Characterization of a high‐affinity Ins‐P₄ (inositol 1,3,4,5‐tetrakisphosphate) receptor from brain by an anti‐peptide antiserum

Abstract

From a high-affinity lns-P4 (inositol 1,3,4,5-P4) receptor purified from pig cerebellum, digested with the protease Lys C peptide sequences were obtained. Synthetic peptide-3 (19 amino acid residues) was used to generate an antiserum. Reaction of the affinity-purified antibodies with the purified pig receptor protein in ELISA or Western blot was completely inhibited by peptide-3. In cerebellar membranes, the antibodies clearly recognized the 42 kDa Ins-P4 receptor protein and two additional proteins (25 kDa, 37 kDa) which still have to be identified. The anti-peptide antibodies could selectively immunoprecipitate the Ius-P4 receptor protein. The antiserum was used (i) to demonstrate that in brain from different species (human, pig, beef, rat, mouse and sheep) a similar 42 kDa Ins-P4 receptor protein is contained, and (ii) to obtain indications for the existence of a related soluble form of the 42 kDa Ins-P4 receptor besides the membrane-associated receptor.

Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3

“…The ligand affinities for Ins(1,3,4,5)P 4 compared to the water-soluble analogues of Ptd-Ins(3,4,5)P 3 show K i values in the same range, 6.0 nm for GroPIns(3,4,5)P 3 , 3.5 nm for DiC8-Ptd-Ins(3,4,5)P 3 . These findings are consistent with our previous results using native p42 IP4 protein [17,30,31]. For rat centaurin-a [20] or the bovine PIP3BP [19], which are highly homologous to the pig p42 IP4 protein, the ligand affinity for Ins(1,3,4,5)P 4 was reported to be lower than for PtdIns(3,4,5)P 3 .…”

“…1B). Recognition by this antiserum confirms unequivocally the identity of the recombinant protein as we have demonstrated previously that the antiserum stains p42 IP4 specifically in Western blots [31] and also in immunohistochemistry of brain sections [35]. The recombinant GST±p42 IP4 fusion protein was also detectable by a rabbit polyclonal anti-GST antiserum (Fig.…”

Centaurin-α1 interacts directly with kinesin motor protein KIF13B

“…A mouse monoclonal anti-centaurin-α 1 was obtained from HyTest. Rabbit polyclonal anti-centaurin-α 1 antiserum was raised against a synthetic peptide derived from the C-terminus of centaurin-α 1 as described previously (Striker et al, 1995) and affinity purified using glutathione S-transferase (GST)-centaurin-α 1 coupled to glutathione beads followed by the adsorption with glutathione beads coupled with GST alone. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulin IgG, and glutathioneSepharose beads were purchased from Amersham Biosciences.…”

From receptor to transporter: insulin signalling to glucose transport

“…Several PIP3 binding proteins have been isolated from rat and pig brain cytosol that differ in apparent molecular size and are therefore unrelated or only distantly related to centaurin-a [97,98]. Presumably the many downstream events mediated by PI 3-kinases are each catalysed by many separate PIP3 binding proteins and therefore identifying the unique PIP3 receptor involved in regulating glucose transport may be a formidable task.…”