Role of soluble adenylyl cyclase in TSH biology

甲状腺癌是最常见的内分泌恶性肿瘤， 年因果因素仍然不明确。促甲状腺激素是甲状腺的主要调节因子，参与甲状腺功能的调节。 生长、分化和甲状腺激素分泌。来自动物模型和新流行病学的证据 研究表明TSH信号通路对于肿瘤进展是至关重要的。因此，充分了解 TSHR下游相关机制可能为治疗提供潜在的新靶点。 TSH通过典型的TSHR-Gs-tmAC单位合成cAMP，cAMP是介导细胞凋亡的第二信使。 许多激素的作用。tmAC是具有G- 蛋白依赖性腺苷酸环化酶活性，负责转导膜受体信号。可溶性 腺苷酸环化酶（adenylyl cyclase，sAC）是腺苷酸环化酶家族中进化上保守的一个成员，是一种相对保守的酶， 新发现的活性不受G蛋白调节，而是由碳酸氢盐和钙激活。的 该建议的前提是基于新的sAC/细胞周期酶相关蛋白1的鉴定 （CAP 1）/Rap 1b复合物。我们将在四个综合的具体目标中解决其在TSH生物学中的参与。 在目标#1中，我们将利用基于FRET的cAMP传感器来评估药理学和遗传学上是否 干扰sAC活性的shRNA/CRISPR-Cas9方法影响TSH介导的cAMP水平; 初步数据表明sAC参与调节PKA-PDE 4单位的TSH介导的cAMP动力学。 在目标#2中，我们将评估sAC-CAP 1复合物在TSH介导的G1/S进展中的参与。 药理学（sAC抑制剂）和遗传学（sh-sAC/shCAP 1）干预可抑制TSH介导的 增殖为了评估CAP 1相关sAC的特定作用，我们将使用shRNA/CRISPR-Cas9 方法下调细胞中的CAP 1，我们将在慢病毒感染后进行拯救试验 编码sh-或Cas9-抗性全长WT和突变体E171 R-CAP 1，sAC结合缺陷。 在目标#3中，我们将讨论TSH介导的G1/S进展中细胞核sAC池的参与。我们将 使用一种新开发的光遗传学工具，将蓝光激活的环化酶（bPAC）融合到一个蓝色的 发射纳米荧光素酶（nLuc）。将bPAC-nLuc构建体靶向不同的区室将使我们能够 直接测试细胞核中产生的cAMP是否能够在sAC下调时挽救增殖。 我们将通过小鼠中sAC的甲状腺特异性缺失和 超声引导甲状腺注射隔室特异性sAC和bPAC-nLuc病毒悬液。 在目标#4中，我们将利用质谱方法来鉴定新的CAP 1相关蛋白， 新开发的试剂和优化的CAP 1-pull down检测方案。 我们的长期目标是提供新的sAC-CAP 1-Rap 1复合物的机制细节， 促甲状腺激素生物学。

Thyroid cancer is the most common endocrine malignancy, with an alarmingly steady increase in the last few years. Causal factors remain ill-defined. TSH is the major regulator of the thyroid gland involved in thyroid growth, differentiation, and thyroid hormone secretion. Evidence from animal models and new epidemiological studies suggest that TSH signaling pathways are critical for tumor progression. Thus, fully understanding the mechanisms involved downstream of the TSHR might provide potential new targets for therapy. TSH acts via the canonical TSHR-Gs-tmAC unit to synthesize cAMP, a second messenger mediating the action of numerous hormones. tmACs are members of a large family of transmembrane proteins with a G- protein-dependent adenylyl cyclase activity responsible for transducing membrane receptor signaling. Soluble adenylyl cyclase (sAC), an evolutionarily conserved member of the adenylyl cyclase family, is a relatively newly discovered activity not modulated by G-proteins, but instead activated by bicarbonate and calcium. The premise of this proposal is based on the identification of a novel sAC/Cyclase-Associated Protein 1 (CAP1)/Rap1b complex. We will address its involvement in TSH biology in four integrated specific aims. In Aim #1 we will utilize FRET-based cAMP sensors to assess whether pharmacological and genetic (shRNA/CRISPR-Cas9) methods that interfere with sAC activity impact TSH-mediated cAMP levels; preliminary data indicate sAC is involved in TSH-mediated cAMP dynamics regulating a PKA-PDE4 unit. In Aim#2 we will assess the involvement of the sAC-CAP1 complex in TSH-mediated G1/S progression. Pharmacological (sAC inhibitors) and genetic (sh-sAC/shCAP1) intervention inhibited TSH-mediated proliferation. To assess a specific role for CAP1-associated sAC, we will use shRNA/CRISPR-Cas9 approaches to downregulate CAP1 in cells and we will perform rescue assays upon infection with lentivirus coding for sh- or Cas9-resistant full length WT and mutant E171R-CAP1, deficient in sAC binding. In Aim #3 we will address the involvement of a nuclear sAC pool in TSH-mediated G1/S progression. We will use a newly developed optogenetic tool incorporating a blue-light activated cyclase (bPAC) fused to a blue- emitting nanoluciferase (nLuc). Targeting the bPAC-nLuc construct to distinct compartments will allow us to directly test whether cAMP generated in the nucleus is able to rescue proliferation upon sAC downregulation. We will complement these cell studies by thyroid-specific deletion of sAC in mice and rescue mediated by ultrasound-guided thyroid injection of compartment-specific sAC and bPAC-nLuc viral suspensions. In Aim #4 we will exploit mass spectrometry approaches to identify new CAP1-associated proteins utilizing newly developed reagents and optimized protocols for CAP1-pull down assays. Our long-term goal is to provide mechanistic details underlying the novel sAC-CAP1-Rap1 complex and its involvement in TSH biology.