Re M , Pampillo M , Savard M , Dubuc C , McArdle CA , Millar RP , Conn PM , Gobeil F , Bhattacharya M , Babwah AV .

HD-18185 NICHD NIH HHS , HD-19899 NICHD NIH HHS , MOP#89832 Canadian Institutes of Health Research , RR-00163 NCRR NIH HHS , P51 OD011092 NIH HHS , R01 HD019899 NICHD NIH HHS , U54 HD018185 NICHD NIH HHS , P51 RR000163 NCRR NIH HHS , R37 HD019899 NICHD NIH HHS , K01 RR000163 NCRR NIH HHS , P30 HD018185 NICHD NIH HHS

	Figure 1. Effect of epitope tag on hGnRH-RI signaling and spatial localization of FLAG-hGnRH-RI.(A) Inositol phosphate (IP) production in response to 100 nM Buserelin was assessed as described in “Materials and Methods”. Data represent three to six independent experiments performed in triplicate and normalized to FLAG-hGnRHRI ± S.E. (B, left) HTR-8/SVneo cells transfected with either FLAG-hGnRH-RI (a) subjected to indirect immunofluorescent staining using affinity purified rabbit anti-FLAG antibody followed by Alexa Fluor 568-conjugated anti-rabbit IgG (red) and counterstained with the nuclear dye, Hoechst (blue). Note the perinuclear localization of the FLAG-tagged hGnRH-RI (a, arrow); (B, right) HTR-8/SVneo cell transfected with GnRH-RI-GFP and counterstained with the nuclear dye, Hoechst (blue). Note the perinuclear localization of the GnRH-RI-GFP. (C) HTR-8/SVneo and HEK 293 cells transfected with FLAG-hGnRH-RI were subjected to indirect immunofluorescent staining using affinity purified rabbit anti-FLAG antibody followed by Alexa Fluor 568-conjugated anti-rabbit IgG (red) and counterstained with Hoechst (blue). Note the perinuclear localization of the FLAG-tagged hGnRH-RI seen in both cell lines (arrows). Bright field images are shown in the second column from the left. (D) Western blot analysis was performed on the lysates of nuclei isolated from HEK 293 cells overexpressing FLAG-GnRH-RI. The results reveal that the full length hGnRH-RI is expressed on the nuclei of HEK 293 cells. (E) Cells expressing FLAG-hGnRH-RI were subjected to indirect immunofluorescent staining for the receptor (red) as well as the nuclear membrane, endoplasmic reticulum and Golgi (all shown in green). Colocalization is seen as yellow staining. Column A: receptor alone; Column B: receptor + Hoescht; Column C: organelle marker (lamin A/C, calnexin or GM130) + Hoescht; Column D: receptor + organelle marker. Column letters and roman numerals used as a coordinate system. hGnRH-RI immunoreactivity co-localized with the nuclear marker, lamin A/C, as seen by the yellow staining (I-D, II-D, arrows) as well as with the endoplasmic reticulum marker, calnexin (III-D, IV-D). Less colocalization was seen between the receptor and the Golgi, as shown by less yellow staining (V-D, VI-D). Scale bar = 10 µm.
	Figure 2. Spatial localization of FLAG-mGluR5a, FLAG-β2AR and FLAG-hGnRH-RI.(A) HTR-8/SVneo cells transfected with either FLAG-mGluR5a or FLAG-β2AR were subjected to indirect immunofluorescent staining for the receptor (red) as well as the nuclear membrane marker lamin A/C (green). Cells were counterstained with Hoechst (blue). Note the perinuclear localization of the FLAG-tagged mGluR5a (arrow) and the plasma membrane localization of both FLAG-mGluR5a and FLAG-β2AR (arrowheads). Bright field images are shown in the second row from top. (B) Isolated nuclei from HEK293 cells expressing FLAG-hGnRH-RI, FLAG-mGluR5a or FLAG-β2AR were subjected to indirect immunofluorescent staining for the receptor (red) as well as the nuclear membrane marker lamin A/C (green). Cells were counterstained with Hoechst (blue). Note the colocalization of both FLAG-hGnRH-RI and FLAG-mGluR5a with the inner nuclear membrane marker lamin A/C (yellow, arrows). FLAG-β2AR was not localized to the nuclear membrane. Bright field images are shown in the second row from top. Scale bar = 10 µm.
	Figure 3. Effect of putative NLS and lysine 191 hGnRH-RI mutants on receptor localization and signaling and spatial localization of FLAG-mGnRH-RI in HTR-8/SVneo cells.(A) Schematic of the human GnRH-RI showing the positions of the lysine 191 residue (triangle) and the location of the putative NLS (square). (B) HTR-8/SVneo cells expressing FLAG-hGnRH-RI in which the putative NLS was deleted were subjected to indirect immunofluorescent staining for the receptor (red) as well as the markers for the nuclear membrane, endoplasmic reticulum and Golgi (shown in green). Cells were counterstained with Hoechst (blue). The NLS deletion mutant showed the same phenotype as the wild-type receptor, showing strong colocalization with lamin A/C and calnexin and less colocalization with GM130. (C) IP formation data represent seven independent experiments performed in triplicate and normalized to FLAG-hGnRHRI ± S.E. ***, p<0.001 versus IP formation of FLAG-hGnRH-RI. (D) HTR-8/SVneo cells expressing FLAG-hGnRH-RI in which lysine 191 was deleted or mutated to a glutamic acid residue (K191E) were subjected to indirect immunofluorescent staining for the receptor (red) as well as the markers for the nuclear membrane, endoplasmic reticulum and Golgi (shown in green). Cells were counterstained with Hoechst (blue). K191 deletion and K191E mutants showed the same phenotype as the wild-type receptor, showing strong colocalization with lamin A/C and calnexin and less colocalization with GM130. (E) IP formation data represent 6–7 independent experiments performed in triplicate and normalized to FLAG-hGnRHRI ± S.E. (F) HTR-8/SVneo cells expressing mGnRH-RI were subjected to indirect immunofluorescent staining for the receptor (red) as well as the markers for the nuclear membrane, endoplasmic reticulum and Golgi (shown in green). Cells were counterstained with Hoechst (blue). FLAG-mGnRH-RI showed similar perinuclear localization (arrows) to FLAG-hGnRH-RI with strong colocalization with lamin A/C and less colocalization with GM130. However, less colocalization was seen between the FLAG-mGnRH-RI and calnexin than was seen with the FLAG-hGnRH-RI. As well, there was evidence of plasma membrane localization (arrowheads) with FLAG-mGnRH-RI that was not seen with FLAG-hGnRH-RI. Bright field images are shown in the second row from top. Scale bar = 10 µm.
	Figure 4. Spatial localization of FLAG-human-Xenopus (HX)-GnRH-RI and FLAG-Xenopus (X)-GnRH-RI in HEK 293 cells using organellar markers.Cells expressing FLAG-GnRH-RI (HX and X) were subjected to indirect immunofluorescent staining for the receptor (red) as well as the nuclear membrane, endoplasmic reticulum and Golgi (all shown in green). Colocalization is seen as yellow staining. Column A: receptor alone; Column B: receptor + Hoescht; Column C: organelle marker (lamin A/C, calnexin or GM130) + receptor; Column D: receptor + organelle marker. Column letters and roman numerals used as a coordinate system. HX-GnRH-RI immunoreactivity co-localized with the nuclear marker, lamin A/C, as seen by the yellow staining (I-D, arrow) as well as the endoplasmic reticulum marker, calnexin (II-D). Less colocalization was seen between the receptor and the Golgi, as shown by less yellow staining (III-D). X-GnRH-RI immunoreactivity strongly localized to the plasma membrane (yellow arrowheads) and weakly in cytoplasm. No visual co-localization detected with lamin A/C (IV-D, arrow), calnexin (V-D) or Golgi (VI-D). Scale bar = 10 µm.
	Figure 5. Effect of GnRH stimulation on Histone 3 acetylation/phosphorylation levels in isolated nuclei from HEK 293 transiently transfected with FLAG-GnRH-RI.(A) Purity assessment of nuclei preparations used in functional assays by bright field microscopy and Western blotting. Left panel: Photomicrograph of a typical nuclei preparation following trypan blue staining. Scale bar = 10 µm. Insert: digital magnification of a single nucleus showing the presence of nucleoles (arrow). Right panel: Immunodetection of the membrane and nuclear markers clathrin and lamin A/C respectively in membrane and isolated nuclei fractions from FLAG-GnRH-R-transfected HEK 293 cells. (B) Time course of Histone H3-Lys9/Lys14 acetylation and -Ser10 phosphorylation in GnRH-stimulated nuclei by Western blot analysis. A representative Western blot is shown. Densitometric values of acetylated and phosphorylated Histone H3 levels were normalized to those of total Histone H3 and the relative intensity ratios were expressed in-fold increase over time zero. Data are means ± S.E.M of four independent experiments. *P<0.05 vs control time zero. Statistical analyses performed using one-way ANOVA followed by Dunnet's ad hoc test.

Arora, Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. 1995, Pubmed

Arora, Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. 1995, Pubmed
Babwah, Protein kinase C isoform-specific differences in the spatial-temporal regulation and decoding of metabotropic glutamate receptor1a-stimulated second messenger responses. 2003, Pubmed
Bhattacharya, Ral and phospholipase D2-dependent pathway for constitutive metabotropic glutamate receptor endocytosis. 2004, Pubmed
Bhattacharya, Nuclear localization of prostaglandin E2 receptors. 1998, Pubmed
Bhattacharya, Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. 1999, Pubmed
Boivin, Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes. 2003, Pubmed
Brothers, Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression. 2006, Pubmed
Brothers, Unexpected effects of epitope and chimeric tags on gonadotropin-releasing hormone receptors: implications for understanding the molecular etiology of hypogonadotropic hypogonadism. 2003, Pubmed
Cao, The composition of the beta-2 adrenergic receptor oligomer affects its membrane trafficking after ligand-induced endocytosis. 2005, Pubmed
Cavanagh, Gonadotropin-releasing hormone-regulated chemokine expression in human placentation. 2009, Pubmed
Chelsky, Sequence requirements for synthetic peptide-mediated translocation to the nucleus. 1989, Pubmed , Xenbase
Chen, A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation. 2000, Pubmed
Cheng, Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. 2000, Pubmed
Chou, Cellular localization of gonadotropin-releasing hormone (GnRH) I and GnRH II in first-trimester human placenta and decidua. 2004, Pubmed
Cornea, Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer. 2001, Pubmed
Eggena, Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA. 1993, Pubmed
Finch, Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines. 2008, Pubmed , Xenbase
Gardner, AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the beta1-adrenergic receptor promotes recycling and functional resensitization of the receptor. 2006, Pubmed
Gobeil, G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigm. 2006, Pubmed
Gobeil, Regulation of eNOS expression in brain endothelial cells by perinuclear EP(3) receptors. 2002, Pubmed
Graham, Establishment and characterization of first trimester human trophoblast cells with extended lifespan. 1993, Pubmed
Grosse, Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. 2000, Pubmed
Hislop, Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization? 2005, Pubmed , Xenbase
Janovick, Evolved regulation of gonadotropin-releasing hormone receptor cell surface expression. 2003, Pubmed
Janovick, Regulation of G protein-coupled receptor trafficking by inefficient plasma membrane expression: molecular basis of an evolved strategy. 2006, Pubmed
Janovick, Refolding of misfolded mutant GPCR: post-translational pharmacoperone action in vitro. 2007, Pubmed
Janovick, Structure-activity relations of successful pharmacologic chaperones for rescue of naturally occurring and manufactured mutants of the gonadotropin-releasing hormone receptor. 2003, Pubmed
Jong, Functional metabotropic glutamate receptors on nuclei from brain and primary cultured striatal neurons. Role of transporters in delivering ligand. 2005, Pubmed
Jong, Nuclear localization of functional metabotropic glutamate receptor mGlu1 in HEK293 cells and cortical neurons: role in nuclear calcium mobilization and development. 2007, Pubmed
Kaiser, Chromosomal localization of the gonadotropin-releasing hormone receptor gene to human chromosome 4q13.1-q21.1 and mouse chromosome 5. 1994, Pubmed
Kakar, Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. 1992, Pubmed
Knollman, Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor. 2008, Pubmed
Leaños-Miranda, Receptor-misrouting: an unexpectedly prevalent and rescuable etiology in gonadotropin-releasing hormone receptor-mediated hypogonadotropic hypogonadism. 2002, Pubmed
Leaños-Miranda, In vitro coexpression and pharmacological rescue of mutant gonadotropin-releasing hormone receptors causing hypogonadotropic hypogonadism in humans expressing compound heterozygous alleles. 2005, Pubmed
Lee, Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors. 2004, Pubmed
Lu, Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons. 1998, Pubmed
Mangia, Gonadotropin releasing hormone receptor expression in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses. 2002, Pubmed
Maudsley, Gonadotropin-releasing hormone (GnRH) antagonists promote proapoptotic signaling in peripheral reproductive tumor cells by activating a Galphai-coupling state of the type I GnRH receptor. 2004, Pubmed
Maya-Núñez, Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression. 2000, Pubmed
Millar, Luteinizing hormone-releasing hormone (LH-RH) binding to purified rat pituitary nuclei. 1983, Pubmed
Morel, Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat. A quantitative autoradiographic study after cryoultramicrotomy. 1987, Pubmed
O'Malley, Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons. 2003, Pubmed
Petrovska, Addition of a signal peptide sequence to the alpha1D-adrenoceptor gene increases the density of receptors, as determined by [3H]-prazosin binding in the membranes. 2005, Pubmed
Petäjä-Repo, Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation. 2002, Pubmed
Pietilä, Inefficient maturation of the rat luteinizing hormone receptor. A putative way to regulate receptor numbers at the cell surface. 2005, Pubmed
Saito, RTP family members induce functional expression of mammalian odorant receptors. 2004, Pubmed
Salahpour, Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting. 2004, Pubmed
Savard, Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation. 2008, Pubmed
Sedgley, Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells. 2006, Pubmed , Xenbase
Spencer, Subcellular localization of prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy. 1998, Pubmed
Szende, The concentration of LH-RH receptors in the nuclei of pancreatic cancer cells. Effect of (D-Trp6)LH-RH on tumor-bearing Syrian golden hamsters. 1994, Pubmed
Tetsuka, The basic residues in the membrane-proximal C-terminal tail of the rat melanin-concentrating hormone receptor 1 are required for receptor function. 2004, Pubmed
Uberti, Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors. 2005, Pubmed
Vadakkadath Meethal, Identification of a gonadotropin-releasing hormone receptor orthologue in Caenorhabditis elegans. 2006, Pubmed
Vandenberghe, Interaction with the unfolded protein response reveals a role for stargazin in biosynthetic AMPA receptor transport. 2005, Pubmed
Venkatesan, A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression. 2001, Pubmed
Wang, NHERF1 regulates parathyroid hormone receptor membrane retention without affecting recycling. 2007, Pubmed
Watson, Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation. 2000, Pubmed
Watson, Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues. 2000, Pubmed