Abstract：
OBJECTIVE: Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and linked symptoms to mGlu1 mutations. However, how mutations alter mGlu1 function remains unknown, as does amenability of receptor function to pharmacological rescue. Here, we explored SCA-associated mutation effects on mGlu1 cell surface expression, canonical signal transduction and allosteric ligand pharmacology.
METHODS: Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery.
RESULTS: mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1.
CONCLUSIONS: Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.
METHODS: Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery.
RESULTS: mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1.
CONCLUSIONS: Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.
摘要：
目的：代谢型谷氨酸受体1（mGlu1）是治疗包括脊髓小脑共济失调（SCAs）在内的神经退行性中枢神经系统疾病的一个有希望的靶点。临床报告已经确定了罕见SCA亚型中天然存在的mGlu1突变，并将症状与mGlu1突变相关联。然而,突变如何改变mGlu1功能仍然未知，受体功能对药理救援的适应性也是如此。这里,我们探索了SCA相关突变对mGlu1细胞表面表达的影响，规范的信号转导和变构配体药理学。
方法：正构激动剂，在表达五种突变mGlu1亚型的FlpInTrexHEK293A细胞系中,在两个功能终点(iCa2+动员和肌醇1-磷酸[IP1]积累)评估了正变构调节剂(PAMs)和负变构调节剂(NAMs)。关键药理学参数，包括配体效力，亲和力和协同性是使用激动和变形金刚的操作模型得出的。
结果：mGlu1突变体对mGlu1表达表现出不同的影响，C末端截短显著降低了表面表达。突变差异影响正位配体亲和力，变构和正构配体之间的功效和功能协同性。功能缺失突变L454F和N885del降低了正构亲和力和功效，分别。功能获得Y792C突变体mGlu1在IP1测定中显示出增强的组成活性，表现为正构激动剂活性降低。相对于野生型mGlu1，mGlu1PAM在iCa2动员中恢复了功能丧失突变的谷氨酸效力，并且mGlu1NAM在Y792C显示出增强的反向激动剂活性。
结论：总的来说，这些数据突出了mGlu1突变影响受体功能的不同机制,并显示变构调节剂可能为恢复罕见SCA亚型中mGlu1异常功能提供治疗策略.
方法：正构激动剂，在表达五种突变mGlu1亚型的FlpInTrexHEK293A细胞系中,在两个功能终点(iCa2+动员和肌醇1-磷酸[IP1]积累)评估了正变构调节剂(PAMs)和负变构调节剂(NAMs)。关键药理学参数，包括配体效力，亲和力和协同性是使用激动和变形金刚的操作模型得出的。
结果：mGlu1突变体对mGlu1表达表现出不同的影响，C末端截短显著降低了表面表达。突变差异影响正位配体亲和力，变构和正构配体之间的功效和功能协同性。功能缺失突变L454F和N885del降低了正构亲和力和功效，分别。功能获得Y792C突变体mGlu1在IP1测定中显示出增强的组成活性，表现为正构激动剂活性降低。相对于野生型mGlu1，mGlu1PAM在iCa2动员中恢复了功能丧失突变的谷氨酸效力，并且mGlu1NAM在Y792C显示出增强的反向激动剂活性。
结论：总的来说，这些数据突出了mGlu1突变影响受体功能的不同机制,并显示变构调节剂可能为恢复罕见SCA亚型中mGlu1异常功能提供治疗策略.
