71 results found
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.
Fanelli F, Hanyaloglu AC, Jonas K, 2019, Integrated structural modeling and super-resolution imaging resolve GPCR oligomers, Progress in Molecular Biology and Translational Science
© 2019 Elsevier Inc. Formation of G protein-coupled receptors (GPCRs) dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signaling activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, in both health and disease, from the study of this unique and complex subfamily of GPCRs with light on the luteinizing hormone receptor (LHR). Focus will be put on the results of an approach relying on the combination of atomistic modeling by protein-protein docking with super-resolution imaging. The latter could resolve single LHR molecules to ~ 8 nm resolution in functional asymmetric dimers and oligomers, using dual-color photoactivatable dyes and localization microscopy (PD-PALM). Structural modeling of functionally asymmetric LHR trimers and tetramers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.
Sposini S, Hanyaloglu AC, 2018, Driving gonadotrophin hormone receptor signalling: the role of membrane trafficking, REPRODUCTION, Vol: 156, Pages: R195-R208, ISSN: 1470-1626
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 Ffnctional 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.
Jones B, Buenaventura T, Kanda N, et al., 2018, Targeting GLP-1 receptor trafficking to improve agonist efficacy, Nature Communications, Vol: 9, ISSN: 2041-1723
Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a novel series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in β-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.
Schilperoort M, van Dam AD, Hoeke G, et al., 2018, The GPR120 agonist TUG-891 promotes metabolic health by stimulating mitochondrial respiration in brown fat, EMBO Molecular Medicine, Vol: 10, ISSN: 1757-4676
Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein-coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT Stimulation of brown adipocytes in vitro with TUG-891 acutely induced O2 consumption, through GPR120-dependent and GPR120-independent mechanisms. TUG-891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG-891 is a promising strategy to increase lipid combustion and reduce obesity.
Gorvin CM, Babinsky VN, Malinauskas T, et al., 2018, A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling, Science Signaling, Vol: 11, ISSN: 1937-9145
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11and Gi/oto stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-functionCASRmutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+iresponses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSRR680Gin HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca2+iresponses. Moreover, this gain of function in MAPK activity occurred independently of Gq/11and Gi/oand was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg680and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg680-Glu767salt bridge in mediating signaling bias.
Jonas KC, Chen S, Virta M, et al., 2018, Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers, Scientific Reports, Vol: 8, ISSN: 2045-2322
Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.
Owens LA, Abbara A, Lerner A, et al., 2018, The direct and indirect effects of kisspeptin-54 on granulosa lutein cell function, Human Reproduction, Vol: 33, Pages: 292-302, ISSN: 1460-2350
STUDY QUESTIONWhat are the in vivo and in vitro actions of kisspeptin-54 on the expression of genes involved in ovarian reproductive function, steroidogenesis and ovarian hyperstimulation syndrome (OHSS) in granulosa lutein (GL) cells when compared with traditional triggers of oocyte maturation?SUMMARY ANSWERThe use of kisspeptin-54 as an oocyte maturation trigger augmented expression of genes involved in ovarian steroidogenesis in human GL cells including, FSH receptor (FSHR), LH/hCG receptor (LHCGR), steroid acute regulatory protein (STAR), aromatase, estrogen receptors alpha and beta (ESR1, ESR2), 3-beta-hydroxysteroid dehydrogenase type 2 (3BHSD2) and inhibin A (INHBA), when compared to traditional maturation triggers, but did not alter markers of OHSS.WHAT IS KNOWN ALREADYhCG is the most widely used trigger of oocyte maturation, but is associated with an increased risk of OHSS. The use of GnRH agonists to trigger oocyte maturation is a safer alternative to hCG. More recently, kisspeptin-54 has emerged as a novel therapeutic option that safely triggers oocyte maturation even in women at high risk of OHSS. Kisspeptin indirectly stimulates gonadotropin secretion by acting on hypothalamic GnRH neurons. Kisspeptin and its receptor are also expressed in the human ovary, but there is limited data on the direct action of kisspeptin on the ovary.STUDY DESIGN SIZE, DURATIONForty-eight women undergoing IVF treatment for infertility consented to kisspeptin-54 triggering and/or granulosa cell collection and were included in the study. Twelve women received hCG, 12 received GnRH agonist and 24 received kisspeptin-54 to trigger oocyte maturation. In the kisspeptin-54 group, 12 received one injection of kisseptin-54 (9.6 nmol/kg) and 12 received two injections of kisspeptin-54 at a 10 h interval (9.6 nmol/kg × 2).PARTICIPANTS/MATERIALS, SETTING, METHODSFollicular fluid was aspirated and pooled from follicles during the retrieval of oocytes for IVF/ICSI. GL cells were iso
Gorvin CM, Rogers A, Hastoy B, et al., 2018, AP2? Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity., Cell Reports, Vol: 22, Pages: 1054-1066, ISSN: 2211-1247
Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 ? subunit (AP2?) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2? mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2? mutations reduced CaSR signaling via G?q/11 and G?i/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by G?q/11. Thus, compartmental bias for CaSR-mediated G?q/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.
Sayers N, Hanyaloglu AC, 2018, Intracellular Follicle-Stimulating Hormone Receptor Trafficking and Signaling., Front Endocrinol (Lausanne), 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, Jean-Alphonse FG, Ayoub MA, et al., 2017, Integration of GPCR signaling and sorting from very early endosomes via opposing APPL1 mechanisms, Cell Reports, Vol: 21, Pages: 2855-2867, ISSN: 2211-1247
Endocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized β2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.
Hanyaloglu AC, Grammatopoulos DK, 2017, Pleiotropic GPCR signaling in health and disease, Molecular and Cellular Endocrinology, Vol: 449, Pages: 1-2, ISSN: 0303-7207
Sposini S, Hanyaloglu AC, 2017, Spatial encryption of G protein-coupled receptor signaling in endosomes; mechanisms and applications, Biochemical Pharmacology, Vol: 143, Pages: 1-9, ISSN: 1873-2968
Within any cellular signaling system membrane trafficking is a critical mechanism for cells to translate complex networks into specific downstream responses, including the signal pathways activated by the superfamily of G protein-coupled receptors (GPCRs). Classically, membrane trafficking is viewed as a mechanism to regulate ligand sensitivity of a target tissue by controlling the level of surface receptors. Recent studies, however, have not only highlighted that GPCR trafficking is a tightly regulated process critical for spatio-temporal control of signaling, but that heterotrimeric G protein signaling can also be reactivated or continue to signal from distinct endocytic compartments, and even endosomal microdomains. The significance of spatio-temporal control will be discussed, not only with respect to how these novel molecular pathways impact our basic understanding of cellular regulation, but also our view of how aberrant signaling can result in disease. Furthermore, these mechanisms offer the potential application for novel therapeutic strategies to identify GPCR compounds with high specificity in their actions
Jonas KC, Hanyaloglu AC, 2017, Impact of G protein-coupled receptor heteromers in endocrine systems, Molecular and Cellular Endocrinology, Vol: 449, Pages: 21-27, ISSN: 0303-7207
The fine-tuning of endocrine homeostasis is regulated by dynamic receptor mediated processes. Thesuperfamily of G protein-coupled receptors (GPCRs) have diverse roles in the modulation of all endocrineaxes, thus understanding the mechanisms underpinning their functionality is paramount for treatmentof endocrinopathies. Evidence over the last 20 years has highlighted homo and heteromerization as a keymode of mediating GPCR functional diversity. This review will discuss the concept of GPCR heteromerizationand its relevance to endocrine function, detailing in vitro and in vivo evidence, and exploringcurrent and potential pharmacological strategies for specific targeting of GPCR heteromers in endocrineheath and disease.
Hanyaloglu AC, Fanelli F, Jonas KC, 2017, Class A GPCR: Di/oligomerization of glycoprotein hormone receptors, Receptors, Pages: 207-231
© Springer International Publishing AG 2017. G protein-coupled receptor (GPCR) dimerization and oligomerization was first described over 2 decades ago, contributing to the recent paradigm shift in GPCR signaling of a simplistic, archetypal view involving single receptors activating specific heterotrimeric G proteins at the cell surface, to one of an increasing complex receptor signaling system. However, our understanding of how dimerization and oligomerization, particularly homomerization, generates functional diversity in GPCR signaling is poorly understood. For the Class A/rhodopsin subfamily of glycoprotein hormone receptors (GpHRs), di/oligomerization has been demonstrated to play a significant role in regulating its signal activity at a cellular and physiological level and even pathophysiologically. Here we will describe and discuss the developments in our understanding of GPCR oligomerization, primarily the role of homomeric receptor complexes, in both health and disease, from the study of this unique and complex subfamily of GPCRs.
Babinsky VN, Hannan FM, Gorvin CM, et al., 2016, Allosteric modulation of the calcium-sensing receptor rectifies signaling abnormalities associated with G-protein Alpha-11 mutations causing hypercalcemic and hypocalcemic disorders, Journal of Biological Chemistry, Vol: 291, Pages: 10876-10885, ISSN: 1083-351X
Germline loss- and gain-of-function mutations of G-protein alpha-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively upregulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK1/2 phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-consti
Jonas KC, Huhtaniemi I, Hanyaloglu AC, 2016, Single-molecule resolution of G protein-coupled receptor (GPCR) complexes, G PROTEIN-COUPLED RECEPTORS: SIGNALING, TRAFFICKING AND REGULATION, Editors: Shukla, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 55-72, ISBN: 978-0-12-803595-5
Kim SH, MacIntyre DA, Hanyaloglu AC, et al., 2015, The oxytocin receptor antagonist, Atosiban, activates pro-inflammatory pathways in human amnion via G(alpha i) signalling, Molecular and Cellular Endocrinology, Vol: 420, Pages: 11-23, ISSN: 1872-8057
West C, Hanyaloglu AC, 2015, Minireview: Spatial Programming of G Protein-Coupled Receptor Activity: Decoding Signaling in Health and Disease., Molecular Endocrinology, Vol: 29, Pages: 1095-1106, ISSN: 1944-9917
Probing the multiplicity of hormone signaling via G protein-coupled receptors (GPCRs) has demonstrated the complex signal pathways that underlie the multiple functions these receptors play in vivo. This is highly pertinent for the GPCRs key in reproduction and pregnancy that are exposed to cyclical and dynamic changes in their extracellular milieu. How such functional pleiotropy in GPCR signaling is translated to specific downstream cellular responses, however, is largely unknown. Emerging data strongly support mechanisms for a central role of receptor location in signal regulation via membrane trafficking. In this review, we discuss current progress in our understanding of the role membrane trafficking plays in location control of GPCR signaling, from organized plasma membrane signaling microdomains, potentially provided by both distinct endocytic and exocytic pathways, to more recent evidence for spatial control within the endomembrane system. Application of these emerging mechanisms in their relevance to GPCR activity in physiological and pathophysiological conditions will also be discussed, and in improving therapeutic strategies that exploits these mechanisms in order to program highly regulated and distinct signaling profiles.
Hanyaloglu A, Jonas K, 2015, Advancing applications of super-resolution imaging: 10 November 2014, Charles Darwin House, London, UK, Biochemist, Vol: 37, Pages: 52-52, ISSN: 0954-982X
Jonas KC, Fanelli F, Huhtaniemi IT, et al., 2014, Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies, Journal of Biological Chemistry, Vol: 290, Pages: 3875-3892, ISSN: 1083-351X
Background: GPCRs form complex oligomers whose role in signaling is poorly understood.Results: Super-resolution imaging of functionally asymmetric oligomers reveals diverse functional and structural organizationsand the ability to alter signal responses.Conclusion: GPCR oligomers may fine-tune receptor signaling by altering the functional role of individual protomers.Significance: Distinct oligomers could be exploited pharmacologically to improve efficacy, selectivity, and/or specificity.
Sposini S, Caltabiano G, Hanyaloglu AC, et al., 2014, Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers, Molecular and Cellular Endocrinology, Vol: 399, Pages: 362-372, ISSN: 1872-8057
The chemokine prokineticin 2 (PK2) activates its cognate G protein-coupled receptor (GPCR) PKR2 to elicit various downstream signaling pathways involved in diverse biological processes. Many GPCRs undergo dimerization that can modulate a number of functions including membrane delivery and signal transduction. The aim of this study was to elucidate the interface of PKR2 protomers within dimers by analyzing the ability of PKR2 transmembrane (TM) deletion mutants to associate with wild type (WT) PKR2 in yeast using co-immunoprecipitation and mammalian cells using bioluminescence resonance energy transfer. Deletion of TMs 5-7 resulted in a lack of detectable association with WT PKR2, but could associate with a truncated mutant lacking TMs 6-7 (TM1-5). Interestingly, TM1-5 modulated the distance, or organization, between protomers and positively regulated Gαs signaling and surface expression of WT PKR2. We propose that PKR2 protomers form type II dimers involving TMs 4 and 5, with a role for TM5 in modulation of PKR2 function.
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