Distinct localization and function of1,4,5IP3 receptor subtypes and the1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Abstract

Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.

Subcellular distribution of the inositol 1,4,5‐trisphosphate receptors: functional relevance and molecular determinants

“…IP 3 R1 is concentrated in a few areas of the cytoplasm whereas IP 3 R2 is homogeneously distributed in the cytoplasm with small, more intense patches near the plasma membrane (Sugiyama et al, 1994). In platelets, IP 3 R3 is expressed at very low levels, but might be located in both intracellular structures and in the plasma membrane whereas IP 3 R1 displays a widespread distribution throughout the cytoplasm and IP 3 R2 was found to be associated to both plasma membranes and inner membranes (Quinton and Dean, 1996;El-Daher et al, 2000).…”

Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted

“…Platelets are reported to express the IP $ R isoforms I, II and III [28,29]. Isoform I has been located in the intracellular membranes [28,29], in contrast to isoform III that has been found exclusively at the PM [29]. IP $ RII exhibits a dual distribution [28,29].…”

An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

“…One second messenger that has been considered as a signal for activating plasma membrane Ca 2+ entry is IP 3 acting on plasma membrane IP 3 receptors (IP 3 R). In fact, a number of studies have provided biochemical evidence for the presence of IP 3 R in the plasma membrane of cells (Guillemette et al ., 1988; Fujimoto et al ., 1992; Khan et al ., 1992a,b, 1996; Feng and Kraus‐Friedmann, 1993; Bush et al ., 1994; Mayrleitner et al ., 1995; Quinton and Dean, 1996; El‐Daher et al ., 2000; Tanimura et al ., 2000). However, there is little physiological evidence that these receptors play a significant role in Ca 2+ signaling at the plasma membrane.…”