Isolation of InsP(4) and InsP(6) binding proteins from human platelets: InsP(4) promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1(IP4BP) protein

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP(4) and InsP(6). It was separated from the bulk of InsP(3)-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP(4) binding sites (K-D = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP(4) (EC(50) 0.6 mu M), but not InsP(3) or InsP(6), released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin-agarose. Most of the InsP(4), and all of the InsP(6), binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP(6) binding activity was chromatographically purified as a 116 kDa protein (K-D for InsP(6) = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP(4) binding protein (K-D for InsP(4) = 12 nM), probably identical to GAP1(IP4BP) described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527-530], was also isolated. This InsP(4) receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP(3) and unloading of internal Ca2+ stores.