SIGNALING PROPERTIES OF THE PTH/PTHRP RECEPTOR

我们最近鉴定了编码PTH/PTHrP受体的cDNA克隆 从大鼠、人、负鼠和小鼠细胞系以及从大鼠和人 肾组织。这些克隆提供了必要的工具研究 这些受体的结构和功能特征。当前数据为 与同一受体在骨骼和肾脏中的表达一致。 然而，Northern印迹分析显示，多个mRNA物种表明 其他具有相关序列的受体。此外，其他受体 与克隆的cdna序列同源性有限(或没有)的亚型 也可能会出现。因此，具体目标1的目标是 通过低严格率杂交分离其他受体亚型 表达克隆；并对不同受体的mRNAs进行鉴定 在Northern印迹分析中可以看到。作为基础的结构特征 受体激活细胞内效应器的能力是 2.利用分子生物学和生物化学 Tools，PTH/PTHrP受体的G蛋白偶联结构域将是 在分子水平上表现出来的。细胞质环的作用 和G蛋白中PTH/PTHrP受体的羧基末端- 将确定耦合和效应器激活。自从第PTH 受体具有激活不止一个效应分子的能力， 我们将确定这是否通过一个或多个G蛋白发生。 因此，特定目标3的目标是确定哪种G蛋白和 哪种效应分子由甲状旁腺素受体激活。自.以来 受体的数量受甲状旁腺素和甲状旁腺素受体的严格调控 受体调节的分子基础将有针对性地进行研究 4.靶细胞模型，稳定、组成性表达An 表位标记的PTH/PTHrP受体，将用于研究 不是由于转录调控而产生的受体蛋白 水平。由于几种G蛋白偶联受体的磷酸化 在受体调节中起着重要作用，磷酸化 甲状旁腺素受体通过已知的蛋白激酶和潜在的甲状旁腺素受体- 将对特定的激酶进行评估。这些分子决定因素 甲状旁腺素受体内化，与G蛋白解偶联， 退化和内化将通过删除和点来映射 突变。这些研究将提供对分子的洞察。 决定靶向甲状旁腺激素受体功能的机制 细胞。这些研究将扩大对甲状旁腺激素早期事件的了解 和PTHrP与常见受体的相互作用，并可能 可能特定地结合每种蛋白质的受体。描述了 甲状旁腺激素/甲状旁腺素受体与G蛋白偶联的特异性及其意义 分子基础，调节这些受体的机制将 两者都定义了这些受体的这些属性(S)，并将作为 这一新型G蛋白连锁受体中其他受体的模型 一家人。对甲状旁腺激素和甲状旁腺素受体功能的进一步了解将 提供对其生理角色的洞察，并与 该计划中的其他项目将导致更合理的药物设计 适用于人类疾病，特别是骨质疏松等骨质流失状态。

We have recently characterized cDNA clones encoding PTH/PTHrP receptors from rat, human, opossum and murine cell lines and from rat and human kidney tissues. These clones provide the necessary tools to study the structural and functional features of these receptors. Current data are consistent with expression of the same receptor in bone and kidney. Northern blot analyses, however, show multiple mRNA species that suggest other receptors with related sequences. Additionally, other receptor subtypes that have limited (or no) sequence homology to the cloned cDNA also may be present. Therefore, the goals of Specific Aim 1 are to isolate other receptor subtypes by low-stringency hybridization and by expression cloning; and also to characterize the different receptor mRNAs seen on Northern blot analysis. the structural features underlying the capacity of the receptor to activate intracellular effectors are the subject of specific Aim 2. Using molecular biological and biochemical tools, G protein coupling domains of the PTH/PTHrP receptor will be characterized at the molecular level. The role of the cytoplasmic loops and the carboxy-terminal tail of the PTH/PTHrP receptor in G protein- coupling and effector-activation will be determined. since the PTH receptor has the capability to activate more than one effector molecule, we will determine whether this occurs through one or multiple G proteins. therefore, the goal of specific aim 3 is to determine which G protein and which effector molecule is activated by the PTH receptor. since the number of receptors is subject to tight regulation by PTH and PTHrP the molecular basis for receptor regulation will be studied in Specific Aim 4. Models of target cells, stably and constitutively expressing an epitope-tagged PTH/PTHrP receptor, will be used to study regulation of the receptor protein that is not due to regulation at the transcriptional level. Since phosphorylation of several G protein-coupled receptors plays an essential role in receptor regulation, phosphorylation of the PTH receptor by known protein kinases and potentially by PTH receptor- specific kinase will be assessed. The molecular determinants that underlie PTH receptor internalization, uncoupling from the G proteins, degradation and internalization will be mapped by both deletion and point mutation. these studies will provide insight into the molecular mechanisms that determine the functions of the PTH receptor in target cells. These studies will expand knowledge of the early events of PTH and PTHrP interactions with common receptors, and potentially of receptors that might bind each protein specifically. Characterizing the specificity of PTH/PTHrP receptor coupling to G proteins and its molecular basis, and the mechanisms that regulate these receptors will both define these properties of these receptor(s), and will serve as models for other receptors in this novel of G protein-linked receptor family. Increased understanding of PTH and PTHrP receptor functions will provide insights into their physiological roles, and together with the other projects in this Program, will lead to more rational drug design for human disease, particularly bone-loss states such as osteoporosis.