Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

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.

Novel Function of Phosphoinositide 3-Kinase in T Cell Ca2+ Signaling

“…To date, published data on the role of Ins(1,3,4,5)P 4 in Ca 2ϩ mobilization across plasma membranes remain inconclusive. Although several reports implicated Ins(1,3,4,5)P 4 in mediating Ca 2ϩ entry in certain types of electrically nonexcitable cells such as sea urchin eggs (42), Xenopus oocytes (43), and platelets (44), other studies indicated that Ins(1,3,4,5)P 4 did not have a significant effect, if any, on potentiating Ca 2ϩ influx in other cells like mouse lacrimal acinar cells (45) and Jurkat T cells (33). The data obtained in this study support the latter view that Ins(1,3,4,5)P 4 does not play a role in anti-CD3-or PI(3,4,5)P 3 -elicited Ca 2ϩ influx.…”

Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca2+ Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca2+Entry Mechanism

“…It was found that microinjection of Ins(1,3,4,5)P 4 into the whole cells caused [Ca 2ϩ ] i increase through a mechanism dependent on external Ca 2ϩ . In addition, several research groups have isolated an Ins(1,3,4,5)P 4 -binding protein, GAP1 IP4BP , from platelet plasma membranes (47)(48)(49)(50). However, the present study refutes the second messenger role of Ins(1,3,4,5)P 4 in regulating FceRI-mediated Ca 2ϩ inflow in mast cells.…”

Up-regulation of ras-GAP genes is reversed by a MEK inhibitor and doxorubicin in v-Ki-ras-transformed NIH/3T3 fibroblasts

“…Though roles of GAP1 are not completely clear, its distinct perinuclear localization [18] leads us to expect that GAP1 m might be involved in the transcriptional process. Physiologically, GAP1 has the G protein (Gα12)- [19] and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 )-binding sites [20], and activates inositol tetrakisphosphate (IP 4 )-gated Ca 2+ influx [21]. The same line of evidence is provided by us that bradykinin and Ins(1,3,4,5)P 4 induce continuous Ca 2+ influx in oncogenic ras-transformed NIH/3T3 fibroblast DT cells [22], predicting the up-regulated GAP1 function [23,24].…”