Abstract
The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) Ca(2+) channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP(3)R forms clusters on the endoplasmic reticulum when cytosolic Ca(2+) concentration ([Ca(2+)](C)) is elevated. However, the molecular mechanism of IP(3)R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP(3)R type 1 (GFP-IP(3)R1), evoked by IP(3)-generating agonists, did not correlate with [Ca(2+)](C) but seemed compatible with cytoplasmic IP(3) concentration. IP(3) production alone induced GFP-IP(3)R1 clustering in the absence of a significant increase in [Ca(2+)](C) but elevated [Ca(2+)](C) without IP(3) production did not. Moreover IP(3)R1 mutants that do not undergo an IP(3)-induced conformational change failed to form clusters. Thus, IP(3)R clustering is induced by its IP(3)-induced conformational change to the open state. We also found that GFP-IP(3)R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.
Highlights
Many extracellular stimuli are received by trimeric G protein- or tyrosine kinase-coupled receptors and relayed to signaling molecules including phospholipase C (PLC),1 which leads to production of a second messenger, inositol 1,4,5trisphosphate (IP3) [1]
green fluorescent protein (GFP)-tagged IP3R1 Forms Clusters following Agonist Stimulation—We utilized GFP-IP3R1 to perform real time imaging of IP3 receptor (IP3R) clustering since we found that GFP-IP3R1 as well as wild-type IP3R1 formed clusters when stimulated with IP3-generating agents
A fine meshwork of IP3R1 and ER was observed before stimulation (Fig. 1A, a), but ϳ1 min after the start of ATP stimulation, GFP-IP3R1 began to form clusters (Fig. 1A, b)
Summary
Many extracellular stimuli are received by trimeric G protein- or tyrosine kinase-coupled receptors and relayed to signaling molecules including phospholipase C (PLC),1 which leads to production of a second messenger, inositol 1,4,5trisphosphate (IP3) [1]. In this study we found that the time course of clustering of green fluorescent protein-tagged IP3R type 1 (GFP-IP3R1), evoked by IP3-generating agonists, did not correlate with [Ca2؉]C but seemed compatible with cytoplasmic IP3 concentration. We found that GFP-IP3R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity.
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