Agonist-evoked inositol trisphosphate receptor (IP₃R) clustering is not dependent on changes in the structure of the endoplasmic reticulum

The size and number of IP₃R (inositol 1,4,5-trisphosphate receptor) clusters located on the surface of the ER (endoplasmic reticulum) is hypothesized to regulate the propagation of Ca²⁺ waves in cells, but the mechanisms by which the receptors cluster are not understood. Using immunocytochemistry, live-cell imaging and heterologous expression of ER membrane proteins we have investigated IP₃R clustering in the basophilic cell line RBL-2H3 following the activation of native cell-surface antigen receptors. IP₃R clusters are present in resting cells, and upon receptor stimulation, form larger aggregates. Cluster formation and maintenance required the presence of extracellular Ca²⁺ in both resting and stimulated cells. Using transfection with a marker of the ER, we found that the ER itself also showed structural changes, leading to an increased number of 'hotspots', following antigen stimulation. Surprisingly, however, when we compared the ER hotspots and IP₃R clusters, we found them to be distinct. Imaging of YFP (yellow fluorescent protein)-IP₃R transfected in to living cells confirmed that IP₃R clustering increased upon stimulation. Photobleaching experiments showed that the IP ₃R occupied a single contiguous ER compartment both before and after stimulation, suggesting a dynamic exchange of IP₃R molecules between the clusters and the surrounding ER membrane. It also showed a decrease in the mobile fraction after cell activation, consistent with receptor anchoring within clusters. We conclude that IP₃R clustering in RBL-2H3 cells is not simply a reflection of bulk-changes in ER structure, but rather is due to the receptor undergoing homotypic or heterotypic protein-protein interactions in response to agonist stimulation.