Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group

Abstract

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 (AbaIP3) 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 (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 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 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative 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)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.

Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia

“…In contrast with the low-Mr proteins labelled in pancreatic cells by 125I-labelled aminoethyl-tethered ASA-InsP3 by the Mayr group (Schafer et al, 1990), the aminopropyl-tethered 125I1 ASA-InsPJ labels the high-Mr (230000) InsP3 receptor in rat cerebellum (Mourey et al, 1991 ; the present work). Interestingly, the iodinated ASA-InsPJ (a mixture of 5-iodoand 3,5-di-iodo-ASA-InsP3) has higher affinity for the rat InsP3 receptor than does uniodinated ASA-InsP3 (Mourey et al, 1991; Marecek, 1991).…”

“…The design of InsP3-receptor photoaffinity labels has focused on modification of the 2-hydroxy (Hirata et al, 1989) or the 1phosphate (Schafer et al, 1990;Prestwich et al, 1991) group. Although steric modification of these positions lowers receptor binding affinity, specificity for the InsP3-binding site is retained.…”

A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-d-myo-inositol 3,4,5-trisphosphate

“…Functional identification of the 42 kDa protein as the highaffinity Ins(1,3,4,5)P4 receptor was not yet possible, since no covalently binding ligand for the receptor was available. Therefore an attempt was made to synthesize a photoaffinity analogue of Ins(1,3,4,5)P4, by a method similar to that already used successfully for synthesis of a photoaffinity analogue of Ins (1,4,5)P3 [20].…”

Synthesis of Modular Headgroup Conjugates Corresponding to All Seven Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation

“…A similar derivatization approach has previously proven effective in the development of tetherable analogs of inositol 1,4,5-trisphosphate (IP 3 ). [39][40][41][42] Finally, a hexyl linker was installed between the headgroup and amino moieties of 1a-g to introduce hydrophobicity and in part mimic the non-polar properties of the lipid backbone. These enantiomerically pure modular headgroup analogs were synthesized as described below.…”