Supplementary Material for: Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling

<b><i>Introduction:</i></b> G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can “rescue” cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. <b><i>Objective:</i></b> Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be “rescued” by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. <b><i>Methods:</i></b> Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. <b><i>Results:</i></b> LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T461^3.47I, L502^4.61P, and S616^7.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131^ECDR and I152^ECDT) or in the hinge region (E354^HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap.<b><i> Conclusions:</i></b> LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.

<b><i>引言：</i></b> G蛋白偶联受体（G protein-coupled receptor, GPCR）突变与多种疾病密切相关。大多数失活突变会导致受体错误折叠，并阻碍其向细胞膜转运。药理学分子伴侣（pharmacological chaperones）可挽救这类突变体的细胞表面表达，其机制推测为稳定新生蛋白的正确折叠。<b><i>研究目的：</i></b> 本研究旨在探究可被药理学分子伴侣LHR-Chap挽救的、在细胞内滞留的黄体生成素受体（luteinising hormone receptor, LHR）突变体的范围，并明确该变构激动剂（allosteric agonist）是否同样能够恢复存在激素结合缺陷或激素诱导信号转导缺陷的突变LHR的功能。<b><i>实验方法：</i></b> 将突变型LHR在HEK 293-T细胞中进行表达。分别通过酶联免疫吸附测定（ELISA）、放射性配体结合实验（radioligand binding）以及肌醇磷酸积累实验（inositol phosphate accumulation assays），检测细胞表面表达/定位情况、激素结合能力以及人绒毛膜促性腺激素（human chorionic gonadotropin, hCG）/LHR-Chap信号转导情况，并通过分子建模（molecular modelling）预测LHR-Chap的相互作用位点。<b><i>实验结果：</i></b> LHR-Chap可提升部分滞留突变体的细胞表面表达水平，这类突变体位于预测可通过LHR-Chap结合实现稳定的跨膜螺旋（transmembrane helices）区域。针对T461^3.47I、L502^4.61P及S616^7.46Y这3种突变体，经LHR-Chap处理后其对hCG的应答水平得到显著提升。位于激素结合位点的C131^ECDR、I152^ECDT突变体，以及位于铰链区（hinge region）的E354^HingeK突变体，其细胞表面表达水平正常，但对激素刺激的应答能力较差，然而却可对LHR-Chap介导的变构激活产生应答。<b><i>研究结论：</i></b> LHR-Chap除了能够挽救在细胞内滞留的LHR突变体的细胞表面表达之外，还可恢复那些细胞表面表达正常但存在激素结合与信号转导缺陷的突变受体的功能。本次研究证实，失活突变所导致的LHR功能异常的多个环节均可被挽救，这为治疗由GPCR突变引发的各类疾病提供了极具前景的潜在治疗策略。