5-trisphosphate (InsP3) in relation to the release of Ca2+ from smooth muscle cells of the canine trachea, InsP3 coupled with p-azidobenzoyl beta-alanine (AB beta A) was synthesized for photoaffinity labelling, using N,N'-carbonyldiimidazole. With subsequent photolysis of the smooth muscle cells following incubation with this derivative (InsP3-AB beta A), the InsP3-induced Ca2+ release from saponin-permeabilized dispersed smooth muscle cells no longer occurred. The irreversible release of Ca2+ induced by InsP3-AB beta A was prevented by adding 10-fold excess amounts of InsP3. These observations strongly suggest that there is a covalent binding of InsP3-AB beta A to a specific "receptor" on the sarcoplasmic reticulum in tracheal smooth muscle cells. To determine the specific binding sites on the endoplasmic reticulum, I synthesized an isotope-labelled arylazide derivative of InsP3; InsP3 was coupled with p-[125I]azidosalicyl beta-alanine, using carbonyldiimidazole as a catalyst. I obtained three proteins with molecular weights of 44K-Da, 27K-Da and 22K-Da, which were preferentially labelled with the [125I]arylazide derivative of InsP3 (InsP3-[125I]AS beta A) in photoirradiated microsomal fractions of canine tracheal smooth muscle cells. The labelling of these proteins was partially blocked by an excess amount of unlabelled InsP3 but not by inositol 1,4-bisphosphate (Ins(1,4)P2). Similar binding proteins with molecular weights of 48K-Da, 32K-Da and 18K-Da were also labelled in the endoplasmic reticulum fraction of the rat liver, but such proteins were not evidenced in the plasma membrane fraction.