FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2572498644> ?p ?o ?g. }

• W2572498644 endingPage "3655" @default.
• W2572498644 startingPage "3637" @default.
• W2572498644 abstract "Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11. Here, we identified a region within the C-terminal domain (amino acids 852–876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+ in situ. Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11. Here, we identified a region within the C-terminal domain (amino acids 852–876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+ in situ. From their position on the cell surface, G-protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCRG-protein-coupled receptorINMinner nuclear membraneERendoplasmic reticulumNESnuclear exclusion signalNLSnuclear localization signalIP3inositol 1,4,5-trisphosphateONMouter nuclear membraneLBRlamin B receptorPMplasma membraneNMnuclear membraneEndo Hendo-β-N-acetylglucosaminidase HROIregion of interestDHPG(S)-3,5-dihydroxyphenylglycinePNGasepeptide:N-glycosidaseMPEP2-methyl-6-(phenylethynyl) pyridinemGluR5metabotropic glutamate receptor 5Quisl-quisqualic acid LY53, LY393053BBSbungarotoxin-binding siteBAPTA AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)FRAPfluorescence recovery after photobleachingRFPred fluorescent protein. can transform external stimuli into a broad range of signaling pathways within the cell. A mounting body of evidence indicates that many GPCRs are also localized inside the cell where they may couple to different signaling systems, display unique desensitization patterns, and/or exhibit distinct patterns of subcellular distribution (1.Jong Y.J. Sergin I. Purgert C.A. O'Malley K.L. Location-dependent signaling of the group 1 metabotropic glutamate receptor mGlu5.Mol. Pharmacol. 2014; 86: 774-785Crossref PubMed Scopus (41) Google Scholar2.Branco A.F. Allen B.G. G protein-coupled receptor signaling in cardiac nuclear membranes.J. Cardiovasc. Pharmacol. 2015; 65: 101-109Crossref PubMed Scopus (25) Google Scholar, 3.Campden R. Audet N. Hébert T.E. Nuclear G protein signaling: new tricks for old dogs.J. Cardiovasc. Pharmacol. 2015; 65: 110-122Crossref PubMed Scopus (28) Google Scholar, 4.Irannejad R. von Zastrow M. GPCR signaling along the endocytic pathway.Curr. Opin. Cell Biol. 2014; 27: 109-116Crossref PubMed Scopus (138) Google Scholar5.Calebiro D. Godbole A. Lyga S. Lohse M.J. Trafficking and function of GPCRs in the endosomal compartment.Methods Mol. Biol. 2015; 1234: 197-211Crossref PubMed Scopus (16) Google Scholar). For example, GPCRs have been found on mitochondria (6.Bénard G. Massa F. Puente N. Lourenço J. Bellocchio L. Soria-Gómez E. Matias I. Delamarre A. Metna-Laurent M. Cannich A. Hebert-Chatelain E. Mulle C. Ortega-Gutiérrez S. Martín-Fontecha M. Klugmann M. et al.Mitochondrial CB(1) receptors regulate neuronal energy metabolism.Nat. Neurosci. 2012; 15: 558-564Crossref PubMed Scopus (371) Google Scholar), endoplasmic reticulum (ER) membranes (7.Meads M.B. Medveczky P.G. Kaposi's sarcoma-associated herpesvirus-encoded viral interleukin-6 is secreted and modified differently than human interleukin-6: evidence for a unique autocrine signaling mechanism.J. Biol. Chem. 2004; 279: 51793-51803Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), lysosomes (8.Rozenfeld R. Devi L.A. Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP-3.FASEB J. 2008; 22: 2311-2322Crossref PubMed Scopus (122) Google Scholar, 9.Oksche A. Boese G. Horstmeyer A. Furkert J. Beyermann M. Bienert M. Rosenthal W. Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor.Mol. Pharmacol. 2000; 57: 1104-1113PubMed Google Scholar), and on nuclear membranes (10.Calebiro D. Nikolaev V.O. Persani L. Lohse M.J. Signaling by internalized G-protein-coupled receptors.Trends Pharmacol. Sci. 2010; 31: 221-228Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 11.Gobeil F. Fortier A. Zhu T. Bossolasco M. Leduc M. Grandbois M. Heveker N. Bkaily G. Chemtob S. Barbaz D. G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigm.Can. J. Physiol. Pharmacol. 2006; 84: 287-297Crossref PubMed Google Scholar12.Tadevosyan A. Vaniotis G. Allen B.G. Hébert T.E. Nattel S. G protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.J. Physiol. 2012; 590: 1313-1330Crossref PubMed Scopus (109) Google Scholar). Certain GPCRs are even found within the nucleoplasm on nuclear bodies and/or nuclear invaginations (13.Lee D.K. Lança A.J. Cheng R. Nguyen T. Ji X.D. Gobeil Jr, F. Chemtob S. George S.R. O'Dowd B.F. Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors.J. Biol. Chem. 2004; 279: 7901-7908Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar14.Morinelli T.A. Raymond J.R. Baldys A. Yang Q. Lee M.H. Luttrell L. Ullian M.E. Identification of a putative nuclear localization sequence within ANG II AT(1A) receptor associated with nuclear activation.Am. J. Physiol. Cell Physiol. 2007; 292: C1398-C1408Crossref PubMed Scopus (39) Google Scholar, 15.Wright C.D. Wu S.C. Dahl E.F. Sazama A.J. O'Connell T.D. Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes.Cell. Signal. 2012; 24: 794-802Crossref PubMed Scopus (41) Google Scholar16.Joyal J.S. Nim S. Zhu T. Sitaras N. Rivera J.C. Shao Z. Sapieha P. Hamel D. Sanchez M. Zaniolo K. St-Louis M. Ouellette J. Montoya-Zavala M. Zabeida A. Picard E. et al.Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.Nat. Med. 2014; 20: 1165-1173Crossref PubMed Scopus (55) Google Scholar). Although many intracellular GPCRs are activated at the cell surface and subsequently trafficked to their intracellular site, others can be activated at their subcellular location via so-called intracrine ligands that can enter cells via diffusion or be made in situ, endocytosed, and/or transported through channels or pores (12.Tadevosyan A. Vaniotis G. Allen B.G. Hébert T.E. Nattel S. G protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.J. Physiol. 2012; 590: 1313-1330Crossref PubMed Scopus (109) Google Scholar, 17.Boivin B. Vaniotis G. Allen B.G. Hébert T.E. G protein-coupled receptors in and on the cell nucleus: a new signaling paradigm?.J. Recept. Signal. Transduct. Res. 2008; 28: 15-28Crossref PubMed Scopus (107) Google Scholar, 18.Barlow C.A. Laishram R.S. Anderson R.A. Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum.Trends Cell Biol. 2010; 20: 25-35Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Intracellular GPCRs can also function independently governing processes such as synaptic plasticity (19.Purgert C.A. Izumi Y. Jong Y.J. Kumar V. Zorumski C.F. O'Malley K.L. Intracellular mGluR5 can mediate synaptic plasticity in the hippocampus.J. Neurosci. 2014; 34: 4589-4598Crossref PubMed Scopus (66) Google Scholar), myocyte contraction (15.Wright C.D. Wu S.C. Dahl E.F. Sazama A.J. O'Connell T.D. Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes.Cell. Signal. 2012; 24: 794-802Crossref PubMed Scopus (41) Google Scholar), and angiogenesis (16.Joyal J.S. Nim S. Zhu T. Sitaras N. Rivera J.C. Shao Z. Sapieha P. Hamel D. Sanchez M. Zaniolo K. St-Louis M. Ouellette J. Montoya-Zavala M. Zabeida A. Picard E. et al.Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.Nat. Med. 2014; 20: 1165-1173Crossref PubMed Scopus (55) Google Scholar). Collectively, the present findings reinforce the notion that intracellular GPCRs play a dynamic role in generating and shaping intracellular signaling pathways. G-protein-coupled receptor inner nuclear membrane endoplasmic reticulum nuclear exclusion signal nuclear localization signal inositol 1,4,5-trisphosphate outer nuclear membrane lamin B receptor plasma membrane nuclear membrane endo-β-N-acetylglucosaminidase H region of interest (S)-3,5-dihydroxyphenylglycine peptide:N-glycosidase 2-methyl-6-(phenylethynyl) pyridine metabotropic glutamate receptor 5 l-quisqualic acid LY53, LY393053 bungarotoxin-binding site 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) fluorescence recovery after photobleaching red fluorescent protein. As many GPCRs are on or in the nucleus, the question arises as to how they get there. Various possibilities exist to transfer membrane proteins from the outer to the inner nuclear membrane (ONM and INM), including vesicle fusion, membrane rupture, and channel-mediated pathways. Most prominently, a diffusion-retention model has been proposed for many INM proteins (e.g. lamin B receptor (LBR)) (20.Zuleger N. Kerr A.R. Schirmer E.C. Many mechanisms, one entrance: membrane protein translocation into the nucleus.Cell. Mol. Life Sci. 2012; 69: 2205-2216Crossref PubMed Scopus (38) Google Scholar, 21.Katta S.S. Smoyer C.J. Jaspersen S.L. Destination: inner nuclear membrane.Trends Cell Biol. 2014; 24: 221-229Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This model suggests that proteins synthesized in the ER rapidly diffuse laterally in the ONM, pass through peripheral channels existing between the nuclear pore complex and the pore membrane, and then become tethered in the INM via nucleoplasmic interactions with nuclear lamins or chromatin (22.Lusk C.P. Blobel G. King M.C. Highway to the inner nuclear membrane: rules for the road.Nat. Rev. Mol. Cell Biol. 2007; 8: 414-420Crossref PubMed Scopus (161) Google Scholar, 23.Hinshaw J.E. Carragher B.O. Milligan R.A. Architecture and design of the nuclear pore complex.Cell. 1992; 69: 1133-1141Abstract Full Text PDF PubMed Scopus (351) Google Scholar24.Zuleger N. Korfali N. Schirmer E.C. Inner nuclear membrane protein transport is mediated by multiple mechanisms.Biochem. Soc. Trans. 2008; 36: 1373-1377Crossref PubMed Scopus (29) Google Scholar). Because the peripheral channels are thought to be about 10 nm, nucleoplasmic domains cannot exceed masses of >60 kDa (25.Reichelt R. Holzenburg A. Buhle Jr, E.L. Jarnik M. Engel A. Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.J. Cell Biol. 1990; 110: 883-894Crossref PubMed Scopus (370) Google Scholar, 26.Holzenburg A. Wilson F.H. Finbow M.E. Ford R.C. Structural investigations of membrane proteins: the versatility of electron microscopy.Biochem. Soc. Trans. 1992; 20: 591-597Crossref PubMed Scopus (12) Google Scholar27.Soullam B. Worman H.J. Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.J. Cell Biol. 1995; 130: 15-27Crossref PubMed Scopus (182) Google Scholar). Although the exact signals are unknown, at least 15 proteins have been shown to translocate through peripheral channels to reside on the INM (20.Zuleger N. Kerr A.R. Schirmer E.C. Many mechanisms, one entrance: membrane protein translocation into the nucleus.Cell. Mol. Life Sci. 2012; 69: 2205-2216Crossref PubMed Scopus (38) Google Scholar, 21.Katta S.S. Smoyer C.J. Jaspersen S.L. Destination: inner nuclear membrane.Trends Cell Biol. 2014; 24: 221-229Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 28.Zuleger N. Kelly D.A. Richardson A.C. Kerr A.R. Goldberg M.W. Goryachev A.B. Schirmer E.C. System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics.J. Cell Biol. 2011; 193: 109-123Crossref PubMed Scopus (77) Google Scholar). Current findings show that a wide range of mechanisms underlie transmembrane INM transport, including use of phenylalanine-glycine (FG) motifs, use of nucleoporins, and use of the peripheral channels or the central channel or both (20.Zuleger N. Kerr A.R. Schirmer E.C. Many mechanisms, one entrance: membrane protein translocation into the nucleus.Cell. Mol. Life Sci. 2012; 69: 2205-2216Crossref PubMed Scopus (38) Google Scholar, 21.Katta S.S. Smoyer C.J. Jaspersen S.L. Destination: inner nuclear membrane.Trends Cell Biol. 2014; 24: 221-229Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Certain INM proteins also contain an NLS, although mutation or deletion does not affect INM localization (20.Zuleger N. Kerr A.R. Schirmer E.C. Many mechanisms, one entrance: membrane protein translocation into the nucleus.Cell. Mol. Life Sci. 2012; 69: 2205-2216Crossref PubMed Scopus (38) Google Scholar). Many peptide-activated GPCRs also use an NLS and karyopherins such as importin-β1 for nuclear import after receptor activation on the cell surface (16.Joyal J.S. Nim S. Zhu T. Sitaras N. Rivera J.C. Shao Z. Sapieha P. Hamel D. Sanchez M. Zaniolo K. St-Louis M. Ouellette J. Montoya-Zavala M. Zabeida A. Picard E. et al.Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis.Nat. Med. 2014; 20: 1165-1173Crossref PubMed Scopus (55) Google Scholar). Inasmuch as these receptors are not associated with the INM but rather appear within the nucleoplasm itself (via unknown mechanisms), the nucleoplasm targeting motifs might be quite different. Thus, unlike karyopherin-mediated transport of soluble proteins, targeting proteins to the INM is more varied and complex. One intracellular GPCR found on the INM is the metabotropic glutamate receptor 5 (mGluR5). mGluR5 plays a key role in normal neurodevelopment as well as in many neurodevelopmental and neurological disorders such as Fragile X syndrome/Autism Spectrum Disorders, anxiety, schizophrenia, drug addiction, Parkinson's disease, dyskinesias, and chronic pain (1.Jong Y.J. Sergin I. Purgert C.A. O'Malley K.L. Location-dependent signaling of the group 1 metabotropic glutamate receptor mGlu5.Mol. Pharmacol. 2014; 86: 774-785Crossref PubMed Scopus (41) Google Scholar). Although mGluR5 is found on the plasma membrane, 60–90% of the receptor is associated with intracellular membranes like the INM (29.Hubert G.W. Paquet M. Smith Y. Differential subcellular localization of mGluR1a and mGluR5 in the rat and monkey substantia nigra.J. Neurosci. 2001; 21: 1838-1847Crossref PubMed Google Scholar30.O'Malley K.L. Jong Y.J. Gonchar Y. Burkhalter A. Romano C. Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons.J. Biol. Chem. 2003; 278: 28210-28219Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 31.Jong Y.J. Kumar V. Kingston A.E. Romano C. O'Malley K.L. Functional metabotropic glutamate receptors on nuclei from brain and primary cultured striatal neurons. Role of transporters in delivering ligand.J. Biol. Chem. 2005; 280: 30469-30480Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32.Hermans E. Challiss R.A. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.Biochem. J. 2001; 359: 465-484Crossref PubMed Scopus (336) Google Scholar33.Vincent K. Cornea V.M. Jong Y.J. Laferrière A. Kumar N. Mickeviciute A. Fung J.S. Bandegi P. Ribeiro-da-Silva A. O'Malley K.L. Coderre T.J. Intracellular mGluR5 plays a critical role in neuropathic pain.Nat. Commun. 2016; 7: 10604Crossref PubMed Scopus (48) Google Scholar). Pharmacological isolation and genetic manipulation have allowed us to determine that INM mGluR5 couples to Gq/11 leading to a large sustained release of luminal Ca2+, phosphorylation of nuclear Erk1/2, and activation of immediate early genes such as Elk-1, Fos, Fosl1, and FosL2, and immediate early effector proteins such as Arc (34.Kumar V. Jong Y.J. O'Malley K.L. Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.J. Biol. Chem. 2008; 283: 14072-14083Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 35.Kumar V. Fahey P.G. Jong Y.J. Ramanan N. O'Malley K.L. Activation of intracellular metabotropic glutamate receptor 5 in striatal neurons leads to up-regulation of genes associated with sustained synaptic transmission including Arc/Arg3.1 protein.J. Biol. Chem. 2012; 287: 5412-5425Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar36.Jong Y.J. Kumar V. O'Malley K.L. Intracellular metabotropic glutamate receptor 5 (mGluR5) activates signaling cascades distinct from cell surface counterparts.J. Biol. Chem. 2009; 284: 35827-35838Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Physiologically, intracellular mGluR5 contributes to long term depression in the hippocampus (19.Purgert C.A. Izumi Y. Jong Y.J. Kumar V. Zorumski C.F. O'Malley K.L. Intracellular mGluR5 can mediate synaptic plasticity in the hippocampus.J. Neurosci. 2014; 34: 4589-4598Crossref PubMed Scopus (66) Google Scholar) and pathological pain in the spinal cord dorsal horn (33.Vincent K. Cornea V.M. Jong Y.J. Laferrière A. Kumar N. Mickeviciute A. Fung J.S. Bandegi P. Ribeiro-da-Silva A. O'Malley K.L. Coderre T.J. Intracellular mGluR5 plays a critical role in neuropathic pain.Nat. Commun. 2016; 7: 10604Crossref PubMed Scopus (48) Google Scholar). To further dissect mGluR5's subcellular functions, defining the sequence motifs responsible for its localization is necessary. Using molecular, immunological, and optical techniques, here we show that 25 amino acids within the mGluR5 nucleoplasmic domain are necessary and sufficient for its localization to the INM. Moreover, mGluR5 appears to be tethered in place via interactions with chromatin. Thus, mGluR5 appears to use a non-canonical signal sequence-retention strategy to anchor itself on the INM where it is poised to regulate transcription (35.Kumar V. Fahey P.G. Jong Y.J. Ramanan N. O'Malley K.L. Activation of intracellular metabotropic glutamate receptor 5 in striatal neurons leads to up-regulation of genes associated with sustained synaptic transmission including Arc/Arg3.1 protein.J. Biol. Chem. 2012; 287: 5412-5425Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), chromosome remodeling, and genomic integrity. Previously, we have shown that mGluR5 can be expressed on the PM and on intracellular membranes, including the ER, ONM, and INM (30.O'Malley K.L. Jong Y.J. Gonchar Y. Burkhalter A. Romano C. Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons.J. Biol. Chem. 2003; 278: 28210-28219Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 31.Jong Y.J. Kumar V. Kingston A.E. Romano C. O'Malley K.L. Functional metabotropic glutamate receptors on nuclei from brain and primary cultured striatal neurons. Role of transporters in delivering ligand.J. Biol. Chem. 2005; 280: 30469-30480Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 33.Vincent K. Cornea V.M. Jong Y.J. Laferrière A. Kumar N. Mickeviciute A. Fung J.S. Bandegi P. Ribeiro-da-Silva A. O'Malley K.L. Coderre T.J. Intracellular mGluR5 plays a critical role in neuropathic pain.Nat. Commun. 2016; 7: 10604Crossref PubMed Scopus (48) Google Scholar). To date, no motifs responsible for maintaining mGluR5 or other INM GPCRs in this location have been described. Because trafficking of GPCRs is often dictated by sequences within the cytoplasmic tail (37.Walther C. Ferguson S.S. Arrestins: role in the desensitization, sequestration, and vesicular trafficking of G protein-coupled receptors.Prog. Mol. Biol. Transl. Sci. 2013; 118: 93-113Crossref PubMed Scopus (37) Google Scholar38.Magalhaes A.C. Dunn H. Ferguson S.S. Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins.Br. J. Pharmacol. 2012; 165: 1717-1736Crossref PubMed Scopus (240) Google Scholar, 39.Dunn H.A. Ferguson S.S. PDZ protein regulation of GPCR trafficking and signaling pathways.Mol. Pharmacol. 2015; 88: 624-639Crossref PubMed Scopus (80) Google Scholar, 40.Dores M.R. Trejo J. Atypical regulation of G protein-coupled receptor intracellular trafficking by ubiquitination.Curr. Opin. Cell Biol. 2014; 27: 44-50Crossref PubMed Scopus (19) Google Scholar41.Shirvani H. Gätà G. Marullo S. Regulated GPCR trafficking to the plasma membrane: general issues and the CCR5 chemokine receptor example.Subcell. Biochem. 2012; 63: 97-111Crossref PubMed Scopus (7) Google Scholar), we hypothesized that the mGluR5 C terminus is the domain required for INM localization. To test this idea, we prepared HA-tagged chimeric constructs derived from mGluR5 and the closely related GABAB2 GPCR. Typically, GABAB2 receptors form heterodimers with GABAB1, masking an ER retention signal (42.Margeta-Mitrovic M. Jan Y.N. Jan L.Y. A trafficking checkpoint controls GABA(B) receptor heterodimerization.Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar), following which the heterodimer is efficiently transported to the PM (43.White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Heterodimerization is required for the formation of a functional GABA(B) receptor.Nature. 1998; 396: 679-682Crossref PubMed Scopus (1013) Google Scholar). Because GABAB2 always traffics to the PM, it serves as a control for PM localization. Thus, chimeric plasmids were created in which the C termini of mGluR5 and GABAB2 were swapped (Fig. 1A). For this experiment and others described below, HA-tagged control and chimeric receptors were transiently transfected into HEK293 cells and subsequently stained for PM expression using antibodies directed against HA on non-permeabilized cells. All constructs showed at least some level of PM expression, although absolute amounts varied as indicated by the line scans and western blots associated with FIGURE 1, FIGURE 2 (and data not shown). Because the HA tag is in the extracellular ligand binding domain, these results suggest that introduced receptors assume the correct orientation within the PM and that regardless of the described manipulation, the receptor could be found on the PM. Immunocytochemical analyses of permeabilized cells revealed that the chimeric receptor, NmG5CGB2, was found predominately at the cell surface whereas NGB2CmG5 was on NM and ER membranes. In particular, NGB2CmG5 co-localized with the NM marker lamin B2 whereas NmG5CGB2 did not (Fig. 1A). Line scan analysis corroborated that mGluR5 and NGB2CmG5 co-localized with lamin B2 on NM (Fig. 1, A–C). We also assessed the intensity of fluorescent signals detected on either the PM or NM across multiple experiments. These results confirmed that the C terminus of mGluR5 prevented the chimeric NGB2CmG5 from going to the cell surface, and in contrast, the GABAB2 C terminus predominantly localized mGluR5 lacking its C terminus at the PM (Fig. 1, A and D). To confirm and extend these results, we used subcellular fractionation. Following transient transfection, mGluR5 was readily visible in NM fractions (Fig. 2A), whereas GABAB2 was not (Fig. 2B). Consistent with the immunocytochemistry, the NmG5CGB2 construct was primarily detected in the PM fraction (Fig. 2C); whereas NGB2CmG5 was detected in both PM and nuclear fractions (Fig. 2D). Quantitation of results from multiple experiments is shown in Fig. 2E. Treatment with 1 mm glutamate or baclofen (mGluR5 and GABAB2 agonists, respectively) did not produce an obvious re-distribution of receptor subtypes in these cultures (Fig. 11B, and not shown). Taken together, these data indicate that the C terminus is necessary and sufficient for NM localization of mGluR5. To further define regions critical for NM localization, we prepared three different constructs to examine the involvement of the proximal regions of the mGluR5 C terminus for NM localization. Because the size of the C-terminal region available to go through the nuclear pore might influence its ability to do so (22.Lusk C.P. Blobel G. King M.C. Highway to the inner nuclear membrane: rules for the road.Nat. Rev. Mol. Cell Biol. 2007; 8: 414-420Crossref PubMed Scopus (161) Google Scholar), additional constructs were deliberately kept within the size range of the original mGluR5 C terminus. Thus, constructs were similar to the NmG5CGB2 construct but carried small C-terminal extensions downstream of the seventh transmembrane domain of mGluR5 before the addition of the GABAB2 C terminus (Fig. 3A). Distribution and orientation of the constructs NmG5_854CGB2, NmG5_896CGB2, and NmG5_966CGB2 were examined by immunostaining and subcellular fractionation methods (Fig. 3, A–G). Lamin B2 co-localization was observed for the NmG5_896CGB2 and NmG5_966CGB2 constructs in HEK293 cells; however, NmG5_854CGB2 was primarily localized on the PM (Fig. 3, A–C). This distribution pattern was further confirmed by subcellular fractionation (Fig. 3, D–G). Occasionally, doublet bands were seen in some PM and NM fractions, which presumably reflect the glycosylation state of the receptor at the time of processing. This interpretation is consistent with results below (Fig. 11A), in which similar doublets were seen prior to PNGase F treatment but not afterward. These results suggest that mGluR5 amino acids 855–896 are required for NM localization. To further define which amino acids were responsible for nuclear targeting, we separately deleted amino acids 852–876 (Δ852–876) and amino acids 877–896 (Δ877–896) from the HA-tagged full-length mGluR5 plasmid (Fig. 4, A and B). HA immunostaining indicated that the Δ852–876 construct was largely in the ER, whereas Δ877–896 still showed strong co-localization with lamin B2 (Fig. 4, A and B). Thus, these results suggest that amino acids 852–876 contain the minimal region for NM localization. To determine whether these sequences were sufficient to target the nucleus on their own, we created a construct with an N-terminal Kozak sequence followed by mGluR5 amino acids 827–992, an HA epitope, and then a stop codon. Because there are no transmembrane domains in this construct, it would not be associated with any membrane. However, if nuclear localization signals are found within this stretch of amino acids, then presumably karyopherins will recognize these motifs and promote the transport of the receptor fragment into the nucleoplasm. We also created new constructs, including the Δ852–876 deletion as well as the Δ877–896 deletion in the mG5(827–992) backbone (Fig. 4C). As shown in Fig. 4, C and D, HA staining is only observed within the nucleus for constructs containing amino acids 852–876. In contrast, the construct missing those amino acids is largely found in the cytoplasm (Fig. 4, C and D). Therefore, these sequences contain sufficient information to be translocated into the nucleus. To confirm and extend these results in a more physiological environment, we tested the localization of constructs in dissociated striatal neurons that endogenously express mGluR5. Thus, rat striatal neurons were transfected with the chimeric constructs, proximal mGluR5 constructs, or deletion constructs (mGluR5, GABAB2, NmG5CGB2, NGB2CmG5, NmG5_854CGB2, NmG5_896CGB2, Δ852–876, and Δ877–896), subsequently immunostained, and then assessed for localization (Fig. 5, A and B). Lamin B2 co-localization was observed for the NGB2CmG5, NmG5_896CGB2, and Δ877–896 constructs, similar to what was observed in HEK293 cells. These data are consistent with mGluR5 requiring amino acids 852–876 for NM localization. To determine whether more distal mGluR5 C-terminal sequences also played a role in NM localization, we prepared additional constructs consisting of the entire GABAB2 receptor fused with distal mGluR5 C-terminal stretches (NGB2CmG5_827–966, NGB2CmG5_967–1036, NGB2CmG5_967–1106, and NGB2CmG5_1107–1171). This strategy kept the total length of the chimeric C terminus a" @default.
• W2572498644 created "2017-01-26" @default.
• W2572498644 creator A5031921543 @default.
• W2572498644 creator A5073942417 @default.
• W2572498644 creator A5081198928 @default.
• W2572498644 creator A5082371543 @default.
• W2572498644 creator A5087730558 @default.
• W2572498644 date "2017-03-01" @default.
• W2572498644 modified "2023-10-15" @default.
• W2572498644 title "Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization" @default.
• W2572498644 cites W102867438 @default.
• W2572498644 cites W1480534106 @default.
• W2572498644 cites W1503184886 @default.
• W2572498644 cites W1517348949 @default.
• W2572498644 cites W1534968983 @default.
• W2572498644 cites W1571586310 @default.
• W2572498644 cites W190223546 @default.
• W2572498644 cites W1966244470 @default.
• W2572498644 cites W1966495757 @default.
• W2572498644 cites W1968778693 @default.
• W2572498644 cites W1974152360 @default.
• W2572498644 cites W1976986632 @default.
• W2572498644 cites W1981719698 @default.
• W2572498644 cites W1982737894 @default.
• W2572498644 cites W1984608574 @default.
• W2572498644 cites W1985153924 @default.
• W2572498644 cites W1986752758 @default.
• W2572498644 cites W1989344763 @default.
• W2572498644 cites W1993655108 @default.
• W2572498644 cites W1994363140 @default.
• W2572498644 cites W1997924950 @default.
• W2572498644 cites W2000187790 @default.
• W2572498644 cites W2002309357 @default.
• W2572498644 cites W2009086729 @default.
• W2572498644 cites W2016336187 @default.
• W2572498644 cites W201847332 @default.
• W2572498644 cites W2018626241 @default.
• W2572498644 cites W2022990675 @default.
• W2572498644 cites W2023313219 @default.
• W2572498644 cites W2026346661 @default.
• W2572498644 cites W2027055752 @default.
• W2572498644 cites W2027056512 @default.
• W2572498644 cites W2027642569 @default.
• W2572498644 cites W2028291889 @default.
• W2572498644 cites W2030796378 @default.
• W2572498644 cites W2032876747 @default.
• W2572498644 cites W2034079946 @default.
• W2572498644 cites W2035681553 @default.
• W2572498644 cites W2036970799 @default.
• W2572498644 cites W2042675248 @default.
• W2572498644 cites W2043485308 @default.
• W2572498644 cites W2043929240 @default.
• W2572498644 cites W2047935630 @default.
• W2572498644 cites W2050393168 @default.
• W2572498644 cites W2058162240 @default.
• W2572498644 cites W2061189653 @default.
• W2572498644 cites W2068006705 @default.
• W2572498644 cites W2068107769 @default.
• W2572498644 cites W2068742572 @default.
• W2572498644 cites W2075168304 @default.
• W2572498644 cites W2075591794 @default.
• W2572498644 cites W2079003718 @default.
• W2572498644 cites W2081040572 @default.
• W2572498644 cites W2085174561 @default.
• W2572498644 cites W2103292884 @default.
• W2572498644 cites W2115352418 @default.
• W2572498644 cites W2116265895 @default.
• W2572498644 cites W2116501553 @default.
• W2572498644 cites W2122778614 @default.
• W2572498644 cites W2124702700 @default.
• W2572498644 cites W2126174089 @default.
• W2572498644 cites W2140192917 @default.
• W2572498644 cites W2152993287 @default.
• W2572498644 cites W2156961771 @default.
• W2572498644 cites W2161584310 @default.
• W2572498644 cites W2165533815 @default.
• W2572498644 cites W2171991181 @default.
• W2572498644 cites W2305551271 @default.
• W2572498644 cites W2333061151 @default.
• W2572498644 cites W2334972854 @default.
• W2572498644 cites W2344982738 @default.
• W2572498644 cites W2406525517 @default.
• W2572498644 cites W2417795456 @default.
• W2572498644 cites W4248835773 @default.
• W2572498644 doi "https://doi.org/10.1074/jbc.m116.757724" @default.
• W2572498644 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5339749" @default.
• W2572498644 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28096465" @default.
• W2572498644 hasPublicationYear "2017" @default.
• W2572498644 type Work @default.
• W2572498644 sameAs 2572498644 @default.
• W2572498644 citedByCount "31" @default.
• W2572498644 countsByYear W25724986442017 @default.
• W2572498644 countsByYear W25724986442018 @default.
• W2572498644 countsByYear W25724986442019 @default.
• W2572498644 countsByYear W25724986442020 @default.
• W2572498644 countsByYear W25724986442021 @default.
• W2572498644 countsByYear W25724986442022 @default.
• W2572498644 countsByYear W25724986442023 @default.

• next