Abstract
A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-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 InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 microM), but not InsP3 or InsP6, 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 InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527-530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.