A rise in the concentration of intracellular free calcium ([Ca2+]i) is a key signal in the initiation of a wide variety of biological processes including cell proliferation and differentiation, hormone and neurotransmitter secretion, cell motility and apoptosis. A new dimension in Ca2+ signaling has recently emerged with the identification and cloning of the G protein-coupled extracellular Ca2+-sensing receptor (CaR). The CaR is an allosteric protein that recognizes and responds to two different agonists, Ca2+ and aromatic amino acids, with the production of differential [Ca2+]i oscillations. Although the amplitude, frequency and duration of [Ca2+]i oscillations are increasingly recognized as encoding important information for a variety of biological processes, the mechanism(s) regulating the activity of the CaR and cellular responses associated to the differential [Ca2+]i oscillations it mediates remain largely undefined. The central hypothesis to be explored in this K22 proposal is that the stimulation of the CaR by aromatic amino acids or by extracellular calcium activates different signal transduction pathways leading to the production of transient or sinusoidal [Ca2+]i oscillations. Accordingly, a major objective of this proposal is to elucidate the mechanism(s) that mediates the differential [Ca2+]i oscillations initiated by the CaR. An increasing amount of experimental evidence indicates that the CaR is a signal transducer of great biological importance implicated in the control of Ca2+ homeostasis, gastric acid secretion, intestinal transport, homing of embryonic cells and in the progression and spread of colorectal, breast, ovary and parathyroid cancers. Despite the extended presence of this receptor in the gastrointestinal (Gl) tract and substantial epidemiological literature demonstrating the chemopreventive properties of Ca2+ in colon carcinogenesis, very little is known on the role of the CaR in Gl cell biology and malignancy. Therefore, a second objective of this proposal is to study the regulation and function of the CaR in normal colon-derived epithelial cells. We plan to accomplish the objectives of this application by pursuing the following Specific Aims: 1) Determine the role of transient receptor potential channel1 in CaR-mediated [Ca2+]i oscillations, 2) Characterize the mechanism(s) responsible for the generation of transient [Ca2+]i oscillations mediated by the CaR, and 3) Characterize the regulation of the CaR in normal colon-derived epithelial cells. This project will explore how two different agonists mediate the activation of unique signaling pathways by stimulating a receptor expressed along the entire Gl tract. It is our expectation that upon the completion of these studies we will have characterized the mechanism mediating production of [Ca2+]i oscillations mediated by the CaR. We also anticipate that these mechanistic studies will be extended to normal colon epithelial cells and thus provide a robust base for the formulation of an R01 award application.
