Gq activity- and ?-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility.
ABSTRACT: Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and ?-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or ?-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that ?-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/?-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.
Project description:In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.
Project description:We performed whole exome sequencing to identify an unknown genetic cause of azoospermia and male infertility in a large Pakistani family. Three infertile males were subjected to semen analysis, hormone testing, testicular histology, ultrasonography, karyotyping, Y-chromosome microdeletion and CFTR testing. The clinical testing suggested a diagnosis of obstructive azoospermia (OA). To identify the cause, we performed whole exome sequencing (WES) for 2 infertile brothers and 2 fertile family members. For segregation analysis and variant confirmation, we performed Sanger sequencing. WES data analysis of the family revealed segregated variants in 3 candidate genes. We considered novel nonsense variant c.2440C?>?T(p.Arg814*) in X-linked gene ADGRG2 as biologically most plausible. It is predicted to truncate the protein by 204 amino acids (aa) at a key transmembrane domain. Adgrg2-knockout male mice show sperm loss due to obstructive fluid stasis, while ADGRG2 mutations cause OA in the infertile male patients. Our analysis of testicular histology reveals secondary severe reduction of spermatogenesis, consistent with human and knockout mouse phenotypes. The ADGRG2 nonsense mutation is absent in the largest population databases, ExAC and gnomAD. Analysis of the novel nonsense mutation in extended family members confirmed co-segregation of the mutation with OA in all affected males. The likely pathogenic nature of the mutation is supported by its truncation effect on the transmembrane domain and distinctive ultrasound results. The study demonstrates effectiveness of WES in discovering a genetic cause of azoospermia.
Project description:The sympathetic nervous system (SNS) accelerates heart rate, increases cardiac contractility, and constricts resistance vessels. The activity of SNS efferent nerves is generated by a complex neural network containing neurons and glia. Gq G protein-coupled receptor (Gq-GPCR) signaling in glial fibrillary acidic protein-expressing (GFAP+) glia in the central nervous system supports neuronal function and regulates neuronal activity. It is unclear how Gq-GPCR signaling in GFAP+ glia affects the activity of sympathetic neurons or contributes to SNS-regulated cardiovascular functions. In this study, we investigated whether Gq-GPCR activation in GFAP+ glia modulates the regulatory effect of the SNS on the heart; transgenic mice expressing Gq-coupled DREADD (designer receptors exclusively activated by designer drugs) (hM3Dq) selectively in GFAP+ glia were used to address this question in vivo. We found that acute Gq-GPCR activation in peripheral GFAP+ glia significantly accelerated heart rate and increased left ventricle contraction. Pharmacological experiments suggest that the glial-induced cardiac changes were due to Gq-GPCR activation in satellite glial cells within the sympathetic ganglion; this activation led to increased norepinephrine (NE) release and beta-1 adrenergic receptor activation within the heart. Chronic glial Gq-GPCR activation led to hypotension in female Gfap-hM3Dq mice. This study provides direct evidence that Gq-GPCR activation in peripheral GFAP+ glia regulates cardiovascular functions in vivo.
Project description:Estrogen receptor alpha (ER alpha) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na(+)/H(+) exchanger-3 (NHE3) and the rate of (22)Na(+) transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ER alpha knockout (alpha ERKO) efferent ductules. Targeted gene-deficient mice were compared with alpha ERKO, and the NHE3 knockout and CAII-deficient mice showed alpha ERKO-like fluid accumulation, but only the NHE3 knockout and alpha ERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in alpha ERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in alpha ERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in alpha ERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na(+) transport. Finally, these data raise the possibility of targeting ER alpha in developing a contraceptive for the male.
Project description:Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (?NTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ?NTF and P622 mutants interact with ?-arrestin1 and ?-arrestins2 and are constitutively internalized in steady states. However, only ?NTF shows exaggerated basal activation of the G?s -cAMP-CRE signaling cascade. Neither ?NTF nor P622 shows constitutive activation of the G?13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ?NTF and P622 to early endosomes, where ?NTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with ?-arrestins and the consequent endocytosis and sustained signaling from endosomes.
Project description:G protein-coupled receptors (GPCRs) respond to diversified extracellular stimuli to modulate cellular function. Despite extensive studies investigating the regulation of single GPCR signaling cascades, the effects of concomitant GPCR activation on downstream signaling and cellular function remain unclear.We aimed to characterize the cellular mechanism by which GPCR crosstalk regulates mitogen-activated protein kinase (MAPK) activation.Adrenergic receptors on cardiac fibroblasts were manipulated to examine the role of arrestin in the spatiotemporal regulation of extracellular signal-regulated kinase (ERK)1/2 MAPK signaling. We show a general mechanism in which arrestin activation by one GPCR is capable of regulating signaling originating from another GPCR. Activation of Gq coupled-receptor signaling leads to prolonged ERK1/2 MAPK phosphorylation, nuclear accumulation, and cellular proliferation. Interestingly, coactivation of these receptors with the beta-adrenergic receptors induced transient ERK signaling localized within the cytosol, which attenuated cell proliferation. Further studies revealed that recruitment of arrestin3 to the beta2-adrenergic receptor orchestrates the sequestration of Gq-coupled receptor-induced ERK to the cytosol through direct binding of ERK to arrestin.This is the first evidence showing that arrestin3 acts as a coordinator to integrate signals from multiple GPCRs. Our studies not only provide a novel mechanism explaining the integration of mitogenic signaling elicited by different GPCRs, but also underscore the critical role of signaling crosstalk among GPCRs in vivo.
Project description:BACKGROUND:Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of obstructive azoospermia and male infertility. Mutations of CFTR caused the majority of CBAVD cases, and ADGRG2 was recently identified as a new pathogenic gene. Yet, most of the genetic evidence came from sporadic cases, and only one mutation in CFTR can be found in patients. METHODS:In present study, we collected two CBAVD pedigrees, each having two affected male siblings. We performed whole exome sequencing on all patients and validated all potential variants by Sanger sequencing. RESULTS:We excluded ADGRG2 variants but identified compound heterozygous variants of CFTR in both families (NM_000492.3:c.1210-33_1210-6GTT and c.4056G>C;p.Gln1352Cys in pedigree 1, c.592G>C;p.Ala198Pro and c.3717G>A;p.Arg1239= in pedigree 2), which were subsequently validated by direct sequencing. c.1210-33_1210-6GTT (also known as IVS8-T5-TG13) was a known disease-causing variant causing the skipping of exon 9 of CFTR and inherited from the proband's mother. p.Gln1352Cys and Ala198Pro were rare or novel in public databases and predicted to be deleterious. The p.Arg1239= was a synonymous variant but located at the end of an exon, which was predicted to alter the splicing pattern. CONCLUSION:Our study, in which compound heterozygous variants were identified in two pedigrees, provides more familial evidence that only recessive variants (homozygous or compound heterozygous) in CFTR cause CBAVD. Furthermore, whole exome sequencing may be utilized as a useful tool for mutation screening of genes causing CBAVD.
Project description:Estrogen receptor-alpha (ERalpha) plays a critical role in male reproductive tract development and fertility. To determine whether estrogen-dependent and -independent ERalpha mechanisms are involved in male fertility, we examined male estrogen nonresponsive ERalpha knock-in mice. These animals have a point mutation (G525L) in the ligand-binding domain of ERalpha that significantly reduces interaction with, and response to, endogenous estrogens but does not affect growth factor activation of ligand-independent ERalpha pathways. Surprisingly, we found that ligand-independent ERalpha signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption. In contrast, estrogen-dependent ERalpha signaling is required for germ cell viability, most likely through support of Sertoli cell function. By treating estrogen nonresponsive ERalpha knock-in (ENERKI) mice with the ERalpha selective synthetic agonist propyl pyrazole triol, which is able to bind and activate G525L ERalpha in vivo, we discovered male fertility required neonatal estrogen-mediated ERalpha signaling. Thus, our work indicates both estrogen-dependent and -independent pathways play separable roles in male murine reproductive tract development and that the role of ERalpha in human infertility should be examined more closely.
Project description:Cardiac ?-adrenergic receptor (?AR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ?ARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists.Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ?ARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled ?1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ?1AR and ?2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled ?2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ?2AR phosphorylation effectively inhibit ?AR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ?2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ?2AR in a ?-arrestin 2 dependent manner without promoting ?2AR endocytosis. The increased binding between ?2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ?2AR, inhibition of PDE4, or genetic ablation of PDE4D or ?-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ?-arrestin 2 or PDE4D gene also rescues ?-adrenergic stimuli-induced myocyte contractile function.These data reveal essential roles of ?-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ?ARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.