Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor
1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês
10.1074/jbc.273.37.24000
ISSN1083-351X
AutoresTomáš Drmota, Gwyn W. Gould, Graeme Milligan,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3–5 min after treatment with 1 × 10−7m TRH, with dramatic reductions in plasma membrane localization achieved within 10–15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist. The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3–5 min after treatment with 1 × 10−7m TRH, with dramatic reductions in plasma membrane localization achieved within 10–15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist. thyrotropin releasing hormone thyrotropin-releasing hormone receptor green fluorescent protein vesicular stomatitis virus phosphate-buffered saline Krebs-Ringer-Hepes. Thyrotropin-releasing hormone (TRH)1 is a hypothalamic tripeptide intimately involved in controlling the production of thyrotropin and prolactin from the anterior pituitary (1Gershengorn M.C. Physiol. Rev. 1996; 76: 175-191Crossref PubMed Scopus (144) Google Scholar, 2Hinkle P.M. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). TRH functions via binding to a seven-transmembrane element-G protein-coupled receptor (3Sellar R.E. Taylor P.L. Lamb R.F. Zabavnik J. Anderson L. Eidne K.A. J. Mol. Endocrinol. 1993; 10: 199-206Crossref PubMed Scopus (42) Google Scholar), which, by interacting selectivity with Gq and G11, causes activation of phospholipase Cβ1 and the hydrolysis of phosphatidylinositol 4,5-bisphosphate (4Hsieh K.P. Martin T.F.J. Mol. Endocrinol. 1992; 6: 1673-1681Crossref PubMed Google Scholar, 5Aragay A.M. Katz A. Simon M.I. J. Biol. Chem. 1992; 267: 24983-24988Abstract Full Text PDF PubMed Google Scholar, 6Kim G.D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar, 7Svoboda P. Kim G.D. Grassie M.A. Eidne K.A. Milligan G. Mol. Pharmacol. 1996; 49: 646-655PubMed Google Scholar). As with other G protein-coupled receptors there has been great interest in the mechanisms of regulation of the TRH receptor (TRHR) (6Kim G.D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar, 7Svoboda P. Kim G.D. Grassie M.A. Eidne K.A. Milligan G. Mol. Pharmacol. 1996; 49: 646-655PubMed Google Scholar, 8Anderson L. Alexander C.L. Faccenda E. Eidne K.A. Biochem. J. 1995; 311: 385-392Crossref PubMed Scopus (27) Google Scholar, 9Yu R. Hinkle P.M. J. Biol. Chem. 1997; 272: 28301-28307Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10Nussenzveig D.R. Heinflink M. Gershengorn M.C. J. Biol. Chem. 1993; 268: 2389-2392Abstract Full Text PDF PubMed Google Scholar, 11Ashworth R.R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar). One focus of studies on G protein-coupled receptors relates to processes contributing to desensitization, the phenomenon by which sustained exposure of a receptor to an agonist ligand results in a waning of cellular response to the ligand (12Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). Elements contributory to this phenomenon include receptor phosphorylation, which can either directly or indirectly result in a diminution of receptor-G protein interactions, internalization of the receptor and/or G protein, and alterations in cellular levels of these proteins (12Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). During studies of the TRHR we have noted that both the rat TRHR and the G proteins Gqα and G11α are internalized in response to agonist (13Drmota T. Novotny J. Kim G.D. Eidne K.A. Milligan G. Svoboda P. J. Biol. Chem. 1998; 273: 21699-21707Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Milligan G. Trends Endocrinol. Metab. 1998; 9: 13-19Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), although internalization of the G proteins proceeds substantially more slowly than the receptor (13Drmota T. Novotny J. Kim G.D. Eidne K.A. Milligan G. Svoboda P. J. Biol. Chem. 1998; 273: 21699-21707Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Milligan G. Trends Endocrinol. Metab. 1998; 9: 13-19Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The G proteins are subsequently down-regulated (7Svoboda P. Kim G.D. Grassie M.A. Eidne K.A. Milligan G. Mol. Pharmacol. 1996; 49: 646-655PubMed Google Scholar). The capacity of ligands to internalize G protein-coupled receptors has been studied using a wide range of approaches (15Petrou C. Chen L. Tashjian Jr., A.H. J. Biol. Chem. 1997; 272: 2326-2333Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 16von Zastrow M. Kobilka B.K J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 17Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar, 18Moore R.H. Sadornikoff N. Hoffenberg S. Liu S. Woodford P. Angelides K Trial J. Carsrud N.D.V. Dickey B.F. Knoll B.J. J. Cell Sci. 1995; 108: 2983-2991Crossref PubMed Google Scholar, 19Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 20Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (856) Google Scholar, 21Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Holtmann M.H. Roettger B.F. Pinon D.I. Miller L.J. J. Biol. Chem. 1996; 271: 23566-23571Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1190) Google Scholar, 24Ruiz-Gomez A. Mayor Jr., F. J. Biol. Chem. 1997; 272: 9601-9604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Roettger B.F. Ghanekar D. Rao T. Toledo C. Yingling J. Pinon D. Miller L.J. Mol. Pharmacol. 1997; 51: 357-362PubMed Google Scholar, 26Gaudriault G. Nouel D. Dal Farra C. Beaudet A. Vincent J.-P. J. Biol. Chem. 1997; 272: 2880-2888Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27Molino M. Bainton D.F. Hoxie J.A. Coughlin S.R. Brass L.F. J. Biol. Chem. 1997; 272: 6011-6017Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), including the use of fluorescent ligands, antireceptor antibodies, and antibodies to epitope tags, which have been introduced to alter the sequence of a cDNA or DNA encoding the receptor. These approaches have been instrumental in providing key information in relation to receptor regulation. However, for many of these, analyses cannot be performed in real time and on intact cells because it is often necessary to fix the cell before visualization. Recently, the green fluorescent protein (GFP) derived fromAequorea victoria has become a powerful adjunct to cell biological research as a molecular marker for gene expression (28Kakinoki Y. Somers J. Brautigan D.L. J. Biol. Chem. 1997; 272: 32308-32314Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 30Zernicka-Goetz M. Pines J. McLean Hunter S. Dixon J.P.C. Siemering K.R. Haseloff J. Evans M.J. Development. 1997; 124: 1133-1137PubMed Google Scholar). Very recently it has begun to be applied for analysis of the subcellular distribution and regulation of G protein-coupled receptors (14Milligan G. Trends Endocrinol. Metab. 1998; 9: 13-19Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 31Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar, 32Tarasova N.I. Stauber R.H. Choi J.K. Hudson E.A. Czerwinski G. Miller J.L. Pavlakis G.N. Michejda C.J. Wank S.A. J. Biol. Chem. 1997; 272: 14817-14824Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 33Kallal L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1998; 273: 322-328Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In the present study we utilize the stable expression of a chimeric protein in which a modified form of GFP (30Zernicka-Goetz M. Pines J. McLean Hunter S. Dixon J.P.C. Siemering K.R. Haseloff J. Evans M.J. Development. 1997; 124: 1133-1137PubMed Google Scholar) was attached to the COOH-terminal tail of the long isoform of the rat TRH receptor to analyze the processes contributing to agonist-mediated internalization and trafficking of this receptor. All materials for tissue culture were supplied by Life Technologies Inc. myo-[3H]Inositol was purchased from Amersham Pharmacia Biotech and [3H]TRH from NEN Life Science Products. Cytochalasin B and nocodazole were from Sigma. Oligonucleotides were purchased from Oswel (Southampton, U. K.). Texas Red transferrin was from Molecular Probes. Production and subcloning of the vesicular stomatitis virus (VSV)-tagged TRHR-GFP fusion protein were done in two separate steps. In the first step the coding sequence of the long isoform of the rat TRH receptor (3Sellar R.E. Taylor P.L. Lamb R.F. Zabavnik J. Anderson L. Eidne K.A. J. Mol. Endocrinol. 1993; 10: 199-206Crossref PubMed Scopus (42) Google Scholar) was modified by polymerase chain reaction amplification. Using the amino-terminal primer 5′-AAAGCTAGCGCCACCATGTACACCGATATAGAGATGAACAGGCTGGGAAAGGAGAATGAAACCGTCAGTGAACTGAAC-3′, an NheI restriction site and the VSV epitope (YTDIEMNRLGK) were introduced adjacent to the sequence of codon 2 of the TRHR. Using the COOH-terminal primer 5′-GCTATCTAGAGTCAAAGCTTCTCCTGTTTGGCAGTCAAA-3′, an XbaI restriction site following the stop codon and aHindIII restriction site just in the front of stop codon were introduced. The HindIII restriction site changed the last four nucleotides of the TRHR coding sequence from 5′-AATA-3′ to 5′-GCTT-3′ and thus altered the last amino acid from Ile to Leu. The amplified fragment of VSV-TRHR digested with NheI and XbaI was ligated to pcDNA3.1(+) expression vector (Invitrogen) digested with NheI and XbaI. The VSV-TRHR functionality was characterized by binding experiments and agonist-mediated inositol phosphate production (see “Results”). To obtain the VSV-TRHR-GFP fusion protein the coding sequence of a modified form of GFP (30Zernicka-Goetz M. Pines J. McLean Hunter S. Dixon J.P.C. Siemering K.R. Haseloff J. Evans M.J. Development. 1997; 124: 1133-1137PubMed Google Scholar) was amplified by polymerase chain reaction. Using the amino-terminal primer 5′-GAGAAGCTTGGAGCTATGAGTAAAGGAGAAGAACTTTTCACT-3′, aHindIII restriction site and a 2-amino acid spacer (Gly-Ala) were introduced in front of the initiator Met of GFP. Using the COOH-terminal primer 5′- TGCTCTAGATTATTTGTATAGTTCATCCATGCCATG-3′, an XbaI restriction site was introduced behind the stop codon of GFP. Finally, the VSV-TRHR construct in pcDNA3.1(+) was digested withHindIII (to remove the stop codon from the VSV-TRHR coding sequence), and XbaI and was ligated together with the polymerase chain reaction product of GFP amplification which was digested with HindIII and XbaI. The open reading frame so produced represents the coding sequence of VSV-TRHR-GFP. This was sequenced fully before its expression and analysis. HEK293 cells were maintained in minimal essential medium (Sigma) supplemented with 0.292 g/liter l-glutamine and 10% newborn calf serum at 37 °C. Cells were grown to 60–80% confluence before transient transfection. Transfection was performed using LipofectAMINE reagent (Life Technology, Inc.) according to the manufacturer's instructions. To generate cell lines stably expressing VSV-TRHR-GFP 2 days after transfection cells were seeded/diluted and maintained in minimal essential medium supplemented with 1 mg/ml Geneticin (Life Technology, Inc.). Medium was replaced every 3 days with minimal essential medium containing 1 mg/ml Geneticin. Clonal expression was examined initially by fluorescence microscopy, and clones for further study were selected and expanded. Transiently transfected HEK293 cells (24 h after transfection) or cell lines stably expressing either the TRHR or VSV-TRHR-GFP were reseeded into 12-well plates and incubated for a further 24 h. They were labeled with [3H]inositol (1 μCi/ml) in inositol-free Dulbecco's modified Eagle's medium supplemented with 2% dialyzed newborn calf serum and 1% glutamate for 24 h. On the day of the experiment cells were washed twice with Krebs-Ringer-Hepes-LiCl buffer (KRH/LiCl) (115 mm NaCl, 5 mm KCl, 15 mm LiCl, 1.2 mm MgSO4, 1.2 mm CaCl2, 20 mm Hepes, 1.2 mm Na2PO4, 10 mmglucose, 0.1% bovine serum albumin, pH 7.4), incubated for 10 min with KRH/LiCl, and stimulation by varying concentrations of TRH was performed in the same buffer for 10 min. All manipulations were done at 37 °C. Reactions were stopped by aspiration of the KRH/LiCl/TRH buffer, and the cells were lysed using 0.75 ml of 20 mmformic acid on ice (30 min). Supernatant fractions were centrifuged (14,000 × g for 3 min). The following steps were carried out according to Ref. 34Conklin B.R. Chabre O. Wong Y.H. Federman A.D. Bourne H.R J. Biol. Chem. 1992; 267: 31-34Abstract Full Text PDF PubMed Google Scholar. Supernatant fractions were loaded onto Dowex (Sigma, Dowex 1 X-8-200) columns followed by the immediate addition of 3 ml of 50 mm NH4OH ([3H]inositol fraction). The columns were then washed with 4 ml of 40 mm ammonium formate followed by 5 ml of 2m ammonium formate ([3H] inositol phosphates fraction). Data are presented as the quotient of [3H]inositol phosphates divided by inositol phosphates plus [3H]inositol. For binding studies on intact cells, they were harvested using 0.5 mm EDTA in PBS, washed, and used directly for experiments. Membranes were prepared as described previously (6Kim G.D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar, 13Drmota T. Novotny J. Kim G.D. Eidne K.A. Milligan G. Svoboda P. J. Biol. Chem. 1998; 273: 21699-21707Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Both types of experiment were performed with 10 nm [3H]TRH and the addition of different concentrations of TRH at 4 °C for 60 min. Free ligand was separated by vacuum filtration through GF/C filters followed by three washes (each 5 ml) with ice-cold buffer A (2 mm MgCl2, 40 mm Tris, pH 7.4). In the case of intact cell binding, cells were incubated in Krebs-Ringer-Hepes buffer (KRH) (130 mm NaCl, 5 mm KCl, 1.2 mmMgSO4, 1.2 mm CaCl2, 20 mm Hepes, 1.2 mmNa2PO4, 10 mm glucose, 0.1% bovine serum albumin, pH 7.4). Membrane binding was performed with 40 μg of membrane protein in buffer A with the addition of 100 μmp[NH]ppG to convert the TRHR into a single class of low affinity sites. Cells were observed using a laser scanning confocal microscope (Zeiss Axiovert 100) using a Zeiss Plan-Apo 63 × 1.40 NA oil immersion objective, pinhole of 35, and electronic zoom 1 or 3. The GFP was exited using a 488 nm argon/krypton laser and detected with 515–540 nm band pass filter. The Texas Red-modified transferrin was exited at 543 nm and detected with a long pass band filter 570 nm. The images were manipulated with Zeiss LSM or MetaMorph software. Two different protocols for preparation of cells were used. When examining the time course of internalization, short time exposures to TRH, and the Texas Red transferrin colocalization studies, live cells were used. Cells were grown on glass coverslips and mounted on the imaging chamber. Cells were maintained in KRH buffer, and temperature was maintained at 37 °C. Cell labeling by Texas Red transferrin was performed by incubation for 10 min in KRH buffer with 10 μg/ml Texas Red transferrin, and after washing with KRH (three times) the cells were used for analysis. In other studies fixed cells were used. Cells on glass coverslips were washed with PBS and fixed for 20 min at room temperature using 4% paraformaldehyde in PBS and 5% sucrose, pH 7.2. After one wash with PBS coverslips were mounted on microscope slides with 40% glycerol in PBS. Cytochalasin B and nocodazole were dissolved as 500× concentrated stock solutions in dimethyl sulfoxide and finally used in concentrations of 4 μg/ml and 10−5m, respectively. Cytochalasin B was added 30 min and nocodazole 60 min before TRH treatment. Cells were seeded to 12-well plates, and the amount of [3H]TRH internalized was measured. On the day of the experiment, the medium was changed to serum-free minimal essential medium, and cells were equilibrated for 1 h in 5% CO2 at 37 °C. In some cases cells were treated with sucrose or with cytoskeletal modifying drugs before the addition of 10 nm [3H]TRH. After incubation, plates were placed on ice, washed three times with 0.15 mNaCl, one time with 0.5 m NaCl and 0.2 m acetic acid, and finally one time with 0.15 m NaCl to remove excess and plasma membrane-bound [3H]TRH. Cells were lysed with 1.5% SDS and 1.5% Triton X-100 and internalized radioactivity measured in this fraction. To estimate nonspecific binding, 10−5m TRH was added. A cDNA encoding the long isoform of the rat TRHR was modified such that an 11-amino acid (YTDIEMNRLGK) VSV epitope tag was added to the NH2 terminus of the encoded protein (VSV-TRHR). This construct was modified further by a polymerase chain reaction-based strategy such that a cDNA encoding a highly fluorescent and thermostabilized form of the A. victoria GFP (30Zernicka-Goetz M. Pines J. McLean Hunter S. Dixon J.P.C. Siemering K.R. Haseloff J. Evans M.J. Development. 1997; 124: 1133-1137PubMed Google Scholar) was linked in-frame with the VSV-TRHR cDNA to generate a VSV-TRHR-GFP cDNA. For convenience of construction this resulted in the alteration of the final amino acid of the TRHR from Ile to Leu and the incorporation of a 2-amino acid (Gly-Ala) linker between the two proteins now contained within the chimeric construct (Fig. 1). Transient expression of the VSV-TRHR-GFP cDNA in HEK293 cells followed by fixation and imaging in a confocal microscope demonstrated expression of the construct. The expressed protein had a predominantly plasma membrane localization (14Milligan G. Trends Endocrinol. Metab. 1998; 9: 13-19Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To characterize the integrity and functionality of VSV-TRHR-GFP it was expressed transiently in HEK293 cells in parallel with both the unmodified TRHR and VSV-TRHR. The cells were labeled subsequently with [3H]inositol (1 μCi/ml, 24 h), and the capacity of varying concentrations of TRH to stimulate the generation of [3H]inositol phosphates was measured. All three TRHR constructs allowed robust stimulation of [3H]inositol phosphate production (Fig. 2 with TRH displaying slightly greater potency at VSV-TRHR-GFP (EC50 = 3.2 × 10−9m) compared with either VSV-TRHR (EC50 = 6.2 × 10−9m) or TRHR (EC50 = 6.9 × 10−9m). However, the fold stimulation of [3H]inositol phosphate production over basal produced by VSV-TRHR-GFP (3.2-fold) was lower than that produced by either VSV-TRHR (6.7-fold) or TRHR (6.2-fold). The capacity of each construct to bind [3H]TRH in membranes prepared from these transiently transfected cells and for this to be competed for by increasing concentrations of TRH allowed approximate assessment of the levels of expression and affinity for TRH by application of the formalisms of DeBlasi et al. (35DeBlasi A. O'Reilly K. Motulsky H.J. Trends Pharmacol. Sci. 1989; 10: 227-229Abstract Full Text PDF PubMed Scopus (235) Google Scholar) to the binding data. Over a series of such transfections the estimated K d for TRH at VSV-TRHR-GFP was 3.3 × 10−8m, for VSV-TRHR it was 3.8 × 10−8m, and for TRHR it was 4.9 × 10−8m. With clear demonstration that the VSV-TRHR-GFP construct was able both to bind TRH with high affinity and to activate G proteins and second messenger responses upon addition of agonist, this construct was expressed stably in HEK293 cells. A single clone, designated VTGP1, was selected for detailed analysis based on expression of the construct with an essentially homogeneous plasma membrane distribution (see later) and maintained contact inhibition of the cells (data not shown). Intact cell binding studies were performed on cells of clone VTGP1 (Fig. 3). These experiments indicated a K d for TRH of 3.8 ± 1.2 × 10−8m with expression of VSV-TRHR-GFP at 1.2 ± 0.2 × 105copies/cell. Clone VTGP1 cells were visualized in a confocal microscope and then exposed to 1 × 10−7m TRH to allow real time and direct visualization of VSV-TRHR-GFP (Fig. 4). Initially the construct was concentrated heavily at the plasma membrane of the cells (Fig. 4 a ). However, within 3–5 min distinct internalization was occurring (Fig. 4 b). This became more pronounced such that within 10–15 min the bulk of the VSV-TRHR-GFP had been relocated away from the plasma membrane (Fig. 4 c), an effect that was maintained at subsequent times up to at least 30 min (Fig. 4 d). To establish that occupation of VSV-TRHR-GFP by TRH was required to produce internalization/sequestration we examined the concentration dependence and pharmacology of the effect. Cells were incubated with or without the low affinity TRHR antagonist/inverse agonist chlordiazepoxide (100 μm) (36Hinkle P.M. Shanshala E.D. II Mol. Endocrinol. 1989; 3: 1337-1344Crossref PubMed Scopus (21) Google Scholar) in the presence of concentrations of TRH ranging from 5 × 10−10m to 5 × 10−8m (Fig. 5) for 30 min and fixed and visualized. Chlordiazepoxide alone did not alter the cellular distribution of VSV-TRHR-GFP (Fig. 5 a). 5 × 10−10m TRH produced little internalization of VSV-TRHR-GFP (data not shown); however, 5 × 10−9m TRH caused strong internalization (Fig. 5 b). Coadministration of chlordiazepoxide (100 μm) along with 5 × 10−9m TRH largely inhibited internalization (Fig. 5 c), but 5 × 10−8m TRH was able to overcome the blockade of internalization caused by the antagonist (Fig. 5 d). The presence of hyperosmotic conditions has been established to block internalization processes that utilize clathrin-coated pits and vesicles (37Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (181) Google Scholar, 38Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (791) Google Scholar). To establish the concentration requirements for sucrose, intact clone VTGP1 cells in medium were exposed to 1 × 10−8m [3H]TRH for 30 min at 37 °C after preexposure in the presence or absence of either 0.3m or 0.4 m sucrose for 20 min. Subsequently the cells were acid washed to remove cell surface-bound and excess [3H]TRH, and the acid-resistant (internalized) pools of specific [3H]TRH binding were determined. In the absence of sucrose, a substantial amount of specific [3H]TRH binding was acid-resistant and thus represented internalized VSV-TRHR-GFP. However, little acid-resistant [3H]TRH binding was detected in the presence of 0.4 m sucrose, and the levels of acid-resistant [3H]TRH binding were reduced substantially in the presence of 0.3 m sucrose (Fig. 6). We wished to equate this biochemical measurement of VSV-TRHR-GFP internalization with direct visualization. Using the same assay conditions, TRH induced strong internalization of VSV-TRHR-GFP in a 30-min period in the absence of sucrose (Fig. 7, a and b). Dramatic differences were observed, however, in the presence of sucrose and indeed between the two concentrations of sucrose used. In the presence of 0.4 m sucrose, little internalization was observed in response to TRH (Fig. 7 c). However, the VSV-TRHR-GFP was now distributed nonuniformly around the plasma membrane with clear punctate concentrations. In the presence of 0.3 m sucrose TRH produced a composite pattern in which a significant fraction of VSV-TRHR-GFP was internalized, but equally a significant fraction was located at punctate but plasma membrane-delineated sites (Fig. 7 d).Figure 7Hyperosmolar sucrose prevents internalization of VSV-TRHR-GFP: imaging studies. The capacity of TRH (1 × 10−7m) ( panels b–d) to cause redistribution and internalization of VSV-TRHR-GFP over a 30-min period was examined in the absence (panels a and b) or presence of sucrose (0.4 m, panel c or 0.3 m, panel d). Where appropriate, the cells had been treated with sucrose for 20 min before the addition of TRH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transferrin is internalized constitutively via clathrin-coated vesicles (39Mayor S. Presley J.F. Maxfield F.R. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (422) Google Scholar, 40Ghosh R.N. Gelman D.L. Maxfield F.R. J. Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar). Dual wavelength scanning allowed concurrent detection of plasma membrane-located VSV-TRHR-GFP and internalized Texas Red transferrin in live, untreated, clone VTGP1 cells. Application of TRH resulted in a time-dependent internalization of VSV-TRHR-GFP into vesicle populations that were either identical with or overlapped those containing Texas Red transferrin as assessed by the merging of the red and green color signals over a 6–20-min period (Fig. 8). Several studies have suggested roles for the cellular cytoskeletal architecture in internalization processes (41Mukhopadhyay A. Funato K. Stahl P.D. J. Biol. Chem. 1997; 272: 13055-13059Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Treatment of cells with cytochalasin B, an inhibitor of microfilament function (4 μg/ml, 30 min), did not interfere with subsequent TRH-mediated internalization (Fig. 9 a). By contrast, pretreatment with the microtubule-disrupting agent nocodazole (1 × 10−5m, 60 min) resulted in TRH producing a punctate plasma membrane and periplasma membrane distribution pattern without resulting in the intense perinuclear, deep, cellular redistribution observed without pretreatment with nocodazole (Fig. 9 b). The acid resistance of the bulk of the intact cell binding of [3H]TRH after nocodazole treatment (Table I) demonstrated that the bulk of the observed VSV-TRHR-GFP was truly internalized rather than simply being bound to plasma membrane-localized TRHR which could be accessed by the low pH wash.Table INocodazole treatment does not block internalization of [H]TRH associated with VSV-TRHR-GFPConditionInternalized [3H]TRHfmol/dishControl369.6 ± 1.2Nocodazole339.7 ± 42.5Sucrose80.8 ± 3.4Intact clone VTGP1 cells were either untreated (control) or pretreated with nocodazole (1 × 10−5m, 60 min) or with hyperosmotic shock (0.4 m sucrose, 20 min). The specific internalization of [3H]TRH was then followed over a 30-min period at 37 °C as under “Experimental Procedures.” Results are from a representative experimen
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