Publications
100 results found
Riaposova L, Kim SH, Hanyaloglu A, et al., 2023, Prostaglandin F2alpha requires activation of calcium-dependent signalling to trigger inflammation in human myometrium, Frontiers in Endocrinology, Vol: 14, Pages: 1-19, ISSN: 1664-2392
Introduction: Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators.Methods: Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR.Results: The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-β. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling.Discussion: In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors – NF-κB, MAP kinases, CREB and C/EBP-β. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight i
Walker A, Larsen C, Kundu S, et al., 2022, Functional rewiring of G protein-coupled receptor signaling in human labo, Cell Reports, Vol: 40, ISSN: 2211-1247
Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.
Mann ON, Kong C-S, Lucas ES, et al., 2022, Expression and function of the luteinizing hormone choriogonadotropin receptor in human endometrial stromal cells., Sci Rep, Vol: 12
The human luteinising hormone choriogonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca2+ and extracellular signal-regulated kinase (ERK) signalling. LHCGR expression has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity, and we identified a small subpopulation of stromal cells with detectable LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca2+ and ERK signals that were absent in wild-type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature.
Sayers S, Anujan P, Yu H, et al., 2022, Follicle-stimulating hormone induces lipid droplets via Gαi/o and β- arrestin in an endometrial cancer cell line, Frontiers in Endocrinology, Vol: 12, Pages: 1-15, ISSN: 1664-2392
Follicle-stimulating hormone (FSH) and its G protein-coupled receptor, FSHR, represents a paradigm for receptor signaling systems that activate multiple and complex pathways. Classically, FSHR activates Gαs to increase intracellular levels of cAMP, but its ability to activate other G proteins, and β-arrestin-mediated signaling is well documented in many different cell systems. The pleiotropic signal capacity of FSHR offers a mechanism for how FSH drives multiple and dynamic downstream functions in both gonadal and non-gonadal cell types, including distinct diseases, and how signal bias may be achieved at a pharmacological and cell system-specific manner. In this study, we identify an additional mechanism of FSH-mediated signaling and downstream function in the endometrial adenocarcinoma Ishikawa cell line. While FSH did not induce increases in cAMP levels, this hormone potently activated pertussis toxin sensitive Gαi/o signaling. A selective allosteric FSHR ligand, B3, also activated Gαi/o signaling in these cells, supporting a role for receptor-mediated activation despite the low levels of FSHR mRNA. The low expression levels may attribute to the lack of Gαs/cAMP signaling as increasing FSHR expression resulted in FSH-mediated activation of the Gαs pathway. Unlike prior reports for FSH-mediated Gαs/cAMP signaling, FSH-mediated Gαi/o signaling was not affected by inhibition of dynamin-dependent receptor internalization. While chronic FSH did not alter cell viability, FSH was able to increase lipid droplet size. The β-arrestins are key adaptor proteins known to regulate FSHR signaling. Indeed, a rapid, FSH-dependent increase in interactions between β-arrestin1 and Gαi1 was observed via NanoBiT complementation in Ishikawa cells. Furthermore, both inhibition of Gαi/o signaling and siRNA knockdown of β-arrestin 1/2 significantly reduced FSH-induced lipid droplet accumulation, implying a role for a
Toufaily C, Fortin J, Alonso CA, et al., 2021, Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice, ELIFE, Vol: 10, ISSN: 2050-084X
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De Pascali F, Ayoub MA, Benevelli R, et al., 2021, Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol: 22
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Bosch E, Alviggi C, Lispi M, et al., 2021, Reduced FSH and LH action: implications for medically assisted reproduction., Hum Reprod, Vol: 36, Pages: 1469-1480
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play complementary roles in follicle development and ovulation via a complex interaction in the hypothalamus, anterior pituitary gland, reproductive organs, and oocytes. Impairment of the production or action of gonadotropins causes relative or absolute LH and FSH deficiency that compromises gametogenesis and gonadal steroid production, thereby reducing fertility. In women, LH and FSH deficiency is a spectrum of conditions with different functional or organic causes that are characterized by low or normal gonadotropin levels and low oestradiol levels. While the causes and effects of reduced LH and FSH production are very well known, the notion of reduced action has received less attention by researchers. Recent evidence shows that molecular characteristics, signalling as well as ageing, and some polymorphisms negatively affect gonadotropin action. These findings have important clinical implications, in particular for medically assisted reproduction in which diminished action determined by the afore-mentioned factors, combined with reduced endogenous gonadotropin production caused by GnRH analogue protocols, may lead to resistance to gonadotropins and, thus, to an unexpected hypo-response to ovarian stimulation. Indeed, the importance of LH and FSH action has been highlighted by the International Committee for Monitoring Assisted Reproduction Technologies (ICMART) in their definition of hypogonadotropic hypogonadism as gonadal failure associated with reduced gametogenesis and gonadal steroid production due to reduced gonadotropin production or action. The aim of this review is to provide an overview of determinants of reduced FSH and LH action that are associated with a reduced response to ovarian stimulation.
Hanyaloglu AC, Reiter E, 2021, Editorial: G protein–coupled receptors: From molecules to medicine, Current Opinion in Endocrine and Metabolic Research, Vol: 16, Pages: iv-vi
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Ulloa-Aguirre A, Janovick JA, Zariñán T, et al., 2021, Intracellular Trafficking of G Protein-Coupled Receptors to the Cell Surface Plasma Membrane in Health and Disease, Cellular Endocrinology in Health and Disease, Second Edition, Pages: 375-412, ISBN: 9780128198018
Proteins are synthesized in the endoplasmic reticulum (ER) of the cell, where a strict quality control system (QCS) guards against aberrant protein structures and non-productive protein aggregation. The ER QCS checks for adequate folding and structural integrity of nascent proteins so that only properly folded and assembled secretory and membrane proteins may reach their final destination within the cell. For this task, the ER QCS relies on different strategies, including the action of members of major molecular chaperone families. As any other protein, G protein-coupled receptors (GPCRs) are subjected to structural and conformational scrutiny at the ER before processing and trafficking to the plasma membrane. Mutations in these integral membrane proteins may provoke misfolding, retention, and eventually degradation of the defective protein receptor, leading to disease. In this chapter, we review the mechanisms governing upward and downward trafficking of this class of membrane receptors, including interactions with molecular chaperones and scaffold molecules, and the role of some structural requirements for proper intracellular trafficking. We also exemplify how misfolding may lead to disease and describe different strategies to rescue the function of misfolded GPCRs in vitro and in vivo.
Casarini L, Lazzaretti C, Paradiso E, et al., 2020, Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) heteromeric complexes promote human ovarian follicle survival, iScience, Vol: 23, ISSN: 2589-0042
Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.
Sposini S, De Pascali F, Richardson R, et al., 2020, Pharmacological programming of endosomal signaling activated by small molecule ligands of the follicle stimulating hormone receptor, Frontiers in Pharmacology, Vol: 11, ISSN: 1663-9812
Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists (benzamide, termed B3 and thiazolidinone, termed T1) were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recyclin
Abbara A, Eng P, Phylactou M, et al., 2020, Kisspeptin receptor agonist has therapeutic potential for female reproductive disorders., Journal of Clinical Investigation, Vol: 130, Pages: 6739-6753, ISSN: 0021-9738
BACKGROUND. Kisspeptin is a key regulator of hypothalamic gonadotropin-releasing hormone (GnRH) neurons and is essential for reproductive health. A specific kisspeptin receptor (KISS1R) agonist could significantly expand the potential clinical utility of therapeutics targeting the kisspeptin pathway. Herein, we investigate the effects of a KISS1R agonist, MVT-602, in healthy women and in women with reproductive disorders.METHODS. We conducted in vivo and in vitro studies to characterize the action of MVT-602 in comparison with native kisspeptin-54 (KP54). We determined the pharmacokinetic and pharmacodynamic properties of MVT-602 (doses 0.01 and 0.03 nmol/kg) versus KP54 (9.6 nmol/kg) in the follicular phase of healthy women (n = 9), and in women with polycystic ovary syndrome (PCOS; n = 6) or hypothalamic amenorrhea (HA; n = 6). Further, we investigated their effects on KISS1R-mediated inositol monophosphate (IP1) and Ca2+ signaling in cell lines and on action potential firing of GnRH neurons in brain slices.RESULTS. In healthy women, the amplitude of luteinizing hormone (LH) rise was similar to that after KP54, but peaked later (21.4 vs. 4.7 hours; P = 0.0002), with correspondingly increased AUC of LH exposure (169.0 vs. 38.5 IU∙h/L; P = 0.0058). LH increases following MVT-602 were similar in PCOS and healthy women, but advanced in HA (P = 0.004). In keeping with the clinical data, MVT-602 induced more potent signaling of KISS1R-mediated IP1 accumulation and a longer duration of GnRH neuron firing than KP54 (115 vs. 55 minutes; P = 0.0012).CONCLUSION. Taken together, these clinical and mechanistic data identify MVT-602 as having considerable therapeutic potential for the treatment of female reproductive disorders.TRIAL REGISTRATION. International Standard Randomised Controlled Trial Number (ISRCTN) Registry, ISRCTN21681316.FUNDING. National Institute for Health Research and NIH.
Shackley M, Ma Y, Brown A, et al., 2020, Short chain fatty acids enhance expression and activity of the umami taste receptor in enteroendocrine cells via a Galphai/o pathway, Frontiers in Nutrition, Vol: 7, ISSN: 2296-861X
The short chain fatty acids (SCFAs) acetate, butyrate and propionate, are produced by fermentation of non-digestible carbohydrates by the gut microbiota and regulate appetite, adiposity, metabolism, glycemic control, and immunity. SCFAs act at two distinct G protein coupled receptors (GPCRs), FFAR2 and FFAR3 and are expressed in intestinal enteroendocrine cells (EECs), where they mediate anorectic gut hormone release. EECs also express other GPCRs that act as nutrient sensors, thus SCFAs may elicit some of their health-promoting effects by altering GPCR expression in EECs and enhance gut sensitivity to dietary molecules. Here, we identify that exposure of the murine EEC STC-1 cell line or intestinal organoids to physiological concentrations of SCFAs enhances mRNA levels of the umami taste receptors TASR1 and TASR3, without altering levels of the SCFA GPCRs, FFAR2 and FFAR3. Treatment of EECs with propionate or butyrate, but not acetate, increased levels of umami receptor transcripts, while propionate also reduced CCK expression. This was reversed by inhibiting Gαi/o signaling with pertussis toxin, suggesting that SCFAs act through FFAR2/3 to alter gene expression. Surprisingly, neither a FFAR3 nor a FFAR2 selective ligand could increase TASR1/TASR3 mRNA levels. We assessed the functional impact of increased TASR1/TASR3 expression using unique pharmacological properties of the umami taste receptor; namely, the potentiation of signaling by inosine monophosphate. Activation of umami taste receptor induced inositol-1-phosphate and calcium signaling, and butyrate pretreatment significantly enhanced such signaling. Our study reveals that SCFAs may contribute to EEC adaptation and alter EEC sensitivity to bioactive nutrients.
Caengprasath N, Gonzalez Abuin N, Shchepinova M, et al., 2020, Internalization-dependent free fatty acid receptor 2 signaling is essential for propionate- induced anorectic gut hormone release, iScience, Vol: 23, ISSN: 2589-0042
The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut.
Dwivedi-Agnihotri H, Chaturvedi M, Baidya M, et al., 2020, Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling, SCIENCE ADVANCES, Vol: 6, ISSN: 2375-2548
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Baidya M, Kumari P, Dwivedi-Agnihotri H, et al., 2020, Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G protein-coupled receptors, Journal of Biological Chemistry, Vol: 295, Pages: 10153-10167, ISSN: 0021-9258
Agonist stimulation of G protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR-βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR-βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.
Shchepinova MM, Hanyaloglu AC, Frost GS, et al., 2020, Chemical biology of noncanonical G protein-coupled receptor signaling: Toward advanced therapeutics, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 56, Pages: 98-110, ISSN: 1367-5931
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Ulloa-Aguirre A, Hanyaloglu AC, Zariñán T, et al., 2020, Protein homeostasis and regulation of intracellular trafficking of G protein-coupled receptors, Protein Homeostasis Diseases: Mechanisms and Novel Therapies, Pages: 247-277, ISBN: 9780128191330
G protein-coupled receptors (GPCRs) regulate a countless number of cellular functions and are currently major protein targets for drug discovery, as they frequently lead to disease. Like any other protein, GPCRs must be properly folded into a mature tertiary conformation compatible with quaternary assembly and endoplasmic reticulum export to the cell surface plasma membrane. Several primary and secondary structural features, including presence of particular amino acid residues and short motifs and, in addition, posttranslational modifications (PTMs), regulate the intracellular trafficking of this class of membrane proteins. In contrast to the paucity in knowledge on the molecular mechanisms underlying GPCR cell surface delivery after biosynthesis (anterograde or upward trafficking), our understanding of the processes following internalization and endocytosis of GPCRs from the plasma membrane has almost exponentially increased during the last decade, and the function of the intracellular compartments and interacting proteins that regulate the fate of the internalized receptors, either to the degradation or recycling pathways, as well as their endosomal signaling after endocytosis, are currently under intense investigation. Knowing how GPCRs traffic within the cell is essential for continuing to pave the way in the discovery of new therapeutic strategies for an array of diseases caused by receptor misrouting.
Fanelli F, Hanyaloglu AC, Jonas K, 2020, Integrated structural modeling and super-resolution imaging resolve GPCR oligomers, OLIGOMERIZATION IN HEALTH AND DISEASE: FROM ENZYMES TO G PROTEIN-COUPLED RECEPTORS, Editors: Giraldo, Ciruela, Publisher: ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD, Pages: 151-179
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Owens LA, Kristensen SG, Lerner A, et al., 2019, Gene expression in granulosa cells from small antral follicles from women with or without polycystic ovaries, Journal of Clinical Endocrinology and Metabolism, Vol: 104, Pages: 6182-6192, ISSN: 0021-972X
CONTEXT: Polycystic ovary syndrome (PCOS) is the commonest cause of anovulation. A key feature of PCOS is arrest of follicles at the small-medium sized antral stage. OBJECTIVE AND DESIGN: To provide further insight into the mechanism of follicle arrest in PCOS, we profiled; (1) gonadotropin receptors; (2) characteristics of aberrant steroidogenesis, and (3) expression of anti-Mullerian hormone (AMH) and its receptor in granulosa cells (GCs) from unstimulated, human small antral follicles (hSAFs) and from granulosa-lutein cells (GLCs). SETTING: GCs from hSAFs were collected at the time of cryopreservation of ovarian tissue for fertility preservation and GLCs collected during oocyte aspiration before IVF/ICSI. PARTICIPANTS: hSAF GCs were collected from 31 women (98 follicles), 10 with polycystic ovaries (PCO) and 21 without. GLCs were collected from 6 women with PCOS and 6 controls undergoing IVF. MAIN OUTCOME MEASURES: Expression of the following genes: LHCGR, FSHR, AR, INSR, HSD3B2, CYP11A1, CYP19, STAR, AMH, AMHR2, FST, INHBA, INHBB in GCs and GLCs were compared between women with PCO and controls. RESULTS: GCs in hSAFs from PCO women showed higher expression of LHCGR in a subset (20%) of follicles. Expression of FSHR (p<0.05), AR (p<0.05), CYP11A1 (P<0.05) was lower, and expression of CYP19A1 (p<0.05), STAR (p<0.05), HSD3B2 (ns), INHBA (p<0.05) higher in PCO GCs. Gene expression in GL cells differed between women with and without PCOS but also differed from that in GCs. CONCLUSIONS: Follicle arrest in PCO is characterised in GCs by differential regulation of key genes involved in follicle growth and function.
Buenaventura T, Bitsi S, Laughlin WE, et al., 2019, Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells., PLoS Biology, Vol: 17, Pages: 1-40, ISSN: 1544-9173
The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.
Jonas KC, Hanyaloglu AC, 2019, Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy., Methods Mol Biol, Vol: 1947, Pages: 337-348
Super-resolution imaging has provided unprecedented insight in the molecular complexities of fundamental cell biological questions. For G protein-coupled receptors (GPCRs), its application to the study of receptor homomers and heteromers have unveiled the diversity of complexes these GPCRs can form at the plasma membrane at a structural and functional level. Here, we describe our methodological approach of photoactivated localization microscopy with photoactivatable dyes (PD-PALM) to visualize and quantify the spatial assembly of GPCR heteromers at the plasma membrane.
Kim SH, Riaposova L, Ahmed H, et al., 2019, Oxytocin receptor antagonists, atosiban and nolasiban, inhibit prostaglandin F2α-induced contractions and inflammatory responses in human myometrium, Scientific Reports, Vol: 9, ISSN: 2045-2322
Oxytocin receptor antagonists (OTR-A) have been developed as tocolytics for the management of preterm labour due to the significant role of oxytocin (OT) in the onset of both term and preterm labour. Similar to OT, prostaglandins (PGs) play key roles in myometrial contractility and cervical ripening. Inhibition of PG synthesis/activity is used to delay preterm birth. Thus, targeting the PG pathway in combination with an OTR-A may be an effective strategy for delaying preterm delivery. In this study, we examined the effects of atosiban and nolasiban on PGF2α-induced contractions and pro-inflammatory responses in human pregnant myometrium. Both OTR-As, atosiban and nolasiban, inhibited PGF2α-induced contractions in a dose-dependent manner (p < 0.001 and p < 0.01, respectively). These inhibitory effects involved the suppression of PGF2α-mediated increase in intracellular calcium levels. In addition, the OTR-As significantly suppressed PGF2α-induced activation of pro-inflammatory pathways such as NF-κB and mitogen activated protein kinases (MAPKs), and the subsequent expression of contraction-associated-protein, COX-2. We have demonstrated that atosiban and nolasiban not only inhibit contractions elicited by OT, but also inhibit contractions and inflammation induced by PGF2α. This suggests a possible crosstalk between OTR and PG receptor signalling and highlights the importance of understanding G protein-coupled receptor interactions/crosstalk in the development of future tocolytics.
Caengprasath N, Hanyaloglu A, 2019, Hardwiring wire-less networks: spatially encoded GPCR signaling in endocrine systems, Current Opinion in Cell Biology, Vol: 57, Pages: 77-82, ISSN: 0955-0674
The pivotal and diverse roles G protein-coupled receptors (GPCRs) play in physiology are matched by the increasingly complex signal systems they activate. Over the past decade, our models of GPCR signaling systems also include a vital role of location in controlling GPCR signaling, whereby plasma membrane, clathrin-associated structures and a diverse endomembrane network provide highly specialized signal platforms for this superfamily of receptors. The aim of this review is to highlight the recent developments in this fast-evolving field, with particular emphasis on endocrine-relevant GPCRs. We will also highlight studies that address the possibility of therapeutic intervention and how this fundamental cell biology can be translated to physiology/pathophysiology and therapeutic interventions.
Sposini S, Hanyaloglu AC, 2018, Driving gonadotrophin hormone receptor signalling: the role of membrane trafficking, REPRODUCTION, Vol: 156, Pages: R195-R208, ISSN: 1470-1626
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Sayers N, Hanyaloglu AC, 2018, Intracellular follicle-stimulating hormone receptor trafficking and signaling, Frontiers in Endocrinology, Vol: 9, ISSN: 1664-2392
Models of G protein-coupled receptor (GPCR) signaling have dramatically altered over the past two decades. Indeed, GPCRs such as the follicle-stimulating hormone receptor (FSHR) have contributed to these new emerging models. We now understand that receptor signaling is highly organized at a spatial level, whereby signaling not only occurs from the plasma membrane but distinct intracellular compartments. Recent studies in the role of membrane trafficking and spatial organization of GPCR signaling in regulating gonadotropin hormone receptor activity has identified novel intracellular compartments, which are tightly linked with receptor signaling and reciprocally regulated by the cellular trafficking machinery. Understanding the impact of these cell biological mechanisms to physiology and pathophysiology is emerging for certain GPCRs. However, for FSHR, the potential impact in both health and disease and the therapeutic possibilities of these newly identified systems is currently unknown, but offers the potential to reassess prior strategies, or unveil novel opportunities, in targeting this receptor.
Sposini S, Hanyaloglu AC, 2018, Evolving View of Membrane Trafficking and Signaling Systems for G Protein-Coupled Receptors., Prog Mol Subcell Biol, Vol: 57, Pages: 273-299, ISSN: 0079-6484
The G protein-coupled receptor (GPCR) superfamily activates complex signal pathways, yet untangling these signaling systems to understand how specificity in receptor signaling pathways is achieved, has been a challenging question. The roles of membrane trafficking in GPCR signal regulation has undergone a recent paradigm shift, from a mechanism that programs the plasma membrane G protein signaling profile to providing distinct signaling platforms critical for specifying receptor function in vivo. In this chapter, we discuss this evolution of our understanding in the endocytic trafficking systems employed by GPCRs, and how such systems play a deeply integrated role with signaling. We describe recent studies that suggest that the endomembrane compartment can provide a mechanism to both specify, and yet also diversify, GPCR signal transduction. These new evolving models could aid mechanistic understanding of complex disease and provide novel therapeutic avenues.
Jonas KC, Hanyaloglu AC, 2018, Super-resolution imaging as a method to Study GPCR dimers and higher-order oligomers, Receptor-receptor interactions in the central nervous system, Publisher: Springer, Pages: 329-343
The study of G protein-coupled receptor (GPCR) dimers and higher order oligomershas unveiled mechanisms for receptors to diversify signaling and potentially uncovernovel therapeutic targets. The functional and clinical significance of these receptorreceptor associations has been facilitated by the development of techniques andprotocols, enabling researchers to unpick their function from the molecularinterfaces, to demonstrating functional significance in vivo, in both health anddisease. Here we describe our methodology to study GPCR oligomerization at thesingle molecule level via super-resolution imaging. Specifically, we have employedphotoactivated localization microscopy, with photoactivatable dyes (PD-PALM) tovisualize the spatial organization of these complexes to <10nm resolution, and thequantitation of GPCR monomer, dimer and oligomer in both homomeric andheteromeric forms. We provide guidelines on optimal sample preparation, imagingparameters and necessary controls for resolving and quantifying single moleculedata. Finally, we discuss advantages and limitations of this imaging technique and itspotential future applications to the study of GPCR function.
Pyle E, Kalli AC, Amillis S, et al., 2018, Structural lipids enable the formation of Functional oligomers of the eukaryotic purine symporter UapA, Cell Chemical Biology, Vol: 25, Pages: 840-848.e4, ISSN: 2451-9456
The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.
Hanyaloglu AC, 2018, Advances in membrane tafficking and endosomal signaling of G protein-coupled receptors, International Review of Cell and Molecular Biology, Vol: 339, Pages: 93-131, ISSN: 1937-6448
The integration of GPCR signaling with membrane trafficking, as a single orchestrated system, is a theme increasingly evident with the growing reports of GPCR endosomal signaling. Once viewed as a mechanism to regulate cell surface heterotrimeric G protein signaling, the endocytic trafficking system is complex, highly compartmentalized, yet deeply interconnected with cell signaling. The organization of receptors into distinct plasma membrane signalosomes, biochemically distinct endosomal populations, endosomal microdomains, and its communication with distinct subcellular organelles such as the Golgi provides multiple unique signaling platforms that are critical for specifying receptor function physiologically and pathophysiologically. In this chapter I discuss our emerging understanding in the endocytic trafficking systems employed by GPCRs and their novel roles in spatial control of signaling. Given the extensive roles that GPCRs play in vivo, these evolving models are starting to provide mechanistic understanding of distinct diseases and provide novel therapeutic avenues that are proving to be viable targets.
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