In both nonexcitable and excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R) is the primary cytosolic target responsible for the initiation of intracellular calcium (Ca(2+)) signaling. To fulfill this function, the IP(3)R depends on interaction with accessory subunits and regulatory proteins. These include proteins that reside in the lumen of the endoplasmic reticulum (ER), such as chromogranin A and B and ERp44, and cytosolic proteins, such as neuronal Ca(2+) sensor 1, huntingtin, cytochrome c, IP(3)R-binding protein released with inositol 1,4,5-trisphosphate, Homer, and 4.1N. Specific interactions between these modulatory proteins and the IP(3)R have been described, making it clear that the controlled modulation of the IP(3)R by its binding partners is necessary for physiological cell regulation. The functional coupling of these modulators with the IP(3)R can control apoptosis, intracellular pH, the initiation and regulation of neuronal Ca(2+) signaling, exocytosis, and gene expression. The pathophysiological relevance of IP(3)R modulation is apparent when the functional interaction of these proteins is enhanced or abolished by mutation or overexpression. The subsequent deregulation of the IP(3)R leads to pathological changes in Ca(2+) signaling, signal initiation, the amplitude and frequency of Ca(2+) signals, and the duration of the Ca(2+) elevation. Consequences of this deregulation include abnormal growth and apoptosis. Complex regulation of Ca(2+) signaling is required for the cell to live and function, and this difficult task can only be managed when the IP(3)R teams up and acts properly with its numerous binding partners.
Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca 2؉ ) release channel. We performed a yeast two-hybrid screen by using the NH 2 terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca 2؉ levels, decreased endoplasmic reticulum (ER) Ca 2؉ stores, and reduced Ca 2؉ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca 2؉ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca 2؉ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca 2؉ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca 2؉ signaling associated with polycystic kidney disease.Ca 2ϩ channel ͉ polycystic kidney disease ͉ t-SNARE ͉ TRP channel A utosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of cysts, occurring over decades, in previously normal appearing kidney tubules (1). A functional hallmark of ADPKD is the loss of a calcium (Ca 2ϩ ) signal that serves to inhibit dysregulated kidney tubule cell proliferation, polarization, and secretory function. Either of two causative genes, PKD1 or PKD2, can initiate cyst formation after homozygous loss-of-function mutations, typically resulting from a combination of germline mutation on one allele followed by somatic second step mutation occurring at the level of individual cells. The respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2), form a receptor-channel complex in the membrane of the apical primary cilia in renal tubular cells (2, 3), as well as in bile and pancreatic duct cells. PC2 (TRPP2), a member of the transient receptor potential (TRP) cation channel family, is abundantly expressed in the endoplasmic reticulum (ER) membrane (4) and has been shown to function as a Ca 2ϩ release channel from intracellular stores (5, 6). Indirect evidence has led to the proposal that PC2 channel activity is required for the rise in cellular Ca 2ϩ in ciliated monolayers of cultured epithelial cells under conditions of laminar shear stress because of fluid flow (7,8). This response to flow requires Ca 2ϩ from both extracellular and ER stores (9).Although variations in the reported channel properties of PC2 exist, it is generally accepted that PC2 is a high conductance cation channel (40-177 pS) whose a...
Ca2ϩ signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP 3 )-mediated intracellular Ca 2ϩ signals, we correlated the subcellular distribution of components of the InsP 3 pathway with measurements of agonist-induced intracellular Ca 2ϩ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKI␥) and chromogranin B (CGB), proteins acting synergistically to increase InsP 3 receptor (InsP 3 R) activity and sensitivity. In contrast, Ca 2ϩ pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum as well as buffers that antagonize the rise in intracellular Ca 2ϩ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKI␥ or disrupting the CGB-InsP 3 R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca 2ϩ signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca 2ϩ signaling in different types of neurons.
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