SYNTHESIS AND APPLICATION OF PHOTOAFFINITY ANALOGS OF INOSITOL 1,4,5-TRISPHOSPHATE SELECTIVELY SUBSTITUTED AT THE 1-PHOSPHATE GROUP

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (K(d) = 66 and 70 nM) than Ins(1,4,5)P3 (K(d) = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticlum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, K(d) = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, K(d) = 4-mu-M; Ins(1,4)P2, K(d) = = 80-mu-M]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [K(d) = 300 nM as compared with Ins(1,4,5)P3, K(d) = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1-mu-M] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 =0.3-mu-M). From the two radioactive labelled analogues ([H-3]AbaIP3 and I-125-AsaIP3) synthesized, the radioiodinated deriative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)-induced Ca2+ release from the ER of rat pancreatic acinar cells.