cDNA cloning of porcine p42^IP4, a membrane‐associated and cytosolic 42 kDa inositol(1,3,4,5)tetrakisphosphate receptor from pig brain with similarly high affinity for phosphatidylinositol (3,4,5)P₃

Abstract

We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C‐terminus than centaurin‐α from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide‐specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane‐associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki=1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment. © 1997 Federation of European Biochemical Societies

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

“…Previously we cloned the cDNA of p42 IP4 a 42-kDa receptor protein for Ins(1,3,4,5)P 4 from pig cerebellum [17] and from rat brain [18]. Porcine and rat p42 IP4 have 374 amino acid residues.…”

“…The endogenous p42 IP4 protein in brain can be found either in the cytosolic fraction or associated with the membrane [17]. Fig.…”

“…The p42 IP4 protein purified from brain tissue binds Ins(1,3,4,5)P 4 and its analogue GroPIns(3,4,5)P 3 with similar affinity [17]. It was important to demonstrate that the recombinant p42 IP4 had retained the characteristics of this binding site.…”

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We have recently cloned the cDNA of p42 IP4 , a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hu Èlser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229±236.]

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“…p42 IP4 is expressed mainly in brain [17,18]; these proteins displaying two PH domains comprise the porcine p42 IP4 [17], the rat p42 IP4 [18], and the bovine Ptd-Ins(3,4,5)P 3 -binding protein (PIP3BP) [19] GST, glutathione-S-transferase; PI3-kinase, phosphoinositide-3-kinase; Ins, inositol; Ptd-Ins, phosphatidylinositol; PH, pleckstrin homology; PLC, phospholipase C; SH2 domain, src homology-2 domain; Btk, Bruton's tyrosine kinase; diC16, dipalmitoyl-l-a; diC8, dioctanly-l-a; GroPIns(3, 4,5)P 3, glycerophosphoinositol 3,4,5-trisphosphate; PIP3BP, Ptd-Ins(3,4,5)P 3 -binding protein; Sf9, Spodoptera frugiperda. Note: Theodor Hanck and Rolf Stricker contributed equally to this work.…”