EXPRESSION AND STRUCTURE OF THE CALCIUM SENSING RECEPTOR

描述（申请人提供）：该R21资助申请是响应项目公告PA-01-127“与肾脏相关的试点和可行性项目”而提交的，其目的是开发用于钙敏感受体（CaSR）的细胞外结构域的表达和结晶的方法，CaSR是一种重要的七跨膜结构域，G蛋白偶联受体，参与控制甲状旁腺激素分泌和肾钙重吸收。他的授权申请的假设是GaSR的细胞外结构域形成同源二聚体，其中Ca结合位点沿着二聚体对的外表面存在.钙（Ca 2+）与同二聚体的结合导致同二聚体的更紧凑的结构。这种Ca 2+诱导的CaSR胞外结构域的结构变化是改变受体活性的信号，并允许调节改变甲状旁腺激素从甲状旁腺细胞释放的细胞内事件。我们的具体目标是：具体目标1：开发用于表达人CaSR的以下结构域的方法-人CaSR的胞外结构域（氨基酸Tyr 20-Glu 610）、胞外结构域的缩写形式（氨基酸Tyr 20-Glu 535）和富含半胱氨酸的结构域（氨基酸Glu 536-Glu 610）。具体目标二：检测表达的CaSR胞外结构域（胞外结构域构建体，氨基酸Tyr 20-Glu 610，以及胞外结构域构建体氨基酸Tyr 20-Glu 535的缩写形式）的Ca 2+结合特性，并确定Ca 2+结合后二级结构是否发生变化。具体目标3：确定在存在和不存在钙的情况下人CaSR胞外结构域结晶的适当条件。重要性：CaSR是一种非常重要的受体，在许多生理相关过程中发挥作用，如甲状旁腺激素的分泌和肾对Ca 2+的重吸收。受体的结构尚不清楚。如果成功，我们的实验将提供所需的新方法，使我们能够确定这种重要受体的胞外结构域的结构。它将使我们能够确定各种激动剂、拮抗剂和变构激活剂或抑制剂如何结合受体并改变受体的活性。野生型和突变型受体的结构之间的比较可能有助于理解与激活或失活突变的CaSR的个人收集的临床数据。此外，这些信息将作为涉及全长受体结构的更广泛实验的基础。

DESCRIPTION (provided by applicant): The objective of this R21 grant application, submitted in response to program announcement, PA-01-127, "Pilot and Feasibility Program Related to the Kidney", is to develop methods for the expression and crystallization of the extracellular domain of the calcium-sensing receptor (CaSR), an important seven- transmembrane domain, G-protein coupled receptor, involved in the control of parathyroid hormone secretion and renal calcium reabsorption. The hypothesis of his grant application is that the extracellular domain of the GaSR forms a homodimer in which Ca -binding sites are present along the outer surface of the dimer pair. The binding of calcium (Ca2+) to the homodimer results in a more compact structure of the homodimer. This Ca2+-induced structural change in the extracellular domain of the CaSR is the signal that alters the activity of the receptor, and allows the modulation of intra-cellular events that alter the release of parathyroid hormone from parathyroid cells. Our specific aims are: Specific Aim 1: To develop methods for the expression of the following domains of the human CaSR - the extracellular domain (amino acids Tyr20-Glu610), an abbreviated form of the extracellular domain (ammo acids Tyr20- Glu535) and the cysteine-rich domain (amino acids Glu536-Glu610) of the human CaSR. Specific Aim 2: To test the Ca2+-binding properties of the expressed extracellular domain of the CaSR (the extracellular domain construct, amino acids Tyr20-Glu610, and the abbreviated form of the extracellular domain construct amino acids Tyr20-Glu535) and determine whether changes in secondary structure occur upon the binding of Ca2+. Specific Aim 3: To determine appropriate conditions for crystallization of the extracellular domain of the human CaSR in the presence and absence of calcium. Significance: The CaSR is an exceptionally important receptor that plays a role in many physiologically relevant processes such as the secretion of parathyroid hormone and the renal reabsorption of Ca2+. The structure of the receptor is unknown. If successful, our experiments will provide required, new methods that will allow us to determine the structure of the extracellular domain of this important receptor. It will allow us to determine how various agonists, antagonists and allosteric activators or inhibitors bind to, and alter the activity of the receptor. Comparison between structures of wild-type and mutant receptors might aid understanding of the clinical data collected on individuals with either activating or inactivating mutations of the CaSR. Furthermore, this information will serve as the basis for more extensive experiments involving the structure of the full-length receptor.