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)P-3

We previously identified a 42 kDa Ins(1,3,4,5)P-4 (InsP(4)) receptor protein (p42(IP4)) in brain membranes from several species. Here the cDNA sequence of p42(IP4) 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 p42(IP4) is 45 amino acids shorter at the C-terminus than centaurin-alpha from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP(4) 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 p42(IP4) since (i) a peptide-specific antiserum recognizing specifically p42(IP4) labels the InsP(4) 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 InsP(4) 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 InsP(4) and specificity for various inositolphosphates are similar for the membrane-associated and for the soluble p42(IP4). The functional importance of p42(IP4) is highlighted by the identical affinity for InsP(4) and for phosphatidylinositol (3,4,5)P-3 (K-i = 1.6 and 0.9 nM, respectively). Thus, the InsP(4) receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP(4) or by PtdInsP(3) between membranes and cytosolic compartment. (C) 1997 Federation of European Biochemical Societies.