INTERACTIONS BETWEEN GABA B AND CA2+-SENSING RECEPTORS

描述（由申请方提供）：G蛋白偶联受体超家族的C家族受体包括代谢型？-氨基丁酸（GABA）-B受体（GABA-B-Rs）、代谢型谷氨酸受体（mGluRs）和Ca 2+敏感受体（汽车）。这些受体以二聚体或多聚体形式起作用，其维持配体结合、运输和偶联至下游效应物的适当构象。功能性GABA-B-R由GABA-B-R1和R2（两种不同的基因产物）之间的异二聚体组成。汽车在靶细胞中起同二聚体的作用（即，甲状旁腺和肾），并且它们也可以与脑中的mGluR异二聚化。我们最近发现，汽车也可以形成异聚体复合物与GABA-B-R 1在HEK-293细胞转染两种受体cDNA。与汽车共表达GABA-B-R1沿着抑制CaR蛋白水平和细胞中磷脂酶C活化对细胞外[Ca 2 +]（[Ca 2 +]e）变化的反应性，从而支持GABA-B-R1通过异聚受体复合物改变CaR表达和功能的观点。这种共缔合是否发生在体内靶细胞中，很少受到关注。汽车和GABA-B-R都存在于许多组织中-脑、肾、甲状旁腺、肠、骨和软骨。在培养的小鼠生长板软骨细胞（mGPCs）中，敲除GABA-B-R1基因显著增加CaR蛋白水平和对高[Ca 2 +]e的信号应答，支持GABA-B-R1在调节mGPCs中汽车的表达和功能中的作用。我们的假设是，GABA-B-R1 s通过在两个分子之间形成异聚体复合物来调节汽车的表达和信号转导，并且这种相互作用影响靶细胞的信号应答和功能。为了验证这一假设，我们提出了以下目标。目的1：探讨汽车与GABA-B-R1之间是否存在相互作用，从而调节CaR的表达和信号转导。我们将通过（a）在HEK-293细胞中共表达GABA-B-R1与汽车，并评估它们的结合是否影响CaR蛋白的合成和/或降解并改变汽车的信号转导特性;（B）测试阻断mGPC中GABA-B-R1表达对CaR蛋白的合成和/或降解以及对CaR介导的信号转导的影响;（c）确定GABA-B-R1是否与mGPCs中的汽车共缔合;和（d）检测阻断GABA-B-R1表达对增殖、凋亡和分化标志物表达的影响--这些过程响应于mGPCs中[Ca 2 +]e的变化。目标2：通过检查GABA-B-R1基因的软骨特异性敲除对小鼠生长板软骨中的CaR表达、形态、细胞增殖、细胞凋亡和分化标志物表达的影响，确定GABA-B-R1在体内软骨中的影响。这项工作的完成将为汽车和GABA-B-R1在生理环境中如何相互作用以及这种相互作用如何影响软骨发育提供见解。汽车控制体内的矿物质平衡，而GABA-B-Rs对神经细胞功能至关重要。生长板提供启动新骨形成的细胞和蛋白质，这最终决定了我们的身高和体型。这项工作可以开辟未来的研究，以开发治疗方法来调节汽车，GABA-B-Rs或这两种受体的异聚复合物的功能。

DESCRIPTION (provided by applicant): Family C receptors of the G-protein coupled receptor superfamily include metabotropic ?-aminobutyric acid (GABA)-B receptors (GABA-B-Rs), metabotropic glutamate receptors (mGluRs), and Ca2+-sensing receptors (CaRs). These receptors function in dimeric or multimeric forms which maintain the proper conformations for ligand binding, trafficking, and coupling to downstream effectors. A functional GABA-B-R consists of a heterodimer between the GABA-B-R1 and R2, two distinct gene products. CaRs function as homodimers in target cells (i.e., parathyroid and kidney), and they also can heterodimerize with mGluRs in the brain. We recently found that CaRs can also form heteromeric complexes with GABA-B-R 1s in HEK-293 cells transfected with both receptor cDNAs. Co-expressing GABA-B-R1s along with CaRs suppressed CaR protein levels and the responsiveness of phospholipase C activation in the cells to changes in the extracellular [Ca2+] ([Ca2+]e), thus supporting the idea that GABA-B-R1s alter CaR expression and function via a heteromeric receptor complex. Whether such co-associations occur in the target cells in vivo has received little attention. Both CaRs and GABA-B-Rs are present in many tissues - brain, kidney, parathyroid, intestine, bone and cartilage. In cultured mouse growth plate chondrocytes (mGPCs), knocking out the GABA-B-R1 genes dramatically increased the level of CaR protein and signaling responses to high [Ca2+]e, supporting a role for GABA-B-R1s in regulating the expression and function of the CaRs in mGPCs. Our hypothesis is that GABA-B-R1s modulate the expression and signal transduction of CaRs by the formation of heteromeric complexes between the two molecules and that this interaction impacts on the signaling responses and function of target cells. To test this hypothesis, we propose the following aims. AIM 1: To determine whether CaRs and GABA-B-R1s interact to modulate CaR expression and signal transduction. We will address this by (a) co-expressing GABA-B-R1s with CaRs in HEK-293 cells and assessing whether their association affects the synthesis and or degradation of CaR protein and alters the signal transduction properties of CaRs; (b) testing the effects of blocking GABA-B-R1 expression in mGPCs on the synthesis and or degradation of CaR protein and on CaR-mediated signal transduction; (c) ascertaining whether GABA-B-R1s co-associate with CaRs in mGPCs; and (d) examining the effects of blocking GABA-B-R1 expression on the proliferation, apoptosis, and expression of markers of differentiation -- processes responsive to changes in the [Ca2+]e in mGPCs. AIM 2: To determine the impact of the GABA- B-R1 in cartilage in vivo by examining the effect of cartilage-specific knockout of the GABA-B-R1 genes on CaR expression, morphology, cell proliferation, apoptosis, and the expression of markers of differentiation in growth plate cartilage in mice. Completion of this work will provide insights into how CaRs and GABA-B-R1s interact in a physiological context and how such an interaction influences cartilage development. CaRs control mineral balance in the body, and GABA-B-Rs are essential to nerve cell function. The growth plate provides the cells and proteins that initiate new bone formation, which ultimately determines our height and body size. This work can open up future studies targeted to developing therapies to modulate the function of CaRs, GABA-B-Rs, or the heteromeric complexes of these two receptors.