Novel Insights into the Role of Junctate in Calcium Homeostasis: Identification of Binding Domain on the InsP3R and Cellular Localization as Determined by TIRF Microscopy

Abstract

Junctate is a 33 kDa calcium binding protein with a single ER/SR membrane spanning domain expressed in excitable and non-excitable tissues. It is generated by alternative splicing from the AsH-J-J locus, which also encodes the enzyme aspartyl-s-hydroxylase, the sarcoplasimc reticulum structural protein junctin and humbug, a truncated version of aspartyl-s-hydroxylase, lacking its catalytic domain, which shares with junctate the high capacity moderate affinity Ca2+ binding domain and is over-expressed in a variety of tumours. We have previously shown that junctate forms a macromolecular complex with the InsP3R and TRPC3 channels and when transiently over-expressed in HEK293 cells, it induces extensive proliferation of the ER resulting in significantly larger and more frequent couplings between the ER and the plasma membrane. In the present work we have mapped the binding domain of the cytoplasmic NH2 terminus of junctate on the InsP3R and show that it binds to the domain involved in InsP3 binding. Such a result is supported by the finding that in the presence of a peptide encompassing the NH2 terminal domain of junctate, the Bmax for InsP3 binding is significantly higher than that obtained in the presence of an unrelated peptide. Transmission electron microscopy revealed that clones stably transfected with junctate-YFP display a significant larger number of junctions between the ER and the plasma membrane compared to control HEK293 cells and this effect is enhanced in clones that also over-express TRCP3. The size and distribution of these punctae however, was not affected by the addition of agonists mobilizing calcium via InsP3R activation.