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

Abstract

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.

Expression and Subcellular Localization of p42IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain

“…We identified a different InsP4 receptor protein, the 42 kDa neural protein which we purified to homogeneity from cerebellum from several species (DoniC and Reiser, 1991;Reiser eta/., 1995a). Moreover, we recently found that the purified native receptor protein from porcine cerebellum recognizes not only InsP4 (G 2.2 nM) but also the water soluble analogue of phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3) glycero-phosphatidylinositol-( 3,4,5)-trisphosphate with the same high affinity (Stricker et al, 1996).…”

Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

“…Other inositol phosphates such as Ins(1,3,4,5)P % and InsP ' are found in many cells, and it has been suggested that they are also involved in signal transduction processes, including the control of cytosolic Ca# + levels and clathrin assembly, but their specific functions are largely either unclear or unknown. InsP % is formed intracellularly by the phosphorylation of InsP $ , and InsP % binding sites have been identified in cerebellum [1,2], HL-60 cells [3], adrenal cortex [4], liver [5] and platelets [6]. A specific InsP % receptor has been purified from pig platelet plasma membranes [7], and recently its cDNA was sequenced and shown to code for a member of the GTPase-activating protein (GAP) protein family, and designated as GAP1 IP%BP [8].…”

Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance

“…have recently purified a pH 5.0-dependent Ins(1,3,4,5)P4 binding protein from pig cerebellum which appears to be a single protein of 42 kDa molecular mass. Further to this, Reiser et al (1991) have recently developed an Ins(1,3,4,5)P4 photoaffinity analogue which, when used to probe pig cerebellum under acidic conditions, binds specifically to a 42 kDa protein. In apparent contradiction to this, Theibert et al (1991) have described and purified rat cerebellar proteins which bind Ins(1,3,4,5)P4 at pH 7.4-8.3.…”