ISOLATION OF INSP(4) AND INSP(6) BINDING-PROTEINS FROM HUMAN PLATELETS - INSP(4) PROMOTES CA2-OUT PLASMA-MEMBRANE VESICLES CONTAINING 104-KDA GAP1(IP4BP) PROTEIN( EFFLUX FROM INSIDE)

A low-density membrane fraction from human platelets contained the pla sma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP(4) and InsP(6). It was separated from the bulk of InsP(3)-re ceptor-containing membranes, but was heterogeneous, probably also cont aining surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalat e 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 vesicl es, which were shown to be inside-out plasma membrane vesicles by a bi otinylation labelling technique and selective removal of right-side-ou t plasma membrane vesicles with streptavidin-agarose. Most of the InsP (4), and all of the InsP(6), binding was present in the much denser ca lcium oxalate-loaded subfractions, which were free of GpIb. InsP(6) bi nding 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 I nsP(4) binding protein (K-D for InsP(4) = 12 nM), probably identical t o GAP1(IP4BP) described by Cullen, Hsuan, Truong, Letcher, Jackson, Da wson and Irvine [(1995) Nature (London) 376, 527-530], was also isolat ed. This InsP(4) receptor may mediate Ca2+ influx in platelets that oc curs subsequent to receptor-stimulated production of InsP(3) and unloa ding of internal Ca2+ stores.