沉浸式翻译Functional characterization of the ADGRG2 K990E mutation linked to CBAVD pathogenesis
与CBAVD发病机制相关的ADGRG2 K990E突变的功能表征
Abstract
抽象
The orphan receptor adhesion G protein-coupled receptor G2 (ADGRG2) has been considered an important genetic factor contributing to congenital bilateral absence of the vas deferens (CBAVD) pathogenesis. Several mutations of ADGRG2 have been isolated from CBAVD patients with their functional roles unknown. ADGRG2 belongs to adhesion G protein-coupled receptor family, which were known to activate both G protein and arrestin signaling. Downstream of ADGRG2, our previous result disclosed that the coupling of ADGRG2 to CFTR mediated by Gq and -arrestin-1 was required for fluid reabsorption and normal male fertility. We therefore characterized the signaling properties of four missense mutation C570Y, N771S, K990E or R1008Q of ADGRG2 by using pharmacological and electrophysiological approaches. We demonstrated that four missense mutations displayed impaired basal and ligand-promoted Gs Gq activity, as well as decreased ligand-promoted -arrestin-1 recruitment, which contribute to a reduction of CFTR-mediated whole cell Cl- current. Our results suggested the impairment of the pathogenetic ADGRG2 C570Y, N771S, K990E or R1008Q mutation in the G protein, arrestin and CFTR coupling maybe highly related to the pathogenetic CBAVD development.
孤儿受体粘附 G 蛋白偶联受体 G2 （ADGRG2） 被认为是导致先天性双侧输精管缺失 （CBAVD） 发病机制的重要遗传因素。已从 CBAVD 患者中分离出 ADGRG2 的几个突变，其功能作用未知。ADGRG2 属于粘附 G 蛋白偶联受体家族，已知该家族可激活 G 蛋白和抑制蛋白信号传导。在 ADGRG2 的下游，我们之前的结果揭示了 ADGRG2 与 Gq 和 -arrestin-1 介导的 CFTR 偶联是液体重吸收和正常男性生育能力所必需的。因此，我们通过药理学和电生理学方法表征了 ADGRG2 的四种错义突变 C570Y、N771S、K990E 或 R1008Q 的信号转导特性。我们证明，四个错义突变显示基础和配体促进的 Gs Gq 活性受损，以及配体促进的 -arrestin-1 募集减少，这有助于减少 CFTR 介导的全细胞 Cl - 电流。我们的结果表明，致病性 ADGRG2 C570Y、N771S、K990E 或 R1008Q 突变在 G 蛋白、抑制蛋白和 CFTR 偶联中的损伤可能与致病性 CBAVD 的发展高度相关。
Key words: adhesion GPCRs; ADGRG2; male infertility; CBAVD; Signal transduction
关键词： 粘附性 GPCRs;ADGRG2的;男性不育症;CBAVD;信号转导
Introduction
介绍
Approximately 15% of couples worldwide are plagued by the infertility problem, with male factors contributing over 50% of all cases [1, 2]. Congenital bilateral absence of vas deferens (CBAVD), which is the most prevalent form of Congenital absence of vas deferens (CAVD), is estimated to account for 1-2% of male infertility by causing obstructive azoospermia [3]. The CBAVD is characterized by abnormal development of vas deferens and epididymis as well as acidic pH and ultralow fructose concentration in semen [4]. Whereas mutations of cystic fibrosis transmembrane conductance regulator (CFTR) are responsible for 80% of CBAVD, the dysfunction of adhesion G protein-coupled receptor G2 (ADGRG2) is suggested to account for 12-20% of CBAVD in CFTR-negative patients, making it the second commonest known genetic factor contributing to CBAVD pathogenesis [5-7].
全世界约有15%的夫妇受到不孕症问题的困扰，其中男性因素占所有病例的50%以上[1,2]。先天性双侧输精管缺失（congenital lack of vas deferens， CBAVD）是先天性输精管缺失（congenital lack of vas deferens， CAVD）最普遍的形式，估计占男性不育症的1%-2%，因为它会引起梗阻性无精子症[3]。CBAVD的特征是输精管和附睾发育异常，以及精液中酸性pH值和超低果糖浓度[4]。囊性纤维化跨膜传导调节因子（CFTR）突变导致80%的CBAVD，而在CFTR阴性患者中，粘附G蛋白偶联受体G2（adgrg2）的功能障碍占CBAVD的12%-20%，使其成为导致CBAVD发病机制的第二大已知遗传因素[5-7]。
The ADGRG2, also named GPR64, belongs to the adhesion GPCRs (aGPCRs) that consist of 33 members and are featured with large N-terminal segment with adhesive potentials. Like most other aGPCRs, ADGRG2 is auto-cleaved at the GPCR proteolytic site (GPS), resulting in an N-terminal fragment that non-covalently binds to the transmembrane domain (TM)-containing C-terminal fragment. ADGRG2 was found to be specifically expressed in the efferent ductules and proximal epididymis, where it regulates fluid homeostasis and sperm maturation [8, 9]. Although the physiological importance of ADGRG2 has been emerging, the functional mechanism remains largely unknown. ADGRG2 was reported to constitutively activate Gs-cAMP pathway [10]. In the male reproduction system, our previous study has demonstrated that the functional coupling of ADGRG2 to CFTR is required for the pH and fluid homeostasis of the efferent ductules and epididymis, which is mediated by both Gq and -arrestin-1 functions [11]. These results collectively reveal the coupling of ADGRG2 to CFTR, as well as the downstream Gq and -arrestin-1 functions, are highly relevant to pH and fluid homeostasis. Conversely, dysfunction of the ADGRG2-CFTR coupling, or impaired Gq or -arrestin-1 functions may play a causal role for impairment of the pH homeostasis in ADGRG2 or CFTR mutations, which finally leads to CBAVD development.
ADGRG2，也称为GPR64，属于粘附GPCR（aGPCR），由33个成员组成，具有具有粘附电位的大N端段。与大多数其他 aGPCR 一样，ADGRG2 在 GPCR 蛋白水解位点 （GPS） 处自动裂解，产生 N 端片段，该片段与含有跨膜结构域 （TM） 的 C 端片段非共价结合。研究发现，ADGRG2在传出导管和附睾近端特异性表达，调节体液稳态和精子成熟[8,9]。尽管ADGRG2的生理重要性已经显现，但其功能机制在很大程度上仍然未知。据报道，ADGRG2组成型激活Gs-cAMP通路[10]。在雄性生殖系统中，我们之前的研究表明，ADGRG2与CFTR的功能偶联对于传出导管和附睾的pH值和体液稳态是必需的，这是由Gq和-arrestin-1功能介导的[11]。这些结果共同揭示了 ADGRG2 与 CFTR 的偶联，以及下游 Gq 和 -arrestin-1 功能与 pH 值和体液稳态高度相关。相反，ADGRG2-CFTR 偶联功能障碍或 Gq 或 -arrestin-1 功能受损可能是 ADGRG2 或 CFTR 突变中 pH 稳态受损的因果作用，最终导致 CBAVD 的发展。
In the present study, one of these missense variants, K990E (rs753694184, NM_001079858.3:c.2968A>G) located at the C-terminal of the receptor, was identified from a 32 years old CBAVD patient with normal vena spermatica, epididymis and kidneys, was selected for detailed functional characterization. By employing pharmacological and electrophysiological approaches, we studied the signaling profiles of the K990E mutant and further characterized the functional coupling between ADGRG2 and CFTR. We demonstrated the functional deficiency of K990E mutant in Gs and Gq signaling as well as ligand-promoted -arrestin-1 recruitment. Moreover, compared with WT ADGRG2, the K990E mutant leads to decreased CFTR-dependent outwardly rectifying whole-cell Cl- current.
在本研究中，从一名 32 岁的 CBAVD 患者中鉴定出其中一种错义变体 K990E （rs753694184， NM_001079858.3：c.2968A>G） 位于受体的 C 末端，用于详细的功能表征。通过采用药理学和电生理学方法，我们研究了K990E突变体的信号传导谱，并进一步表征了ADGRG2和CFTR之间的功能偶联。我们证明了 K990E 突变体在 Gs 和 Gq 信号转导以及配体促进的 -arrestin-1 募集中的功能缺陷。此外，与 WT ADGRG2 相比，K990E 突变体导致 CFTR 依赖性向外整流全细胞 Cl - 电流降低。
Materials and Methods
材料与方法
Constructs
构建
The ADGRG2 full-length (ADGRG2FL) plasmid was gifted by Professor Xu Z. G. at Shandong University School of Life Sciences. The ADGRG2FL and ADGRG2 N-terminal truncation (ADGRG2) gene was cloned into pcDNA3.1 expression vector with an N-terminal Flag tag. The mutants ADGRG2FL-K99E and ADGRG2-K990E were cloned using a QuikChange Mutagenesis Kit (Stratagene) with the following primers: forward, CAATGCATATGTTTAGTGACGAAGAGGACTCCTGCAATG; and reverse, CATTGCAGGAGTCCTCTTCGTCACTAAACATATGCATTG, and verified by DNA sequencing.
ADGRG2全长（ADGRG2FL）质粒由山东大学生命科学学院的徐志刚教授捐赠。将 ADGRG2FL 和 ADGRG2 N 端截断 （ADGRG2） 基因克隆到带有 N 端 Flag 标签的 pcDNA3.1 表达载体中。使用QuikChange诱变试剂盒（Stratagene）克隆突变体ADGRG2FL-K99E和ADGRG2-K990E，引物如下：正向，CAATGCATATGTTTAGTGACGAAGAGGACTCCTGCAATG;反向，CATTGCAGGAGTCCTCTTCGTCACTAAACATATGCATTG，并通过DNA测序验证。
Cell culture and transfection
细胞培养和转染
HEK293 cells were cultured at 37 ℃ and 5% CO2 in DMEM with 10% FBS and penicillin (100 IU/ml)/streptomycin (100 mg/ml). The cells were transfected with different constructs of ADGRG2 (ADGRG2-FL-WT, ADGRG2-FL-K990E, ADGRG2-WT or ADGRG2-K990E) plasmids using Lipofectamine 2000 (Invitrogen).
将HEK293细胞在37°C和5%CO 2 的DMEM中用10%FBS和青霉素（100IU / ml）/链霉素（100mg / ml）培养。使用 Lipofectamine 2000 （Invitrogen） 用不同的 ADGRG2 构建体（ADGRG2-FL-WT、ADGRG2-FL-K990E、ADGRG2-WT 或 ADGRG2-K990E）质粒转染细胞。
Measurement of cell surface receptor expression by ELISA
通过ELISA测定细胞表面受体表达
HEK293 cells were transfected with plasmid encoding WT or K990E ADGRG2. After transfection for 48 hours, the cells were fixed in 4% paraformaldehyde for 5 min. Next, the cells were blocked with 5% BSA for 1 hour at room temperature. Cells with over-expressing WT or ADGRG2-K990E were incubated with anti-Flag M2 monoclonal antibody (Sigma-Aldrich, 1:10000) for 1 hour at room temperature, and followed by incubation with an anti-mouse HRP conjugated antibody (1:2000) for another 1 hour at room temperature. After washing, the HRP activity was detected by incubating cells with TMB (3,3’,5,5’-tetramethylbenzidine) solution (EMD Millipore, Billenca, MA, USA). 0.25M HCl was added into the solution to stop the reaction and the absorbance was measured at 450 nm using an Infinite M200 PRO microplate reader (Tecan).
用编码 WT 或 K990E ADGRG2 的质粒转染 HEK293 细胞。转染 48 小时后，将细胞固定在 4% 多聚甲醛中 5 分钟。接下来，在室温下用 5% BSA 封闭细胞 1 小时。将过表达WT或ADGRG2-K990E的细胞与抗Flag M2单克隆抗体（Sigma-Aldrich，1：10000）在室温下孵育1小时，然后与抗小鼠HRP偶联抗体（1：2000）在室温下再孵育1小时。洗涤后，通过用TMB（3,3'，5,5'-四甲基联苯胺）溶液（EMD Millipore，Billenca，MA，USA）孵育细胞来检测HRP活性。将0.25M HCl加入溶液中以停止反应，并使用Infinite M200 PRO酶标仪（Tecan）在450nm处测量吸光度。
Whole-cell patch-clamp recording
全细胞膜片钳记录
To detect the effect of K990E mutation on ADGRG2 mediated CFTR current. HEK293 cells were transfected with ADGRG2-WT or ADGRG2-K990E with or without CFTR plasmids. Before the patch-clamp recording, the cells were cultured on coverslips. The ionic current was recorded with a data acquisition system (Patchmaster, HEKA) and an amplifier (EPC10, HEKA). Whole-cell Cl- current measurements are detected when the cells were immersed in a buffer solution with a pH of 7.4 and an osmolarity of 310 (5 mM KCl, 2.5 mM CaCl2, 130 mM NaCl, 20 mM HEPES and1 mM MgCl2) and the pipette was filled with a buffering solution with a pH of 7.2 and an osmolarity of 290 (10 mM Hepes, 10 mM EGTA, 2 mM MgATP, 101 mM CsCl, 5.8 mM glucose, 20 mM TEACl and 2 mM MgCl2). The whole-cell current of over-expressing ADGRG2-WT or ADGRG2-K990E with or without CFTR was measured by voltage-clamping command voltages from -100 mV to +100 mV in 20 mV increments.
检测 K990E 突变对 ADGRG2 介导的 CFTR 电流的影响。用 ADGRG2-WT 或 ADGRG2-K990E 转染 HEK293 细胞，有或没有 CFTR 质粒。在膜片钳记录之前，将细胞培养在盖玻片上。离子电流是用数据采集系统（Patchmaster、HEKA）和放大器（EPC10、HEKA）记录的。 - 当将细胞浸入pH值为7.4、渗透压为310（5mM KCl、2.5 mM CaCl 2 、130 mM NaCl、20 mM HEPES和1 mM MgCl 2 ）的缓冲溶液中，并在移液管中填充pH值为7.2、渗透压为290（10 mM Hepes， 10 mM EGTA、2 mM MgATP、101 mM CsCl、5.8 mM 葡萄糖、20 mM TEACl 和 2 mM MgCl 2 ）。通过-100 mV至+100 mV的电压钳位命令电压以20 mV的增量测量过表达ADGRG2-WT或ADGRG2-K990E的全电池电流，无论是否具有CFTR。
GloSensor cAMP assay
GloSensor cAMP检测
HEK293 cells were co-transfected with the receptor (ADGRG2-WT or ADGRG2-K990E) plasmids and the GloSensor plasmid incubated for 24 h at 37oC in 5% CO2. Next, the cells were seeded into 96-well plates at a cell density of 20,000 cells/well and incubated at 37℃ in 5% CO2 for another 24 hours, and then pre-incubated with 100 μl of serum-free medium containing 2% GloSensor cAMP reagent (Promega, Madison, WI) for 2 h. The cAMP signal was directly measured using a Mithras LB 940 multimode microplate reader (Berthold Technologies).
将HEK293细胞与受体（ADGRG2-WT或ADGRG2-K990E）质粒共转染，并将GloSensor质粒在37 o °C的5%CO 2中孵育24小时。接下来，将细胞接种到细胞密度为20,000个细胞/孔的96孔板中，并在37°C的5%CO 2 中再孵育24小时，然后用含有2%GloSensor cAMP试剂（Promega，Madison，WI）的100μl无血清培养基预孵育2小时。使用Mithras LB 940多功能酶标仪（Berthold Technologies）直接测量cAMP信号。
NFAT dual-luciferase reporter assay
NFAT双荧光素酶报告基因检测
HEK293 cells were co-transfected with WT or K990E ADGRG2, pGL4.16-NFAT luciferase, and pRL-TK Renilla (Promega) plasmids incubated for 48 h at 37oC in 5% CO2. The cells were harvested by adding 1x passive lysis buffer for 15 min at room temperature and collected by centrifugation at 12,000 rpm for 10 minutes at 4℃. The activity of NFAT-DLR was measured by a standard luciferase reporter gene assay with normalizing to Renilla luciferase activity.
将 HEK293 细胞与 WT 或 K990E ADGRG2、pGL4.16-NFAT 荧光素酶和 pRL-TK Renilla （Promega） 质粒共转染，在 37 o C 下在 5% CO 2 中孵育 48 小时。通过在室温下加入 1x 被动裂解缓冲液 15 分钟来收获细胞，并通过在 4°C 下以 12,000 rpm 离心 10 分钟收集细胞。通过标准荧光素酶报告基因测定法测量 NFAT-DLR 的活性，并归一化为肾荧光素酶活性。
BRET assay
BRET检测
HEK293 cells were transfected with βarrestin1/2-Rluc alone or together with ADGRG2β-YFP or ADGRG2β-K990E-YFP plasmids. 48 hours after transfection, the cells were re-distributed into 96-well plates with a density of 25,000 cells/well and incubated for 10 min with or without ligand stimulation at 37 ℃, the BRET signal between YFP (530/20 nM) and Rluc (485/20 nM) was measured after the addition of luciferase substrate coelenterazine-h (5μM, Promega) by a Mithras LB940 microplate reader (Berthold Technologies). The net BRET value was defined as the difference between the calculated BRET signal and the one obtained in the cells transfected with luciferase (βarrestin1/2-Rluc) alone.
单独或与 ADGRG2β-YFP 或 ADGRG2β-K990E-YFP 质粒一起转染 HEK293 细胞。转染后48小时，将细胞重新分配到密度为25,000个细胞/孔的96孔板中，并在37°C下孵育10分钟，在加入荧光素酶底物腔肠嗪-h（5μM，Promega）后，通过Mithras LB940酶标仪（Berthold Technologies）测量YFP（530/20nM）和Rluc（485/20nM）之间的BRET信号。净BRET值定义为计算出的BRET信号与单独用荧光素酶（βarrestin1/2-Rluc）转染的细胞中获得的BRET信号之间的差异。
Statistics
统计学
Data were presented as mean ± SEM from three or more independent experiments and statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc.). All experimental data were analyzed using standard parametric statistics including Student's t-test and one-way ANOVA. Statistical values are represented significant when p-value was below 0.05.
数据以来自三个或更多独立实验的平均±SEM表示，并使用GraphPad Prism 7（GraphPad Software，Inc.）进行统计分析。所有实验数据均采用标准参数统计量进行分析，包括学生t检验和单因素方差分析。当 p 值低于 0.05 时，统计值表示为显著。
Results
结果
Structural characterization of four missense mutation impairs both Gs and Gq signaling downstream of constitutive ADGRG2 activity
四种错义突变的结构表征损害了组成型 ADGRG2 活性下游的 Gs 和 Gq 信号转导
Up till now, 9 mutations of ADGRG2 have been identified from different cohorts of
到目前为止，已经从不同的队列中鉴定出 9 个 ADGRG2 突变CBAVD patients, 5 of which are nonsense or frameshift mutations that cause premature truncations of ADGRG2 and resultant activity disruption
CBAVD 患者，其中 5 例是无义突变或移码突变，可导致 ADGRG2 过早截断并导致活动中断[6, 7, 12]. In contrast, the other
.相比之下，另一个human
人4 missense mutations
错义突变 were
是 characterized with single amino acid substitution
以单氨基酸取代为特征and homologous
和同源with
跟mouse ADGRG2
小鼠 ADGRG2, C570Y/C562Y, N771S/N763S, K990E/K982E
C570Y/C562Y、N771S/N763S、K990E/K982E or R1008Q/R1000Q were located
R1008Q/R1000Q 已找到 at the
在 GPCR autoproteolysis-inducing
GPCR自蛋白水解诱导 (GAIN) domain,
（GAIN）域，Extracellular loop (
细胞外环 （ECL) or C-terminus
C-末端 of the ADGRG2
ADGRG2 的, respectively
分别 (Fig. 1A-1
1A-1型C). Whether these mutations cause loss-of-function or molecular defects remains to be explored.
这些突变是否会导致功能丧失或分子缺陷仍有待探索。 In particular,
特别ADGRG2
ADGRG2型was known to be activated by
已知被激活 a tethered agonist, and our recent study
拴系激动剂，以及我们最近的研究have
有also revealed the structural basis of
也揭示了tethered
栓 系 activation mechanism
激活机制. Interestingly
非常, we found a disulfide bond interaction between C562 and C593
我们发现 C562 和 C593 之间存在二硫键相互作用 in GAIN
在收益中 domain
域 based on the alpha fold structure of mouse
ASED on 小鼠的 alpha 折叠结构 ADGRG2, which is disrupted by C mutation to Y
ADGRG2，被 C 突变为 Y 而破坏 (Fig
（图. 1D). We
.1D）. 我们have
有define
定义d the interaction between
两者之间的相互作用stachel
斯塔切尔 peptide and ECL2
肽和ECL2 through computational simulation using the Lamarckian Genetic Algorithm (LGA) and Auto Dock 4
通过使用 Lamarckian 遗传算法 （LGA） 和 Auto Dock 4 的计算模拟 according to
根据ADGRG2-β–Gs complex
ADGRG2-β–Gs复合物 (PDB: 7WUQ)
（PDB：7WUQ）, found that
，发现a hydrophobic interaction between N763 and
N763 和*** (Fig. 1E).
（图 1E）。Firstly,
斯特利，we examin
检查ed whether
是否4 missense mutations
错义突变 affects the trafficking of ADGRG2 to
影响 ADGRG2 的贩运 the plasma
血浆 membrane.
膜。We generated
生成eight missense mutant plasmids in
8 个错义突变质粒both the full length
两者都是全长mouse
鼠ADGRG2 (
ADGRG2 （mADGRG2)
ADGRG2）or human ADGRG2
人类ADGRG2(mADGRG2)
ADGRG2）. . A Flag tag
标记标签 was added at the N-terminal
添加到 N 端子of receptors for the detection of cell
用于检测细胞的受体 surface expression using ELISA methods. Our result indicated that
使用ELISA方法进行表面表达。我们的结果表明C570Y/C562Y, N771S/N763S, K990E/K982E or R1008Q/R1000Q
C570Y/C562Y、N771S/N763S、K990E/K982E 或 R1008Q/R1000Q showed similar cell surface expression levels when compared with
与hADGRG2/mADGRG2(Supplemental Fig. 1A-1B
补充图1A-1B). Therefore, the
因此，four
四missense
错义 mutation
突变s did not affect the
不影响expression level and the
表达水平和 trafficking of ADGRG2 to
将 ADGRG2 贩运到the plasma membrane.
质膜。
ADGRG2 was known to constitutively activate both Gs and Gq signaling [10, 11]. Whereas Gq signaling is crucial for ADGRG2-mediated fluid reabsorption in the efferent ductules and sperm maturation, Gs pathway is finely tuned in these processes [11]. To thoroughly delineate the signaling profiles of 4 missense mutations, we investigated both the Gs and Gq activities downstream of the wild type ADGRG2 and mutant receptors using G protein trimer dissociation. Importantly, four pairs of disease mistranslation mutations have impaired the Gs constitutive activity of mADGRG2 or hADGRG2 (Fig.*** and Supplemental Fig. 1**). Gq dissociation essay and NFAT-dual-luciferase reporter measurement have revealed that the constitutive Gq activity downstream of K990E/K982E and R1008Q/R1000Q were significantly decreased compared with that of wild type receptor (Fig. ***). Collectively, these results indicated the functional deficiency of K990E and R1008Q mutants in both basal Gs and Gq signaling pathways but C570Y and N771S only affected the basal Gs signaling.
已知ADGRG2组成型激活Gs和Gq信号转导[10,11]。虽然Gq信号转导对于ADGRG2介导的传出导管中的液体重吸收和精子成熟至关重要，但Gs通路在这些过程中被微调[11]。为了彻底描述 4 个错义突变的信号传导谱，我们使用 G 蛋白三聚体解离研究了野生型 ADGRG2 和突变受体下游的 Gs 和 Gq 活性。重要的是，四对疾病误译突变损害了 mADGRG2 或 hADGRG2 的 Gs 组成型活性（图***和补充图 1**）。Gq解离论文和NFAT-双荧光素酶报告基因测量显示，与野生型受体相比，K990E/K982E和R1008Q/R1000Q下游的组成型Gq活性显著降低（图***）。总的来说，这些结果表明K990E和R1008Q突变体在基础Gs和Gq信号通路中均存在功能缺陷，但C570Y和N771S仅影响基础Gs信号通路。
Four missense mutations impaired VPM-p15-induced Gs or Gq activity of ADGRG2
4 个错义突变损害了 VPM-p15 诱导的 ADGRG2 的 Gs 或 Gq 活性
To further investigate the effect of ligand-induced Gs or Gq activity of four ADGRG2 missense mutations, we used a Stachel sequence derived peptidomimetic agonist (VPM-p15) of ADGRG2 developed by our laboratory to examine these mutants. Interestingly, although all of the four mutations have decreased ligand-induced cAMP accumulation in a concentration-dependent manner compared with wild type hADGRG2 or mADGRG2 (Fig. 2A-2E), there were differences in Gq dissociation signaling. Mutations of Gain domain or ECL2, which include C570Y/C562Y and N771S/N763S, did not affect the ligand promoted ADGRG2 coupling to Gq, but mutations of C-terminus, including K990E/K982E and 1008Q/R1000Q significantly impaired the efficacy of VPM-p15-induced Gq activity of ADGRG2 in a concentration-dependent manner (Fig. 2F-2G). Furthermore, we performed experiments with mutating four human ADGRG2 residues to alanine to corroborate the effect of the mutation. Mutations of C570A, N771A, K990A, R1008A of hADGRG2 significantly impaired both VPM-p15-induced cAMP accumulation and efficacy of Gq dissociation in overexpressing cells (Fig. 2A-2C and 2F). These result confirmed that the functional deficiency of K990E and R1008Q mutants in both ligand-promoted Gs and Gq coupling to ADGRG2 but C570Y and N771S only affected the ligand-induced Gs signaling.
为了进一步研究配体诱导的 Gs 或 Gq 活性对四个 ADGRG2 错义突变的影响，我们使用我们实验室开发的 ADGRG2 的 Stachel 序列衍生的拟肽激动剂 （VPM-p15） 来检查这些突变体。有趣的是，尽管与野生型hADGRG2或mADGRG2相比，所有四个突变都以浓度依赖性方式减少了配体诱导的cAMP积累（图2A-2E），但Gq解离信号传导存在差异。Gain 结构域或 ECL2 的突变（包括 C570Y/C562Y 和 N771S/N763S）不影响配体促进 ADGRG2 与 Gq 偶联，但 C 末端的突变，包括 K990E/K982E 和 1008Q/R1000Q 以浓度依赖性方式显着损害了 VPM-p15 诱导的 ADGRG2 Gq 活性的功效（图 2F-2G）。此外，我们进行了将四个人类 ADGRG2 残基突变为丙氨酸的实验，以证实突变的影响。hADGRG2 的 C570A、N771A、K990A、R1008A 突变显著损害了 VPM-p15 诱导的 cAMP 积累和过表达细胞中 Gq 解离的功效（图 2A-2C 和 2F）。这些结果证实了K990E和R1008Q突变体在配体促进的Gs和Gq与ADGRG2偶联中的功能缺陷，而C570Y和N771S仅影响配体诱导的Gs信号转导。
Four missense mutations affected the selectivity of -arrestin subtype recruitment to ADGRG2
4个错义突变影响了 -arrestin 亚型募集到 ADGRG2 的选择性
In addition to G proteins, -arrestin regulates diverse functions downstream of aGPCRs[13]. Our previous results revealed the essential role of -arrestin-1 in scaffolding the ADGRG2/Gq/CFTR signaling complex in the apical membrane of non-ciliated cells of the efferent ductules, which is necessary for maintaining the ion-water homeostasis of the male reproduction system. Herein, we directly quantified the recruitment of both subtypes of -arrestins to the WT or mutant ADGRG2 by BRET assay. Using the HEK293 cells transfected only with Rluc--arrestin as a control, we found that the K990E mutation did not significantly affect the basal BRET signal between ADGRG2β and both -arrestin subtypes (Fig. 3A-B). We further examined the agonist stimulated -arrestin recruitment to the receptor by using a Stachel sequence-derived peptide. Intriguingly, whereas the -arrestin-1 recruitment to the K990E mutant induced by ligand stimulation is dramatically decreased compared to that of wild type ADGRG2, the -arrestin-2 recruitment to the K990E is increased by approximately 25% (Fig. 3C-D). Therefore, the Glu substitution for Lys at the C-terminus of ADGRG2 differentially regulates the interaction between the receptor and -arrestin subtypes.
除G蛋白外，-arrestin还调节aGPCR下游的各种功能[13]。我们之前的研究结果揭示了 -arrestin-1 在传出导管非纤毛细胞顶膜中构建 ADGRG2/Gq/CFTR 信号复合物中的重要作用，这对于维持雄性生殖系统的离子-水稳态是必要的。在此，我们通过BRET测定直接量化了两种亚型的-arrestins向WT或突变体ADGRG2的募集。使用仅转染 Rluc--arrestin 的 HEK293 细胞作为对照，我们发现 K990E 突变对 ADGRG2β 和两种 -arrestin 亚型之间的基础 BRET 信号没有显着影响（图 3A-B）。我们进一步检查了激动剂刺激的 -抑制蛋白募集到受体，方法是使用 Stachel 序列衍生的肽。有趣的是，与野生型ADGRG2相比，配体刺激诱导的K990E突变体的-arrestin-1募集显著减少，而K990E的-arrestin-2募集增加了约25%（图3C-D）。因此，ADGRG2 C 末端 Lys 的 Glu 取代差异调节受体和 -arrestin 亚型之间的相互作用。
K990E mutation decreases CFTR-dependent outwardly rectifying whole-cell Cl- current
K990E 突变降低 CFTR 依赖性向外整流的全细胞 Cl - 电流
CFTR plays a critical role in efferent ductule fluid reabsorption by regulating Cl- and pH homeostasis. We previously showed that ADGRG2 mediate CFTR activation in non-ciliated cells through Gq-PKC pathway and -arrestin-1 scaffolding. Based on our observation of Gq and -arrestin-1 signaling deficiency of K990E mutant, we sought to explore whether the CFTR activity was affected by the ADGRG2 mutation. Whole-cell recordings and I-V analysis were performed to examine the membrane current in the cells overexpressing CFTR and WT/mutant ADGRG2. Consistent with our previous data, co-expression of ADGRG2FL and CFTR increased the amplitude and slope of the whole cell Cl- current (Fig. 4A-C). However, the current response was significantly decreased in cells transfected with CFTR and ADGRG2FL-K990E (Fig. 4A-C). Considering the similar cell surface expression levels of K990E and WT ADGRG2, the impaired CFTR activation in ADGRG2-K990E in Gs, Gq and -arrestin-1 signaling, as well as the CFTR activation and improved -arrestin-2 recruitment, may highly be correlated to the functional deficiency of the mutant receptor in male reproduction system.
CFTR 通过调节 Cl - 和 pH 稳态在传出导管液重吸收中起关键作用。我们之前表明，ADGRG2 通过 Gq-PKC 通路和 -arrestin-1 支架介导非纤毛细胞中的 CFTR 激活。基于对 K990E 突变体 Gq 和 ا ال 抑制蛋白-1 信号缺失的观察，我们试图探讨 CFTR 活性是否受 ADGRG2 突变的影响。进行全细胞记录和 I-V 分析以检查过表达 CFTR 和 WT/突变体 ADGRG2 的细胞中的膜电流。与我们之前的数据一致，ADGRG2FL和CFTR的共表达增加了整个细胞Cl - 电流的振幅和斜率（图4A-C）。然而，在用 CFTR 和 ADGRG2FL-K990E 转染的细胞中，当前反应显着降低（图 4A-C）。考虑到 K990E 和 WT ADGRG2 的细胞表面表达水平相似，ADGRG2-K990E 在 Gs、Gq 和 -arrestin-1 信号传导中 CFTR 激活受损，以及 CFTR 激活和 -arrestin-2 募集改善，可能与雄性生殖系统中突变受体的功能缺陷高度相关。
Discussion
讨论
The orphan receptor ADGRG2 is mainly distributed in the reproductive system, has been identified as an essential regulator of fluid reabsorption and sperm maturation. Recently, several ADGRG2 naturally occurring mutations have been identified from CBAVD patients as well as the infertile phenotype of the Adgrg2 knockout mice, highlighting its essential role in male fertility regulation [8, 11]. However, the functional properties of these mutations, especially nonsense mutations by single amino acid substitution, remains unknown.
孤儿受体ADGRG2主要分布在生殖系统中，已被确定为液体重吸收和精子成熟的重要调节因子。最近，从CBAVD患者中发现了几种ADGRG2自然发生的突变以及Adgrg2敲除小鼠的不育表型，突出了其在男性生育能力调节中的重要作用[8,11]。然而，这些突变的功能特性，特别是通过单氨基酸替换的无义突变，仍然未知。
In the present study, we systemically characterized the functional properties of one nonsense mutants of ADGRG2, K990E, this mutant displayed impaired constitutive Gq signaling as well as decreased ligand-induced -arrestin-1 recruitment. Not surprisingly, the CFTR-mediated whole cell Cl- current was substantially decreased in a heterologous system since the CFTR activity is mainly regulated by the Gq-PKC pathway downstream of ADGRG2 and the ADGRG2-CFTR functional complex is scaffolded by -arrestin-1 (Fig. 4E). Considering the functional importance of CFTR in the regulation of male fertility by maintaining water and pH homeostasis, we reasoned that the CFTR deficiency caused by ADGRG2 mutation is a potential mechanism underlying K990E-induced CBAVD pathogenesis. Importantly, the Adgrg2 knockout mice have normal vas deferens whereas the patient harboring K990E mutation shows congenital absence of vas deferens. This discrepancy is might due to the short life span of the mice compared to the much longer life span of the human male. Notably, the dysfunction between the ADGRG2 and CFTR or CFTR dysfunction all resulted in imbalance of the pH homeostasis in efferent ductules, which phenotype copied the CBAVD patients harboring Adgrg2 mutants. Thus, the impaired pH homeostasis and the dysfunction of the fluid absorption caused by the Adgrg2 mutation and the decreased CFTR currents may be highly correlated to the later CBAVD development.
在本研究中，我们系统地表征了 ADGRG2 的一个无义突变体 K990E 的功能特性，该突变体表现出组成型 Gq 信号传导受损以及配体诱导的 -arrestin-1 募集减少。毫不奇怪，CFTR 介导的全细胞 Cl - 电流在异源系统中显着降低，因为 CFTR 活性主要受 ADGRG2 下游的 Gq-PKC 通路调节，并且 ADGRG2-CFTR 功能复合物由 -arrestin-1 支架支撑（图 4E）。考虑到CFTR通过维持水分和pH稳态调节男性生育能力的功能重要性，我们推断ADGRG2突变引起的CFTR缺乏是K990E诱导的CBAVD发病机制的潜在机制。重要的是，Adgrg2敲除小鼠具有正常的输精管，而携带K990E突变的患者则显示先天性输精管缺失。这种差异可能是由于小鼠的寿命较短，而人类男性的寿命要长得多。值得注意的是，ADGRG2 和 CFTR 之间的功能障碍或 CFTR 功能障碍都导致传出导管中 pH 稳态失衡，其表型复制了携带 Adgrg2 突变体的 CBAVD 患者。因此，Adgrg2突变引起的pH稳态受损和液体吸收功能障碍以及CFTR电流降低可能与CBAVD后期的发展高度相关。
Our previous data revealed that specific residues in intracellular loops 2 and 3 of ADGRG2 are crucial for Gs and/or Gq coupling [11]. Herein, we showed that the K990E mutant of ADGRG2 impairs both Gs and Gq signaling, suggesting a significant role of ADGRG2 C-tail in G protein activation. However, it is worth noting that the cytoplasmic helix 8 located proximity to C-tail of class B GPCRs, which share similarity with aGPCRs, directly interacts with G subunit and contributes to Gs protein activation [14, 15]. Therefore, the mutation of K to E at the C-tail of ADGRG2 might disrupt this interaction and impair G protein activation. However, the detailed mechanism still awaits investigation and is expected to be deciphered with the future determination of the structures of the ADGRG2-transducer complexes.
我们之前的数据显示，ADGRG2 的细胞内环 2 和 3 中的特定残基对 Gs 和/或 Gq 偶联至关重要 [11]。在此，我们发现 ADGRG2 的 K990E 突变体会损害 Gs 和 Gq 信号传导，这表明 ADGRG2 C 尾在 G 蛋白激活中起重要作用。然而，值得注意的是，细胞质螺旋 8 位于 B 类 GPCR 的 C 尾附近，与 aGPCR 具有相似性，直接与 G 亚基相互作用并有助于 Gs 蛋白激活 [14， 15]。因此，ADGRG2 C 尾部 K 到 E 的突变可能会破坏这种相互作用并损害 G 蛋白活化。然而，详细的机制仍有待研究，并有望在未来确定ADGRG2-换能器复合物的结构时被破译。
One fascinating concept in GPCR-targeted therapeutic development is biased signaling, which describes the ability of the same receptor selectively initiating distinct signaling pathways when stabilized at different active conformational states [16, 17]. The K990E mutant of ADGRG2 selectively impairs G protein signaling while promotes -arrestin-2 interaction, suggesting that it is a biased mutant receptor with an active conformation distinct from WT receptor. Moreover, we revealed functional selectivity of -arrestin subtypes by K990E mutant, which showed decreased -arrestin-1 interaction but increased -arrestin-2 recruitment. Both -arrestins interact with the negatively charged residues in the cytoplasmic loop of receptor by different phosphate-binding pockets, transforming the phospho-barcode information on the receptor into distinct arrestin conformation and divergent signaling outputs [18-20]. However, the-arrestin subtypes play non-redundant roles in many aspects of GPCR signaling and functions[21]. Whereas the -arrestin-1 is critical for the functional coupling of ADGRG2 to CFTR, the -arrestin-2 remains enigmatic in epididymis and ductus deferens. Therefore, it will be of interest to investigate the functional roles of -arrestin-2-mediated signaling downstream of ADGRG2, which might provide insights for the development of novel contraceptive strategy or therapeutics targeting male infertility.
GPCR靶向治疗开发中一个有趣的概念是偏向信号转导，它描述了同一受体在不同活性构象状态稳定时选择性启动不同信号通路的能力[16,17]。ADGRG2 的 K990E 突变体选择性地损害 G 蛋白信号传导，同时促进 -arrestin-2 相互作用，表明它是一种偏向突变受体，具有不同于 WT 受体的活性构象。此外，我们揭示了 K990E 突变体对 -arrestin 亚型的功能选择性，其显示 -arrestin-1 相互作用减少，但 -arrestin-2 募集增加。两种 -抑制蛋白通过不同的磷酸盐结合口袋与受体细胞质环中带负电荷的残基相互作用，将受体上的磷酸化条形码信息转化为不同的抑制蛋白构象和不同的信号转导输出 [18-20]。然而，-arrestin亚型在GPCR信号传导和功能的许多方面起着非冗余的作用[21]。虽然 -arrestin-1 对 ADGRG2 与 CFTR 的功能偶联至关重要，但 -arrestin-2 在附睾和输精导管中仍然是个谜。因此，研究 -arrestin-2 介导的 ADGRG2 下游信号转导的功能作用将很有意义，这可能为开发针对男性不育症的新型避孕策略或治疗方法提供见解。
In conclusion, we found that the K990E mutant of ADGRG2 shows normal cell surface expression but impaired Gs and Gq signaling as well as decreased ligand-stimulated -arrestin-1 recruitment. The Gq and -arrestin-1 signaling deficiency contributes to a reduction of CFTR-mediated whole cell Cl- current, which should lead to impaired pH homeostasis and fluid resorption. This dysfunction may contribute to the CBAVD development. Our results collectively provide a detailed molecular mechanism for the dysfunction of ADGRG2 K990E mutant in G protein and arrestin signaling, which may be highly correlated to the pathogenic role of ADGRG2 K990E mutation in CBAVD.
总之，我们发现 ADGRG2 的 K990E 突变体表现出正常的细胞表面表达，但 Gs 和 Gq 信号传导受损，以及配体刺激的 -arrestin-1 募集减少。Gq 和 -arrestin-1 信号转导缺陷有助于降低 CFTR 介导的全细胞 Cl - 电流，这应导致 pH 稳态和液体吸收受损。这种功能障碍可能导致 CBAVD 的发展。我们的研究结果共同为ADGRG2 K990E突变体在G蛋白和arrestin信号传导中的功能障碍提供了详细的分子机制，这可能与ADGRG2 K990E突变在CBAVD中的致病作用高度相关。
Acknowledgements
确认
We acknowledge support from the National Key Basic Research Program of China (2018YFC1003600 to X.Y. and J.-P.S.), the National Natural Science Foundation of China (81901548 to D.-L.Z. and 81773704 to J.-P.S.), the National Science Fund for Distinguished Young Scholars (81825022 to J.-P.S.), the National Science Fund for Excellent Young Scholars (81822008 to X. Y.), the Shandong Provincial Natural Science Foundation (ZR2019BC078 to D.-L.Z., ZR2021QC111 to Y.-J.S.), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13028).
感谢国家重点基础研究计划（2018YFC1003600至X.Y.和J.-P.S.）、国家自然科学基金（81901548至D.-L.Z.和81773704至J.-P.S.）、国家杰出青年科学基金（81825022-J.-P.S.）、国家优秀青年科学基金（81822008-X.Y.）、山东省自然科学基金（ZR2019BC078-D.-L.Z.、 ZR2021QC111 至 Y.-J.S.）和大学长江学者和创新研究团队项目（IRT13028）。
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Figure legends
图例
Figure 1. The K990E mutation does not alter cell surface expression of ADGRG2. (A) Schematic representation of ADGRG2 structure the location of K990E indicated. (B-C) Cell surface expression levels of WT and K990E ADGRG2FL (B) or ADGRG2 (C) determined by ELISA assay. Cells transfected with pcDNA vector serve as the control. ***p < 0.001, overexpressing-ADGRG2FL or ADGRG2 cells were compared with control; n.s. no significant difference, K990E ADGRG2FL or ADGRG2 transfected cells were compared with corresponding WT ADRGR2 transfected cells. Values are the mean ± SEM of at least three independent experiments (n=3).
图 1.K990E 突变不会改变 ADGRG2 的细胞表面表达。（A）ADGRG2结构示意图，K990E的位置。（B-C）通过ELISA测定法测定WT和K990E ADGRG2FL（B）或ADGRG2（C）的细胞表面表达水平。用pcDNA载体转染的细胞作为对照。p < 0.001，将过表达ADGRG2FL或ADGRG2细胞与对照组进行比较;K990E ADGRG2FL 或 ADGRG2 转染细胞与相应的 WT ADRGR2 转染细胞相比差异无统计学意义。值是至少三个独立实验 （n=3） 的平均 SEM ±值。
Figure 2. The K990E mutation downregulates basal cAMP and NFAT signaling of ADGRG2. (A-B) Real-time measurement of basal cAMP accumulation of WT and K990E ADGRG2FL (A) or ADGRG2 (B) in Glosensor-expressing cells using pcDNA vector as the control. (C-D) Area under the curve (A.U.C) was calculated for (A) and (B), respectively. Data were normalized to the cAMP response in cells transfected with WT ADGRG2FL (C) or ADGRG2 (D). (E) The luciferase activity of NFAT-DLR in HEK293 cells transfected with WT or K990E ADGRG2FL. Cells transfected with pcDNA vector serve as the control. ***p < 0.001, ADGRG2FL or ADGRG2 transfected cells were compared with control; #p < 0.05, ##p < 0.01, K990E ADGRG2FL or ADGRG2 transfected cells were compared with corresponding WT ADRGR2 transfected cells. Values are the mean ± SEM of at least three independent experiments (n=3).
图2.K990E 突变下调 ADGRG2 的基础 cAMP 和 NFAT 信号转导。（A-B）以 pcDNA 载体为对照，实时测量 WT 和 K990E ADGRG2FL （A） 或 ADGRG2 （B） 在 Glosensor 表达细胞中的基础 cAMP 积累。（C-D）分别计算了（A）和（B）的曲线下面积（A.U.C）。在转染WT ADGRG2FL （C） 或 ADGRG2 （D） 的细胞中，将数据归一化为cAMP反应。（E）用WT或K990E ADGRG2FL转染的HEK293细胞中NFAT-DLR的荧光素酶活性。用pcDNA载体转染的细胞作为对照。p < 0.001，将 ADGRG2FL 或 ADGRG2 转染细胞与对照组进行比较; # p < 0.05，p ## < 0.01，将 K990E ADGRG2FL 或 ADGRG2 转染细胞与相应的 WT ADRGR2 转染细胞进行比较。值是至少三个独立实验 （n=3） 的平均 SEM ±值。
Figure 3. The K990E mutation reduces -arrestin-1 recruitment of ADGRG2. (A-B) Basal -arrestin-1 (A) and -arrestin-2 (B) recruitment of WT or K990E ADGRG2 measured by BRET assay which detects the energy transference from Rluc--arrestin to ADGRG2-YFP. Cells transfected only with Rluc--arrestin-1 or Rluc--arrestin-2 serve as control. (C-D) Ligand-induced -arrestin-1 (C) and -arrestin-2 (D) recruitment of WT or K990E ADGRG2 measured by BRET assay. **p < 0.01, WT or K990E ADGRG2-YFP transfected cells were compared with control; #p < 0.05, ##p < 0.01, n.s. no significant difference, K990E ADGRG2-YFP transfected cells were compared with WT ADGRG2-YFP transfected cells. Values are the mean ± SEM of at least three independent experiments (n=3).
图3.K990E 突变减少了 ADGRG2 的 -arrestin-1 募集。（A-B）通过BRET测定法测量WT或K990E ADGRG2的基础抑制蛋白-1（A）和抑制蛋白-2（B）募集，该测定法检测从Rluc--arrestin到ADGRG2-YFP的能量转移。仅用 Rluc--arrestin-1 或 Rluc--arrestin-2 转染的细胞作为对照。（C-D）通过BRET测定测量的配体诱导的WT或K990E ADGRG2的-arrestin-1（C）和-arrestin-2（D）募集。**p < 0.01，将WT或K990E ADGRG2-YFP转染细胞与对照组进行比较; # p < 0.05，p ## < 0.01，n.s. 无显著差异，将K990E ADGRG2-YFP转染细胞与WT ADGRG2-YFP转染细胞进行比较。值是至少三个独立实验 （n=3） 的平均 SEM ±值。
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Figure 4. The K990E mutation leads to reduction of CFTR-mediated whole cell Cl- current. (A) The whole cell Cl- currents of whole cell were recorded by voltage steps between -100 mV and +100 mV in HEK293 cells transfected with WT or K990E ADGRG2FL and/or CFTR. (B) Corresponding I-V curves of the whole cell Cl- currents recorded in (A). (C) Bar graph representation of average current densities (pA/pF) measured at 100 mV according to (A-B). (D) Corresponding bar graph of average reversal potential (Erev) (n = 3 for each condition) recorded in (A–B) and calculated Nernst potential. (E) Schematic description of the molecular mechanism underlying K990E-induced functional deficiency. *p < 0.05, ***p < 0.001, WT or K990E ADGRG2FL and CFTR co-transfected cells were compared with CFTR transfected cells; #p < 0.05, K990E ADGRG2FL transfected cells were compared with WT ADRGR2FL transfected cells; n.s. no significant difference, the Erev were compared with calculated Nernst potential. Values are the mean ± SEM of at least six independent experiments (n=6).
图4.K990E 突变导致 CFTR 介导的全细胞 Cl- 电流降低。（A） 在转染 WT 或 K990E ADGRG2FL 和/或 CFTR 的 HEK293 细胞中，通过 -100 mV 和 +100 mV 之间的电压步进记录全细胞的全细胞 Cl-电流。（B）（A）中记录的全电池Cl - 电流的对应I-V曲线。（C） 根据 （A-B） 在 100 mV 下测量的平均电流密度 （pA/pF） 的条形图表示。（D） （A-B） 中记录的平均反转电位 （Erev）（每个条件的 n = 3）的相应条形图，并计算出 Nernst 电位。（E） K990E诱导的功能缺陷分子机制的示意图描述。*p < 0.05，***p < 0.001，将 WT 或 K990E ADGRG2FL 和 CFTR 共转染细胞与 CFTR 转染细胞进行比较; # p < 0.05，将 K990E ADGRG2FL 转染细胞与 WT ADRGR2FL 转染细胞进行比较;n.s. 无显著差异，将 Erev 与计算出的能斯特电位进行比较。值是至少六个独立实验 （n=6） 的平均 SEM ±值。