Role of soluble adenylyl cyclase in TSH biology

甲状腺癌是最常见的内分泌恶性肿瘤，在过去的几年中有惊人的稳步上升。 好几年了。因果因素仍然定义不清。促甲状腺激素是甲状腺的主要调节器，与甲状腺有关。 生长、分化和甲状腺激素分泌。来自动物模型的证据和新的流行病学 研究表明，促甲状腺激素信号通路在肿瘤进展中起关键作用。因此，充分理解 TSHR下游的机制可能为治疗提供潜在的新靶点。 TSH通过经典的TSHR-Gs-TMAC单位合成cAMP，第二信使介导 多种荷尔蒙的作用。TmACs是一个跨膜蛋白大家族的成员，具有G- 负责转导膜受体信号的蛋白质依赖的腺酰环化酶活性。可溶的 腺酰环化酶(SAC)是腺酰环化酶家族中进化保守的成员，是一种相对保守的 新发现的活性不受G蛋白的调节，而是由碳酸氢盐和钙激活。这个 这一提议的前提是鉴定了一种新的SAC/环化酶相关蛋白1 (Cap1)/Rap1b复合体。我们将在四个综合的具体目标中解决它对TSH生物学的参与。 在目标1中，我们将使用基于FRET的cAMP传感器来评估药理学和遗传学 (shRNA/CRISPR-Cas9)干扰SAC活性的方法影响TSH介导的cAMP水平； 初步数据表明，SAC参与了TSH介导的cAMP动态调节PKA-PDE4单位。 在目标2中，我们将评估SAC-CAP1复合体在促甲状腺激素介导的G1/S进展中的作用。 药物(SAC抑制剂)和遗传(sh-SAC/shCAP1)干预抑制TSH介导的 扩散。为了评估CAP1相关SAC的特定作用，我们将使用shRNA/CRISPR-Cas9 下调细胞中Cap1的方法，我们将在感染慢病毒时进行救援检测 编码抗sh或Cas9的全长WT和突变体E171R-cap1，SAC结合缺陷。 在目标3中，我们将讨论核囊细胞池在促甲状腺激素介导的G1/S进展中的作用。我们会 使用一种新开发的光遗传工具，该工具将蓝光激活的环化酶(BPAC)融合到蓝色- 释放纳米荧光素酶(NLuc)。将bpac-nluc结构定位于不同的隔间将使我们能够 直接检测在胞核产生的cAMP是否能够在SAC下调时挽救增殖。 我们将在小鼠中通过甲状腺特异性缺失SAC来补充这些细胞研究，并通过 超声引导下甲状腺注射房室特异性SAC和BPAC-nLuc病毒悬液。 在目标#4中，我们将利用质谱学方法来鉴定新的cap1相关蛋白 用于cap1-down分析的新开发的试剂和优化的程序。 我们的长期目标是提供新的SAC-Cap1-Rap1复合体和它的 参与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.