Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Abstract

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP 3 ), which operates as an InsP 3 -gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (Fc⑀R1) leads to activation of phospholipase C ␥ isoforms via tyrosine kinase-and phosphatidylinositol 3-kinase-dependent pathways, release of InsP 3 -sensitive intracellular Ca 2ϩ stores, and a sustained phase of Ca 2ϩ influx. These events are accompanied by a redistribution of type II InsP 3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP 3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP 3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations Ͼ1 M, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP 3 receptor aggregation may be a characteristic cellular response to Ca 2ϩ -mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4 -2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36 M3R cells. Stimulation of these three cell types leads to a reduction in InsP 3 receptor levels only in AR4 -2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP 3 receptors may contribute to the previously observed changes in affinity for InsP 3 in the presence of elevated Ca 2ϩ and/or may establish discrete regions within refilled stores with varying capacity to release Ca 2ϩ when a subsequent stimulus results in production of InsP 3 . INTRODUCTIONCross-linking the immunoglobulin E (IgE)-primed Fc⑀ receptor 1 (Fc⑀R1) of rat basophilic leukemia (RBL-2H3) cells leads to Lyn-mediated phosphorylation of immunoreceptor tyrosine activation motifs within the cytoplasmic tails of Fc⑀R1 ␤ and ␥ subunits, followed by recruitment and activation of the tyrosine kinase Syk (reviewed in Benhamou, 1997). This initial kinase activation results in stimulation of two isoforms of phospholipase C-␥, PLC␥1 and PLC␥2, and leads to elevated levels of inositol 1,4,5-trisphosphate (InsP 3 ) that are sustained over prolonged periods (Ͼ10 -15 min) of cross-linking (reviewed in Wilson et al., 1997). Previous evidence has shown that phosphatidylinositol 3-kinase supports the activation and phosphorylation of PLC␥1 and is required for maximal InsP 3 synthesis (Barker et al., 1995 (Fasolato, et al., 1993), although there is evidence that a second Ca 2ϩ influx pathway also participates in Ca...

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

“…6). 3 These results are consistent with the results of the previous study, in which Ca 2ϩ -mobilizing stimulus-induced clustering of endogenous IP 3 R2 expressed in RBL-2H3 and AR4-2J cells and endogenous IP 3 R3 expressed in E36 M3R cells was observed (36). In the present study, we found the following points: 1) the size and shape of stimulus-induced clusters differ among the three types of IP 3 R; 2) IP 3 R2 SI m2 ϩ forms clusters even in resting cells; 3) IP 3 R2 SI m2…”

p110β and p110δ Phosphatidylinositol 3-Kinases Up-regulate FcεRI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production

“…We showed previously that the type II Ins(1,4,5)P 3 receptors of RBL cells cluster in the endoplasmic reticulum within minutes of calcium mobilization induced by antigen, inonomycin, or thapsigargin treatment (43). We speculate that this dramatic redistribution results in the formation of Ca 2ϩ pools within the ER whose efflux is limited by distance from the Ins(1,4,5)P 3 receptor clusters.…”

Derivation of Ca 2+ signals from puff properties reveals that pathway function is robust against cell variability but sensitive for control

“…Indeed, clustering in cells is a dynamic process (5,(26)(27)(28). During this control of spiking, the positive slope of the σ-T av relation and the existence of the minimal ISI guarantee that faster spiking is also more regular despite the stochastic character of spike generation.…”