DOCSLIB.ORG

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/08/05/mol.116.104828.DC1 1521-0111/90/3/214–224$25.00 http://dx.doi.org/10.1124/mol.116.104828 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:214–224, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist s Hannah M. Stoveken, Laura L. Bahr, M. W. Anders, Andrew P. Wojtovich, Alan V. Smrcka, and Gregory G. Tall Department of Pharmacology and Physiology (H.M.S., L.L.B., M.W.A., A.P.W., A.V.S.), and Department of Anesthesiology (L.L.B., A.P.W.), University of Rochester Medical Center, Rochester, New York; and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (G.G.T.) Received April 21, 2016; accepted June 10, 2016 Downloaded from ABSTRACT Adhesion G protein–coupled receptors (aGPCRs) have emerging response element (SRE)-luciferase–based screening approach roles in development and tissue maintenance and is the most to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000- prevalent GPCR subclass mutated in human cancers, but to compound library comprising known drugs and natural products date, no drugs have been developed to target them in any was screened for GPR56-dependent SRE activation inhibitors molpharm.aspetjournals.org disease. aGPCR extracellular domains contain a conserved that did not inhibit constitutively active Ga13-dependent SRE subdomain that mediates self-cleavage proximal to the start of activation. Dihydromunduletone (DHM), a rotenoid derivative, the 7-transmembrane domain (7TM). The two receptor proto- was validated using cell-free aGPCR/heterotrimeric G protein mers, extracellular domain and amino terminal fragment (NTF), guanosine 59-3-O-(thio)triphosphate binding reconstitution as- and the 7TM or C-terminal fragment remain noncovalently says. DHM inhibited GPR56 and GPR114/ADGRG5, which have bound at the plasma membrane in a low-activity state. We similar tethered agonists, but not the aGPCR GPR110/ADGRF1, recently demonstrated that NTF dissociation liberates the 7TM M3 muscarinic acetylcholine, or b2 adrenergic GPCRs. DHM N-terminal stalk, which acts as a tethered-peptide agonist inhibited tethered peptide agonist-stimulated and synthetic permitting receptor-dependent heterotrimeric G protein activa- peptide agonist-stimulated GPR56 but did not inhibit basal tion. In many cases, natural aGPCR ligands are extracellular activity, demonstrating that it antagonizes the peptide agonist. at ASPET Journals on October 2, 2021 matrix proteins that dissociate the NTF to reveal the tethered DHM is a novel aGPCR antagonist and potentially useful agonist. Given the perceived difficulty in modifying extracellular chemical probe that may be developed as a future aGPCR matrix proteins to create aGPCR probes, we developed a serum therapeutic. Introduction event in which the P19 residue becomes the N terminus of the ∼17–26 amino acid tethered-agonist stalks (Lin et al., 2004; – Adhesion G protein coupled receptors (aGPCRs) are a Arac et al., 2012; Liebscher et al., 2014; Stoveken et al., 2015). – 33-member subclass of family B G protein coupled receptors Self-cleavage is thought to be constitutive, and the resultant (GPCRs) that have critical roles in tissue specification and receptor protomers or fragments remain noncovalently bound replenishment and human cancers (Langenhan et al., 2013; while in residence at the plasma membrane. In this state, the Hamann et al., 2015). It was recently discovered that the N terminus of the postcleaved tethered-agonist stalk binds extracellular peptide stalks emanating from the first firmly within a hydrophobic b-strand network that would transmembrane-spanning helix of multiple adhesion GPCRs preclude it from engaging the carboxy-terminal fragment are tethered-peptide agonists that activate aGPCR-mediated (CTF)/seven-transmembrane domain (7TM) orthosteric site. heterotrimeric G protein signaling (Liebscher et al., 2014; A current hypothesis which we favor that describes the Demberg et al., 2015; Stoveken et al., 2015; Wilde et al., 2016). dynamism of receptor activation consists of two components: 1) Tethered-agonist regulation is intimately linked to the ability anchored protein ligands bind aGPCR N-terminal fragment of aGPCRs to execute a precise autocatalytic, self-cleavage (NTF) binding determinants, and 2) the action of shear force created by cell movement serves to dissociate the NTF from the This work was supported by a PhRMA Foundation predoctoral fellowship in CTF to release or decrypt the tethered agonist (Karpus et al., pharmacology to H.M.S. The production of recombinant G proteins used for this work was supported by the National Institutes of Health National 2013; Langenhan et al., 2013; Scholz et al., 2015; Stoveken et al., Institute of General Medical Sciences [Grant GM088242 to G.G.T.]. 2015). We reconstituted aGPCRs, GPR56 (ADGRG1) and dx.doi.org/10.1124/mol.116.104828. s This article has supplemental material available at molpharm. GPR110 (ADGRF1), with purified G protein heterotrimers and aspetjournals.org. provided a biochemical demonstration that experimentally ABBREVIATIONS: aGPCR, adhesion G protein–coupled receptor; CTF, carboxy-terminal fragment; DHM, dihydromunduletone; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; ETC, electron transport chain; FBS, fetal bovine serum; GPCR, G protein–coupled receptor; GTPgS, 59-3-O-(thio)triphosphate; HA, hemagglutinin; HEK293, human embryonic kidney 293; NTF, amino terminal fragment; PAR, protease activated receptors; [35S]GTPgS, 59-O-(3-[35S]thio)triphosphate; SRE, serum response element; 7TM, seven-transmembrane domain. 214 A Class-Selective Small-Molecule Adhesion GPCR Antagonist 215 induced NTF dissociation dramatically enhanced aGPCR- were purchased individually as powders from MicroSource Discovery mediated G protein activation (Stoveken et al., 2015). The known Systems, Inc. (Gaylordsville, CT). Rotenone and polyclonal hemag- protein ligands of aGPCRs are fibrillar extracellular matrix glutinin (HA) antibody were purchased from Sigma-Aldrich (St. Louis, proteins or, in some instances, proteins presented by neighboring MO). Latrunculin B was from Calbiochem (San Diego, CA). Quant-iT cells (Hamann et al., 1996; Sugita et al., 1999; Stacey et al., 2003; PicoGreen double stranded DNA reagent was from Invitrogen (Carls- bad, CA). Streptavidin Sepharose High Performance was from GE Xu et al., 2006; Bolliger et al., 2011; Chiang et al., 2011; Luo et al., ’ Healthcare (Chicago, IL). Sulfo-NHS-Biotin was from Thermo Scien- 2011; Silva et al., 2011; Boucard et al., 2012; O Sullivan et al., tific (Waltham, MA). 59-O-(3-[35S]thio)triphosphate ([35S]GTPgS) and 2012; Paavola and Hall, 2012; Paavola et al., 2014; Petersen the phRLuc-N1 plasmid were from PerkinElmer (Waltham, MA). The et al., 2015; Scholz et al., 2015). Based on the proposed two- pGL4.33 [luc2/SRE/Hygro] plasmid was purchased from Promega component mode of ligand-mediated aGPCR activation, we (Madison, WI). anticipate that natural NTF ligands or ligand-based NTF- High-Throughput Screen. HEK293T cells were maintained in binding mimetics will be of limited, stand-alone pharmacological Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, use to manipulate receptor activities. Indeed, application of dilute, NY) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were transiently transfected with GPR56 7TM (Met-Thr383 to Ile693) soluble extracellular matrix protein ligands to aGPCR cell Q226L culture–based signaling assays most likely does not recapitulate pcDNA3.1 or GNA13 pcDNA3.1 (cDNA.org) and the SRE- luciferase reporter using a polyethylenimine transfection method authentic aGPCR pharmacology. Hence, there is a critical need (Wang et al., 2010; Oner et al., 2013; Stoveken et al., 2015). Five to develop small-molecule modulators that can bypass the hours after transfection, cells were trypsinized, counted, and seeded at Downloaded from two-component ligand-mediated regulation process and directly 15,000 cells per well in 384-well plates in 20 ml of phenol red–free modulate receptor activities. A number of adhesion GPCR small- DMEM buffered with 25 mM Hepes, pH 7.4, and incubated overnight molecule screening efforts are ongoing, but to our knowledge, only with ∼3–5 mM compounds in dimethylsulfoxide (DMSO); Spectrum a few candidate small-molecule activators were proposed to Collection, at the URMC High Throughput Screening Core (Micro- regulate GPR97 (Gupte et al., 2012; Southern et al., 2013). source Discovery Systems, Inc. Gaylordsville, CT). The next morning, We conducted a chemical-screening effort to identify small- 20 ml of SteadyLite luciferase reagent (PerkinElmer) was robotically molpharm.aspetjournals.org molecule inhibitors of GPR56, an attractive therapeutic target dispensed into each well and incubated for 15 minutes at room due to its proposed roles in neurogenesis, neuromaintenance, temperature. Luminescence was read on a PerkinElmer Envision Plate Reader. and cancer progression (Piao et al., 2004; Shashidhar et al., Directed Dual SRE-Luciferase Assay. HEK293T cells main- 2005; Xu et al., 2006, 2010; Yang et al., 2011; Saito et al., 2013; tained in DMEM 110% (v/v) FBS were transiently transfected in Hamann et al., 2015). GPR56 signals through G13 and multiple 24-well format using the polyethylenimine reagent with 200 ng of laboratories showed
Load more

Recommended publications
  • Mechanisms of Acetylcholine Receptor Loss in Myasthenia Gravis
    J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.43.7.601 on 1 July 1980. Downloaded from Journal of Neurology, Neurosurgery, and Psychiatry, 1980, 43, 601-610 Mechanisms of acetylcholine receptor loss in myasthenia gravis DANIEL B DRACHMAN, ROBERT N ADAMS, ELIS F STANLEY, AND ALAN PESTRONK From the Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA SUMMARY The fundamental abnormality affecting the neuromuscular junctions of myasthenic patients is a reduction of available AChRs, due to an autoimmune attack directed against the receptors. Antibodies to AChR are present in most patients, and there is evidence that they have a predominant pathogenic role in the disease, aided by complement. The mechanism of antibody action involves acceleration of the rate of degradation of AChRs, attributable to cross-linking of the receptors. In addition, antibodies may block AChRs, and may participate in producing destructive changes, perhaps in conjunction with complement. The possibility that cell-mediated mechanisms may play a role in the autoimmune responses of some myasthenic patients remains to be explored. Although the target of the autoimmune attack in myasthenic patients is probably always the acetyl- Protected by copyright. choline receptors, it is not yet clear which of these immune mechanisms are most important. It is likely that the relative role of each mechanism varies from patient to patient. One of the goals of future research will be to identify the relative importance of each
    [Show full text]
  • Edinburgh Research Explorer
    Edinburgh Research Explorer International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list Citation for published version: Davenport, AP, Alexander, SPH, Sharman, JL, Pawson, AJ, Benson, HE, Monaghan, AE, Liew, WC, Mpamhanga, CP, Bonner, TI, Neubig, RR, Pin, JP, Spedding, M & Harmar, AJ 2013, 'International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligands', Pharmacological reviews, vol. 65, no. 3, pp. 967-86. https://doi.org/10.1124/pr.112.007179 Digital Object Identifier (DOI): 10.1124/pr.112.007179 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Pharmacological reviews Publisher Rights Statement: U.S. Government work not protected by U.S. copyright General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 02. Oct. 2021 1521-0081/65/3/967–986$25.00 http://dx.doi.org/10.1124/pr.112.007179 PHARMACOLOGICAL REVIEWS Pharmacol Rev 65:967–986, July 2013 U.S.
    [Show full text]
  • CXCL13/CXCR5 Interaction Facilitates VCAM-1-Dependent Migration in Human Osteosarcoma
    International Journal of Molecular Sciences Article CXCL13/CXCR5 Interaction Facilitates VCAM-1-Dependent Migration in Human Osteosarcoma 1, 2,3,4, 5 6 7 Ju-Fang Liu y, Chiang-Wen Lee y, Chih-Yang Lin , Chia-Chia Chao , Tsung-Ming Chang , Chien-Kuo Han 8, Yuan-Li Huang 8, Yi-Chin Fong 9,10,* and Chih-Hsin Tang 8,11,12,* 1 School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei City 11031, Taiwan; [email protected] 2 Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, Puzi City, Chiayi County 61363, Taiwan; [email protected] 3 Department of Nursing, Division of Basic Medical Sciences, and Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Puzi City, Chiayi County 61363, Taiwan 4 Research Center for Industry of Human Ecology and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Guishan Dist., Taoyuan City 33303, Taiwan 5 School of Medicine, China Medical University, Taichung 40402, Taiwan; [email protected] 6 Department of Respiratory Therapy, Fu Jen Catholic University, New Taipei City 24205, Taiwan; [email protected] 7 School of Medicine, Institute of Physiology, National Yang-Ming University, Taipei City 11221, Taiwan; [email protected] 8 Department of Biotechnology, College of Health Science, Asia University, Taichung 40402, Taiwan; [email protected] (C.-K.H.); [email protected] (Y.-L.H.) 9 Department of Sports Medicine, College of Health Care, China Medical University, Taichung 40402, Taiwan 10 Department of Orthopedic Surgery, China Medical University Beigang Hospital, Yunlin 65152, Taiwan 11 Department of Pharmacology, School of Medicine, China Medical University, Taichung 40402, Taiwan 12 Chinese Medicine Research Center, China Medical University, Taichung 40402, Taiwan * Correspondence: [email protected] (Y.-C.F.); [email protected] (C.-H.T.); Tel.: +886-4-2205-2121-7726 (C.-H.T.); Fax: +886-4-2233-3641 (C.-H.T.) These authors contributed equally to this work.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Receptor-Arrestin Interactions: the GPCR Perspective
    biomolecules Review Receptor-Arrestin Interactions: The GPCR Perspective Mohammad Seyedabadi 1,2 , Mehdi Gharghabi 3, Eugenia V. Gurevich 4 and Vsevolod V. Gurevich 4,* 1 Department of Toxicology & Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48471-93698, Iran; [email protected] 2 Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48167-75952, Iran 3 Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA; [email protected] 4 Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-615-322-7070; Fax: +1-615-343-6532 Abstract: Arrestins are a small family of four proteins in most vertebrates that bind hundreds of different G protein-coupled receptors (GPCRs). Arrestin binding to a GPCR has at least three functions: precluding further receptor coupling to G proteins, facilitating receptor internalization, and initiating distinct arrestin-mediated signaling. The molecular mechanism of arrestin–GPCR interactions has been extensively studied and discussed from the “arrestin perspective”, focusing on the roles of arrestin elements in receptor binding. Here, we discuss this phenomenon from the “receptor perspective”, focusing on the receptor elements involved in arrestin binding and empha- sizing existing gaps in our knowledge that need to be filled. It is vitally important to understand the role of receptor elements in arrestin activation and how the interaction of each of these elements with arrestin contributes to the latter’s transition to the high-affinity binding state. A more precise knowledge of the molecular mechanisms of arrestin activation is needed to enable the construction of arrestin mutants with desired functional characteristics.
    [Show full text]
  • Protease Effects on the Structure of Acetylcholine Receptor Membranes from Torpedo Californica
    PROTEASE EFFECTS ON THE STRUCTURE OF ACETYLCHOLINE RECEPTOR MEMBRANES FROM TORPEDO CALIFORNICA MICHAEL W. KLYMKOWSKY, JOHN E . HEUSER, and ROBERT M. STROUD From the Department of Biochemistry & Biophysics, University of California at San Francisco, San Francisco, California 94143 . Dr . Klymkowsky's present address is MRC Neuroimmunology Project, Department of Zoology, University College London, London WC IE, 6BT, England ABSTRACT Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule . Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-lm-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy . Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al ., 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure . Trypsin digestion of alkali-stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles
    [Show full text]
  • Interplay Between Gating and Block of Ligand-Gated Ion Channels
    brain sciences Review Interplay between Gating and Block of Ligand-Gated Ion Channels Matthew B. Phillips 1,2, Aparna Nigam 1 and Jon W. Johnson 1,2,* 1 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA; [email protected] (M.B.P.); [email protected] (A.N.) 2 Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA * Correspondence: [email protected]; Tel.: +1-(412)-624-4295 Received: 27 October 2020; Accepted: 26 November 2020; Published: 1 December 2020 Abstract: Drugs that inhibit ion channel function by binding in the channel and preventing current flow, known as channel blockers, can be used as powerful tools for analysis of channel properties. Channel blockers are used to probe both the sophisticated structure and basic biophysical properties of ion channels. Gating, the mechanism that controls the opening and closing of ion channels, can be profoundly influenced by channel blocking drugs. Channel block and gating are reciprocally connected; gating controls access of channel blockers to their binding sites, and channel-blocking drugs can have profound and diverse effects on the rates of gating transitions and on the stability of channel open and closed states. This review synthesizes knowledge of the inherent intertwining of block and gating of excitatory ligand-gated ion channels, with a focus on the utility of channel blockers as analytic probes of ionotropic glutamate receptor channel function. Keywords: ligand-gated ion channel; channel block; channel gating; nicotinic acetylcholine receptor; ionotropic glutamate receptor; AMPA receptor; kainate receptor; NMDA receptor 1. Introduction Neuronal information processing depends on the distribution and properties of the ion channels found in neuronal membranes.
    [Show full text]
  • Cellular Trafficking of Nicotinic Acetylcholine Receptors
    npg Acta Pharmacol Sin 2009 Jun; 30 (6): 656–662 Review Cellular trafficking of nicotinic acetylcholine receptors Paul A ST JOHN* Department of Cell Biology and Anatomy, University of Arizona College of Medicine, Tucson, AZ 85724, USA Nicotinic acetylcholine receptors (nAChRs) play critical roles throughout the body. Precise regulation of the cellular loca- tion and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs. Keywords: membrane traffic; protein traffic; biosynthesis; endocytosis; endoplasmic reticulum-associated degradation Acta Pharmacologica Sinica (2009) 30: 656–662; doi: 10.1038/aps.2009.76 Introduction ways, but two particular perturbations have been especially well studied and exert their effects at least in part by altering Nicotinic acetylcholine receptors (nAChRs) mediate the trafficking of nAChRs: 1) denervation changes the total synaptic transmission in the CNS, in autonomic ganglia, and number, the distribution, and the turnover rate of nAChRs in at neuromuscular junctions and other peripheral synapses. skeletal muscle; 2) prolonged exposure to nicotine increases The functional properties of these synapses differ, but in each the total number of nAChRs in neurons. Several of the stud- case, properly functional signaling requires cellular control ies reviewed here addressed the mechanisms by which these of the number, type, and location of nAChRs. Trafficking treatments alter nAChR trafficking. Other authors in this of nAChRs – the movement of nAChRs between compart- special issue will address other aspects of the effects of nico- ments of a cell, including the cell's biosynthetic and degrada- tine on nAChRs.
    [Show full text]
  • G-Protein-Coupled Receptor Signaling and Polarized Actin Dynamics Drive
    RESEARCH ARTICLE elifesciences.org G-protein-coupled receptor signaling and polarized actin dynamics drive cell-in-cell invasion Vladimir Purvanov, Manuel Holst, Jameel Khan, Christian Baarlink, Robert Grosse* Institute of Pharmacology, University of Marburg, Marburg, Germany Abstract Homotypic or entotic cell-in-cell invasion is an integrin-independent process observed in carcinoma cells exposed during conditions of low adhesion such as in exudates of malignant disease. Although active cell-in-cell invasion depends on RhoA and actin, the precise mechanism as well as the underlying actin structures and assembly factors driving the process are unknown. Furthermore, whether specific cell surface receptors trigger entotic invasion in a signal-dependent fashion has not been investigated. In this study, we identify the G-protein-coupled LPA receptor 2 (LPAR2) as a signal transducer specifically required for the actively invading cell during entosis. We find that 12/13G and PDZ-RhoGEF are required for entotic invasion, which is driven by blebbing and a uropod-like actin structure at the rear of the invading cell. Finally, we provide evidence for an involvement of the RhoA-regulated formin Dia1 for entosis downstream of LPAR2. Thus, we delineate a signaling process that regulates actin dynamics during cell-in-cell invasion. DOI: 10.7554/eLife.02786.001 Introduction Entosis has been described as a specialized form of homotypic cell-in-cell invasion in which one cell actively crawls into another (Overholtzer et al., 2007). Frequently, this occurs between tumor cells such as breast, cervical, or colon carcinoma cells and can be triggered by matrix detachment (Overholtzer et al., 2007), suggesting that loss of integrin-mediated adhesion may promote cell-in-cell invasion.
    [Show full text]
  • When 7 Transmembrane Receptors Are Not G Protein–Coupled Receptors
    When 7 transmembrane receptors are not G protein–coupled receptors Keshava Rajagopal, … , Robert J. Lefkowitz, Howard A. Rockman J Clin Invest. 2005;115(11):2971-2974. https://doi.org/10.1172/JCI26950. Commentary Classically, 7 transmembrane receptors transduce extracellular signals by coupling to heterotrimeric G proteins, although recent in vitro studies have clearly demonstrated that they can also signal via G protein–independent mechanisms. However, the physiologic consequences of this unconventional signaling, particularly in vivo, have not been explored. In this issue of the JCI, Zhai et al. demonstrate in vivo effects of G protein–independent signaling by the angiotensin II type 1 receptor (AT1R). In studies of the mouse heart, they compare the physiologic and biochemical consequences of transgenic cardiac-specific overexpression of a mutant AT1R incapable of G protein coupling with those of a wild-type receptor. Their results not only provide the first glimpse of the physiologic effects of this newly appreciated mode of signaling but also provide important and previously unappreciated clues as to the underlying molecular mechanisms. Find the latest version: https://jci.me/26950/pdf commentaries gene, spastin, regulates microtubule stability to 16. Wittmann, C.W., et al. 2001. Tauopathy in Dro- 21. Chan, Y.B., et al. 2003. Neuromuscular defects in modulate synaptic structure and function. Curr. sophila: neurodegeneration without neurofibril- a Drosophila survival motor neuron gene mutant. Biol. 14:1135–1147. lary tangles. Science. 293:711–714. Hum. Mol. Genet. 12:1367–1376. 12. Sherwood, N.T., Sun, Q., Xue, M., Zhang, B., and 17. Shapiro, W.R., and Young, D.F.
    [Show full text]
  • G-Protein-Coupled Receptor Heteromer Dynamics
    Commentary 4215 G-protein-coupled receptor heteromer dynamics Jean-Pierre Vilardaga1,2,*, Luigi F. Agnati3, Kjell Fuxe4 and Francisco Ciruela5 1Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261, USA 2Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA 3IRCCS, San Camillo, Lido Venezia, Italy 4Department of Neuroscience, Karolinska Institute, Stockholm SE-17177, Sweden 5Pharmacology Unit, Department of Pathology and Experimental Therapy, Faculty of Medicine, University of Barcelona, 08907 Barcelona, Spain *Author for correspondence ([email protected]) Journal of Cell Science 123, 000-000 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.063354 Summary G-protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors, and have evolved to detect and transmit a large palette of extracellular chemical and sensory signals into cells. Activated receptors catalyze the activation of heterotrimeric G proteins, which modulate the propagation of second messenger molecules and the activity of ion channels. Classically thought to signal as monomers, different GPCRs often pair up with each other as homo- and heterodimers, which have been shown to modulate signaling to G proteins. Here, we discuss recent advances in GPCR heteromer systems involving the kinetics of the early steps in GPCR signal transduction, the dynamic property of receptor–receptor interactions, and how the formation of receptor heteromers modulate the kinetics of G-protein signaling. Key words: G-protein-coupled receptors, Heterodimers, Signaling Introduction the signaling and trafficking mechanisms of GPCRs is thus central G-protein-coupled receptors (GPCRs) constitute the main family for the development of new and safer therapies for many of cell surface receptors for a large variety of chemical stimuli physiological and psychological disorders.
    [Show full text]
  • Supplemental Material For
    Supplemental material for Epithelial to mesenchymal transition rewires the molecular path to PI3-Kinase-dependent proliferation Megan B. Salt1,2, Sourav Bandyopadhyay1,3 and Frank McCormick1 Authors Affiliations: 1-Helen Diller Family Comprehensive Cancer Center; 2-Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, California; 3-California Institute for Quantitative Biosciences, San Francisco, California. Supplemental Figure Legends Table S1. EMT associated changes in gene expression. Significantly (A) up- and (B) down- regulated genes in comparing H358-TwistER cells to control H358-GFP expressing cells treated with 100nM 4OHT for 12 days. The four columns on the right are associated with significantly upregulated genes, while the four columns furthest left described significantly downregulated genes. The first column for the up- and downregulated genes shows the Probe ID associated with each gene from the Affymetric Human Gene 1.0 ST microarrays. The gene name are shown in the next column, followed by the log2(fold change) in expression for each gene after 4OHT treatment in the H358-TwistER cells. The final column shows the p-value for each gene adjusted for the false discovery rate. Figure S1. Akt inhibition reduces serum-independent proliferation. (A) Proliferation of H358- TwistER cells with increasing concentrations (uM) of GSK-690693 (top) or MK-2206 (bottom). (B) Lysates from H358-TwistER cells treated for 48hrs in serum free media with indicated concentrations of GSK-690693 (top), or MK-2206 (bottom). Figure S2. The effects of TGFβ1 are specific to epithelial cells and lead to reduced serum- independent proliferation. (A) Proliferation of untreated (top) or TGFβ1 pre-treated (bottom) H358 and H441 cells in the presence (10%) or absence (SS) of serum.
    [Show full text]