Calcium sensing receptor and scaffolds

描述(申请人提供)：钙感应受体(CAR)将细胞外钙和代谢物(ANRNA酸、多胺)的局部变化转化为细胞内信号，包括细胞代谢和分泌的急性变化，以及细胞增殖和分化的长期变化。CAR通过控制甲状旁腺甲状旁腺激素的分泌和肾钙排泄/吸收的平衡，对维持全身钙水平至关重要。CAR信号的细胞特异性变异可能部分是由于CAR与支架蛋白的相互作用(S)以及细胞特异性信号复合体的形成。细胞骨架蛋白细丝蛋白A是CAR的支架，是CAR介导的激活MARK信号和减缓CAR降解所必需的。初步数据表明，CAR与arrestins-1和-2的相互作用调节细胞内钙反应和Mark信号。为了了解蛋白质支架如何调节CAR介导的信号，我们将检验以下假设：(1)状态依赖的arrestin-1结合是否调节CAR的反应性？(2)CAR介导的Mark信号是否由细丝A和arrestin-2组织，信号通路是否包括EGF受体的反式激活？(3)细丝A是否通过保护CAR免受蛋白酶体介导的降解而起到稳定细胞CAR水平的分子护航作用？我们将使用人类CAR和突变和缺失结构，将生化方法与细胞内钙和/或共聚焦成像结合起来。拟议的实验将增加我们对蛋白质支架arrestin-1和-2以及细丝素A在增强CAR信号特异性方面的作用的理解。人类CAR的40多个突变与疾病有关，彻底改变了甲状旁腺功能亢进症的临床解剖。CAR的变构调节剂可用于治疗原发性甲状旁腺机能亢进症和甲状旁腺功能减退症、肾脏疾病引起的继发性甲状旁腺功能改变和骨质疏松。CAR刺激细胞增殖和PTHrP的分泌，提示CAR在加强恶性肿瘤的病理生理后果中的作用。CAR表达和/或急性或长期信号的失调可能部分是与支架蛋白相互作用改变的结果，这种相互作用有助于增强CAR信号的特异性和调节CAR的稳定性。了解支架在CAR功能中的作用可能为钙离子处理疾病的治疗提供新的、特定的药物干预场所。

DESCRIPTION (provided by applicant): The calcium sensing receptor (CaR) transduces local changes in extracellular calcium and metabolites (anrtino acids, polyamines) into intracellular signals, which include acute alterations in cell metabolism and secretion, and long term changes in cell proliferation and differentiation. CaR is critical to maintenance of systemic calcium levels, by controlling parathyroid PTH secretion and the balance of renal calcium excretion/resorption. Cell-specific variability in CaR signaling may in part be due to interaction(s) of CaR with scaffold proteins and the formation of cell-specific signaling complexes. The cytoskeletal protein filamin A is a scaffold for CaR, required for CaR-mediated activation of MARK signaling, and slowing CaR degradation. Preliminary data suggests CaR interactions with arrestins-1 and -2 modulate intracellular calcium responses and MARK signaling. To understand how protein scaffolds regulate CaR-mediated signaling, we will test the following hypotheses: (1) Does state-dependent arrestin-1 binding regulate CaR responsiveness? (2) Is CaR-mediated MARK signaling organized by filamin A and arrestin-2, and does the signaling pathway include transactivation of the EGF receptor? (3) Does filamin A act as a molecular escort to stabilize cellular CaR levels by protecting CaR from proteasome-mediated degradation? We will combine biochemical approaches with intracellular calcium and/or confocal imaging, using human CaR and mutant and deletion constructs. The proposed experiments will increase our understanding of the role of the protein scaffolds arrestin-1 and -2 and filamin A in enhancing the specificity of CaR signaling. More than 40 mutations in human CaR have been linked to disease, revolutionizing the clinical dissection of parathyroidisms. Allosteric modulators of CaR have clinical utility for treatment of primary hyper- and hypo- parathyroidism, secondary changes in parathyroid function resulting from renal disease, and osteoporosis. CaR stimulates cell proliferation as well as PTHrP secretion, suggesting role for CaR in potentiating the pathophysiological consequences of malignancies. Dysregulation of CaR expression and/or acute or long term signaling may, in part, be the result of altered interactions with scaffold proteins which serve to enhance CaR signaling specificity and to regulate CaR stability. Understand the role of scaffolds in CaR function may provide novel, specific sites for pharmacological intervention in treatment of calcium handling diseases.