Yusheng Jiang

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca2+]o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.

Identification of an L-Phenylalanine Binding Site Enhancing The Cooperative Responses of The Calcium Sensing Receptor to Calcium

Background: The calcium-sensing receptor (CaSR) is a key mediator of Ca2+ homeostasis in vivo. Results: An l-Phe binding site at the CaSR hinge region globally enhances its cooperative activation by Ca2+. Conclusion: Communication between the binding sites for Ca2+ and l-Phe is crucial for functional cooperativity of CaSR-mediated signaling. Significance: The results provide important insights into the molecular basis of Ca2+ sensing by the CaSR. Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and l-Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The heterocommunication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events.

Application of Designed Calcium Sensors with Fast Kinetic Responses

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium store, as well as membrane calcium channels. To probe fast calcium responses in high calcium environments, there is a pressing need to develop calcium sensors to overcome the limitation of relatively slow kinetics of current GECIs with τ value around several hundred milliseconds. We have developed single green fluorescence protein-based calcium sensors, with tunable calcium binding affinity and fast kinetics. In this study, we first report our further development of a red calcium sensor using our design strategy. We then report our applications of the developed calcium sensors to monitor endoplasmic reticulum (ER) calcium release in several cell lines responding to perturbations of extracellular calcium signaling. The effects of various drugs as channel and pump inhibitors and activators have also been examined using our developed calcium sensors targeted to calcium channels in the ER membrane.

Differential Activation of Intracellular Calcium Oscillations by Multiple Calcium and L-Phenylalanine Binding Sites Located at the Extracellular Domain of Calcium-Sensing-Receptor

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSRs extracellular domain using our developed computational algorithms based on geometric factors and surface electrostatic potentials. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using both a cell population assay and single cell imaging. We then identify a potential amino acid binding site using computational methods and site-directed mutagenesis. The effect of amino acid binding in altering intracellular calcium responses, especially calcium oscillations, and its synergistic interaction with the effects of extracellular calcium on these parameters are also investigated. A common activation mechanism for CaSR and other family C GPCRs, such as mGluR1 by extracellular calcium and amino acid is been proposed. These results could have important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--one an inorganic divalent cation, the other a nutrient--and how the receptor senses these agonists in health and in disease states.