Alteration of intracellular histamine H2 receptor cycling precedes antagonist-induced upregulation
Satoshi Osawa,1
Masayoshi Kajimura,1
Seiji Yamamoto,2
Mutsuhiro Ikuma,1
Chihiro Mochizuki,1
Hirohiko Iwasaki,1
Akira Hishida,1 and
Susumu Terakawa2
1First Department of Medicine and 2Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
Submitted 2 December 2004
; accepted in final form 10 June 2005
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ABSTRACT
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Long-term administration of a histamine H2 receptor (H2R) antagonist (inverse agonist) induces upregulation of H2R in parietal cells, which may be relevant to the rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H2R upregulation could be related to receptor trafficking and used H2R-green fluorescent protein (H2R-GFP) to test the hypothesis. Human H2R-GFP was generated and functionally expressed in HEK-293 cells. Binding of the H2R antagonist [3H]tiotidine was performed to quantify H2R expression, and H2R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [3H]tiotidine was not significantly different between H2R-GFP- and wild-type H2R-expressing HEK-293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H2R-GFP revealed that the agonist-induced H2R internalization and the antagonist-induced recycling of the internalized H2R from the recycling endosome within 2 h. Long exposure to the antagonist increased GFP fluorescence in the plasma membrane and also induced upregulation of H2R-GFP estimated by the binding assay, whereas long exposure to the agonist enhanced degradative trafficking of H2R-GFP. We examined whether the upregulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H2R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on H2R upregulation. These findings suggested that upon exposure to an antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered before H2R upregulation, probably via suppressing H2R degradation.
endocytosis; recycling; inverse agonist; internalization; constitutive activity
THE HISTAMINE H2 RECEPTOR (H2R) belongs to the large family of G protein-coupled receptors (GPCRs) and plays important physiological roles in the regulation of gastric acid secretion, cell differentiation and proliferation, the immunological response, and central nervous system function (4, 9, 11, 25). H2R antagonists have been widely used to treat acid-related diseases such as peptic ulcer and reflux esophagitis. Long-term administration of H2R antagonists leads to tachyphylaxis, which is a tolerance to the inhibition of acid secretion, and to rebound acid hypersecretion after any abrupt withdrawal of treatment; both outcomes have important clinical implications (28). Several clinical studies have confirmed that these phenomena can occur in patients receiving a H2R antagonist (2224) irrespective of concurrent use of a proton pump inhibitor (6).
We (37) previously demonstrated that prolonged treatment with the H2R antagonist famotidine induced an upregulation of H2R and an increase in adenylate cyclase activity in rabbit parietal cells and that this may cause rebound acid hypersecretion. H2R acts as a constitutively active GPCR in vitro, and most of the H2R antagonists that have been widely used to treat acid-related diseases act as pharmacologically characterized "inverse agonists," which reduce the constitutive activity of the receptor (1, 31). Upregulation of other GPCRs may be induced by prolonged treatment with antagonists in constitutively active mutants, whereby the antagonist acts as an inverse agonist, but this is not seen with constitutively inactive receptors, for which the antagonists act as a neutral agonist (14, 15, 18, 36). This antagonist-induced upregulation of H2R may be due to positive feedback effects of the inverse agonism of H2R antagonists, although a possible mechanism for this has not been demonstrated.
One important physiological mechanism of the regulation of GPCR signaling is receptor endocytosis to endosomes, from where they may be recycled back to the plasma membrane or trafficked to lysosomes for proteolytic degradation (7, 19, 39, 43). Because different GPCR subtypes show distinct endocytic trafficking patterns (38, 40), it is crucial to characterize the intracellular trafficking of individual GPCR subtypes. Previous studies into H2R regulation have revealed that upon agonist stimulation, H2R is desensitized via receptor phosphorylation by G protein receptor kinases (26, 30), which leads to endocytosis of the receptor (8, 33, 34). Persistent treatment with an agonist induced H2R downregulation in CHO cells expressing rat H2R, and this was suggested to be mediated by posttranscriptional mRNA instability (32). In endothelial cells, persistent treatment with an agonist induced downregulation of both cell surface and mRNA expression of H2R (29). Because most of this evidence is based on binding assays, Western blot analysis of cell lysates, and the second messenger response, little is known about the intracellular trafficking and localization of H2R, especially during treatment with a H2R antagonist.
The aim of the present study was to investigate the role of receptor trafficking in antagonist-induced upregulation of H2R. We therefore sought to characterize the intracellular localization patterns of H2R and to examine the effect of a H2R antagonist on receptor trafficking in living cells using functional green fluorescent protein (GFP)-tagged human H2R in HEK-293 cells. We monitored the trafficking of GFP-tagged H2R with various markers for intracellular organelles and evaluated changes in the localization of H2R upon antagonist exposure. We also examined whether the receptor upregulation is dependent on the augmentation of de novo receptor synthesis.
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MATERIALS AND METHODS
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Materials.
Histamine dihydrochloride, cycloheximide (CHX), and isobutylmethylxanthine (IBMX) were obtained from Sigma (St. Louis, MO). Famotidine was kindly donated by Yamanouchi (Tokyo, Japan), and cimetidine and ranitidine were kindly donated by GlaxoSmithKline (Tokyo, Japan). [3H]tiotidine was purchased from Perkin-Elmer Life Science (Boston, MA). A genomic DNA clone of human H2R was kindly provided by Dr. M. Futai (Department of Organic Chemistry and Biochemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan).
Construction of GFP-tagged form of human H2R.
The entire coding region of the human H2R gene with the exception of the stop codon was amplified by PCR using an EcoRI forward primer (5'-CCGGAATTCGTCCCAGGATGGCACCC-3') and an ApaI reverse primer (5'-GAAGGGCCCACCTGTCTGTGGCTCCCTGG-3') from a
gt11 clone template, which contained a 12.3-kb 5'-upstream sequence, the 1,080-bp coding region, and a 245-bp 3'-downstream sequence (21). The PCR product (containing nucleotide positions 8 to 1,077) was digested with EcoRI and ApaI, and the resultant fragment was ligated into the multiple cloning site of the pEGFP-N1 vector (Clontech Laboratories; Palo Alto, CA) to generate an expression vector for a fusion protein of human H2R and GFP (pH2R-EGFP). The insert for the wild-type human H2R expression vector was constructed as follows: a human H2R clone containing the full coding region and the stop codon was amplified by PCR using an EcoRI forward primer (5'-CCGGAATTCGTCCCAGGATGGCACCC-3') and a NotI reverse primer (5'-AAAGCGGCCGCTTACCTGTCTGTGGCTCCC-3'). The PCR product (containing nucleotide positions 8 to 1,080) was digested with EcoRI and NotI, and the resultant fragment was ligated into the pEGFP-N1 vector, in which the open reading frame of enhanced GFP (EGFP) had been previously removed (pH2R-wild).
Cell culture and transfection.
HEK-293 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin at 37°C in 5% CO2-95% air. The cells were grown to 7080% confluence and transiently transfected with 1 µg of plasmid DNA per 0.5 ml medium using LipofectAMINE 2000 reagent (Life Technologies; Carlsbad, CA) according to the manufacturer's instructions.
Measurement of cAMP production.
HEK-293 cells seeded onto 24-well plates were transfected with pH2R-EGFP, pH2R-wild, or pEGFP-N1 vector (mock transfection) and incubated for 24 h. The cells were incubated in the presence or absence of various ligands in DMEM-HEPES supplemented with 300 µM IBMX, a phosphodiesterase inhibitor, for 10 min at 37°C. After rapid aspiration of the incubation medium, the reaction was stopped by the addition of 200 µl of 0.1 M ice-cold HCl. The cells were kept on ice and disrupted by sonication. The resultant homogenate was neutralized with 1 M NaOH and assayed for cAMP content by radioimmunoassay using 125I-labeled cAMP with a Yamasa Cyclic AMP Assay Kit (Yamasa Shoyu; Chiba, Japan). cAMP levels were expressed as picomoles of cAMP per milligram of protein per 10 min.
H2R binding.
HEK-293 cells transfected with pH2R-EGFP or pH2R-wild were trypsinized and removed from the plates. A total of 1 x 105 cells was transferred to a 0.6-ml centrifuge tube and incubated at 37°C for 80 min with various concentrations of [3H]tiotidine, a radiolabeled H2R antagonist, in sodium phosphate buffer A (132.4 mM NaCl, 5.4 mM KCl, 5.0 mM Na2HPO4, 1.0 mM NaH2PO4, 1.2 mM MgSO4, 1.0 mM CaCl2, 10 mM HEPES, 0.2% bovine serum albumin, and 2 mg/ml glucose; pH 7.4) in a total volume of 200 µl. The cell suspension was cooled on ice and centrifuged for 5 min at 15,000 g at 4°C. The supernatant was aspirated, and the resultant pellet was rinsed once with 500 µl of ice-cold PBS. The radioactivity was measured with a liquid scintillation counter (LSC-5100, Aloka; Tokyo, Japan). Nonspecific binding was defined as that observed in the presence of 10 µM famotidine and estimated by Scatchard plot analysis. Assays were performed in triplicate in time course experiments and in duplicate in the others.
Imaging by confocal and evanescent wave microscopy.
Time-lapse images of H2R-GFP were obtained using a confocal laser microscope (IX 70, Olympus; Tokyo, Japan) equipped with a microlens-attached Nipkow-disk scanner (CSU-10, Yokokawa Electric; Tokyo, Japan), using an Olympus Apo x100 [1.65 numerical aperture (NA)] oil-immersion objective lens. The confocal fluorescence images were recorded with a charge-coupled device camera (C2400, Hamamatsu Photonics; Hamamatsu, Japan) combined with an image intensifier (C2400-21SV, Hamamatsu Photonics) and analyzed with MetaMorph software (Universal Imaging; Downingtown, PA). To avoid losing the object in the single confocal section, three-dimensional confocal images were recorded at 1-µm z-intervals, starting at the bottom of the cell using IP Lab (Scanalysis; Fairfax, VA). Cells grown on 35-mm glass-base dishes were used for the experiments in medium without phenol red and antibiotics at 37°C in 5% CO2-95% air.
To determine the intracellular localization of H2R-GFP and rhodamine-transferrin (Molecular Probes; Eugene, OR), cells were incubated in medium lacking fetal bovine serum for 30 min, followed by an incubation with 40 µg/ml rhodamine-transferrin for the indicated time at 37°C in 5% CO2-95% air, and the cells were then quickly rinsed three times to remove all excess rhodamine-transferrin and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature before imaging analysis was started. Merged images of H2R-GFP and rhodamine-transferrin were captured by a Bio-Rad MRC-600 (Hemmelholsteadt, UK) using a Nicon Plan-Apo x60 (1.40 NA) oil-immersion objective. We used the 504- to 524-nm laser line with a 527- to 565-nm band-pass filter and 600-nm long-pass filter for the detection of GFP and rhodamine, respectively.
To assess fluorescence of H2R-GFP localized in the plasma membrane, we also used evanescent wave microscopy. The incident light for evanescent illumination was introduced from the objective lens [PlanApo x60 (1.45 NA), Olympus]. To observe the GFP fluorescence image, we used a 473-nm laser for the evanescent wave excitation and a long-pass filter (515 nm) for emission. The capture and analysis of the images were performed in the same manner as with the confocal microscope.
Immunocytochemistry.
HEK-293 cells grown in 35-mm glass-base dishes were transiently transfected with H2R-GFP for 24 h. Cells stimulated with 100 µM histamine for the indicated times were washed with PBS, fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature, and then permeabilized with 0.2% Triton X-100 in PBS for 8 min. The cells were then incubated in blocking solution for 5 min before incubation in primary antibody for 1 h at room temperature. Antibodies against early endosomal antigen (EEA)-1 (Transduction Laboratories; San Diego, CA), Rab11 (Zymed Laboratories; San Francisco, CA), Golgin-97 (Molecular Probes), and lysosomal-associated membrane protein (LAMP)-1 (BD Biosciences-Pharmigen; San Diego, CA) were diluted 1:100, 1:200, 1:200, and 1:200, respectively. After three washes in PBS, cells were incubated in Alexa 594-conjugated secondary antibody (Molecular Probes) for 1 h. Images were acquired with a Bio-Rad MRC-600 confocal laser scanning microscope using the 504- to 524-nm laser line with a 527- to 565-nm band-pass filter and 600-nm long-pass filter for the detection of GFP and Alexa 594, respectively.
Quantification of mRNA levels of H2R.
mRNA expression levels of H2R in HEK-293 cells were quantified and normalized against
-actin by real-time quantitative PCR using a Light Cycler rapid thermal cycler system (Roche Diagnostics; Lewes, UK). Isolation of total RNA and cDNA synthesis was performed using a Cells-to-cDNA II kit (Ambion; Austin, TX). The primers used were for H2R [5'-CAATGTGGTCGTCTGTCTGG-3' (forward) and 5'-AGGCTGTGCAGAGCATCAC-3' (reverse)] and for
-actin [5'-GCACCACACCTTCTACAATGAG-3' (forward) and 5'-ATAGCACAGCCTGGATAGCAAC-3' (reverse)]. Product specificity was confirmed in the initial experiment by agarose gel electrophoresis and routinely performed by melting curve analysis.
Statistical analysis.
Data are expressed as means ± SE. A paired Student's t-test was used for comparisons of increases in H2R binding sites between control and treated groups. An unpaired Student's t-test was used for comparisons of the H2R binding characteristics between wild-type H2R and H2R-GFP groups and for comparisons of the H2R binding sites between two groups at each time point in the time course experiment and in the experiment using CHX. A level of P < 0.05 was considered to be statistically significant.
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RESULTS
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Functional expression of human H2R-GFP fusion protein in HEK-293 cells.
Wild-type human H2R and human H2R-GFP fusion protein, in which the COOH-terminus of H2R was directly linked to the NH2-terminus of EGFP, were transiently transfected into HEK-293 cells (Fig. 1A). Expression of wild-type H2R or H2R-GFP was characterized using [3H]tiotidine, a H2R antagonist, at 24 h after transfection. The presence of GFP did not change the estimated dissociation constant, and the number of [3H]tiotidine binding sites (Bmax) was not different between cells expressing wild-type H2R and H2R-GFP (Table 1). Both overexpressed H2R-GFP and wild-type H2R in HEK-293 cells showed the same degree of agonist-independent constitutive activity for adenylate cyclase, which was measured as any elevation of basal cAMP levels. The specific H2R antagonists famotidine and cimetidine decreased the basal production of cAMP, indicating that both acted as inverse agonists. Histamine (100 µM) markedly increased the levels of cAMP in both H2R-GFP- and wild-type H2R-transfected cells (Table 1).

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Fig. 1. DNA constructs expressed in HEK-293 cells and cellular localization of histamine H2 receptor (H2R)-green fluorescent protein (GFP) fusion protein. A: schematic diagram of the DNA constructs expressed in HEK-293 cells. B: HEK-293 cells expressing H2R-GFP imaged by confocal microscopy. C: HEK-293 cells expressing H2R-GFP imaged by evanescent wave microscopy. Typical findings at 24 h after transfection are represented. Bars = 10 µm.
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We then examined whether persistent treatment with various H2R antagonists for 24 h could induce upregulation of H2R-GFP or wild-type H2R in HEK-293 cells. No effect on the affinity for [3H]tiotidine was recorded in either of the transfected cells (data not shown). Bmax increased by approximately twofold irrespective of the presence of GFP (Table 1). These results showed that both H2R-GFP and wild-type H2R were functionally coupled to intracellular signaling and could be regulated by ligand treatment.
In resting HEK-293 cells, H2R-GFP was predominantly observed at the plasma membrane by confocal microscopy (Fig. 1B). Evanescent wave microscope (41) also showed that H2R-GFP diffusely but prominently localized to the plasma membrane of resting cells in culture dishes (Fig. 1C).
Agonist-induced internalization of H2R-GFP in HEK-293 cells.
To determine the intracellular localization of H2R in detail, we first examined the intracellular trafficking of H2R-GFP during agonist exposure. To assess the change in intracellular localization of H2R-GFP, the three-dimensional distribution of internalized H2R-GFP was determined using confocal time-lapse imaging in sequential z-axis planes. Imaging in living cells expressing H2R-GFP after treatment with 100 µM histamine revealed internalized GFP in discrete intracellular vesicles at 30 min and thereafter in the perinuclear region up to 120 min (Fig. 2A). We also examined whether antagonist treatment causes any internalization of H2R-GFP. There was no internalization of H2R-GFP during treatment, up to 120 min, with 100 µM famotidine or 100 µM cimetidine (Fig. 2, B and C).

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Fig. 2. Agonist-induced internalization of H2R-GFP in HEK-293 cells The three-dimensional distribution of intracellular H2R-GFP was determined in a living cell using confocal time-lapse imaging in sequential z-axis planes. The images were recorded at 1-µm z-intervals, starting at the bottom of the cell, and 10 selected planes were represented at each period after drug application. A: application of 100 µM histamine induced internalization of H2R-GFP. B and C: application of 100 µM famotidine (B) or 100 µM cimetidine (C) did not induce internalization of H2R-GFP. Typical findings are represented. Bars = 10 µm.
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To confirm that the agonist-induced internalization of H2R-GFP corresponded to receptor endocytosis, we examined the colocalization of H2R-GFP with rhodamine-transferrin, a fluorescent marker of endocytosis. Internalized H2R-GFP showed overlapping staining with transferrin in endosomal compartments at both 30 and 60 min after histamine treatment (Fig. 3).

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Fig. 3. Intracellular colocalization of H2R-GFP with rhodamine (R)-transferrin during histamine treatment. HEK-293 cells expressing H2R-GFP were incubated with 40 µg/ml rhodamine-transferrin and treated with 100 µM histamine for the indicated times. Images for GFP (green) and rhodamine (red) were obtained by confocal microscopy. Typical findings are represented. Note that H2R-GFP was colocalized with rhodamine-transferrin in endosomes. Bars = 10 µm.
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Characterization of H2R-GFP-positive intracellular compartments.
To determine the specific intracellular organelle involved in H2R internalization, cells were stained with markers for early endosomes (EEA-1) (3, 20), perinuclear recycling endosomes (Rab11) (35, 42, 45), the trans-Golgi network (Golgin-97), and lysosomes (LysoTracker red) and then observed for the localization of H2R-GFP. After 30 min in the presence of 100 µM histamine, H2R-GFP translocated to the early endosome, stained with EEA-1 (Fig. 4E), and by 60 min was localized in the perinuclear recycling endosome, identified by Rab11 (Fig. 4F). There was little or no colocalization of H2R-GFP with Golgin-97 (Fig. 4G) or LysoTracker red (Fig. 4H) during internalization.

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Fig. 4. Intracellular colocalization of H2R-GFP with markers of various organelles during histamine treatment. HEK-293 cells expressing H2R-GFP were fixed, permeabilized, and immunolabeled for either early endosomal antigen (EEA)-1 (A and E), Rab11 (B and F), or Golgin-97 (C and G). Images for GFP (green) and Alexa 594 (red) fluorescence were obtained by confocal microscopy. HEK-293 cells expressing H2R-GFP were incubated with LysoTracker red in the presence or absence of histamine (D and H). Images for GFP (green) and LysoTracker red (red) were obtained in living cells by confocal microscopy. Typical findings are represented. AD: H2R-GFP localized mainly to the plasma membrane in the absence of histamine (control experiments). E: H2R-GFP was observed in early endosomes at 30 min in the presence of histamine, where it colocalized with EEA-1. F: H2R-GFP was trafficked to perinuclear recycling endosomes at 60 min in the presence of histamine and was colocalized with Rab11 at these sites. G: a small amount of H2R-GFP colocalized with Golgin-97 at 60 min in the presence of histamine. H: a small amount of H2R-GFP colocalized with LysoTracker red at 60 min in the presence of histamine. Bars = 10 µm.
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Short exposure to antagonist (inverse agonist) induces recycling of internalized H2R-GFP.
To examine whether antagonist induces trafficking of the internalized H2R, we performed time-lapse imaging of living cells expressing H2R-GFP in sequential z-axis planes. In the presence of 100 µM famotidine, histamine (100 µM) could not induce the internalization of H2R-GFP (Fig. 5A). The further addition of 100 µM famotidine after pretreatment with 100 µM histamine recycled the internalized H2R-GFP to the plasma membrane within 120 min (Fig. 5B). When the antagonist (famotidine) was not added, histamine-induced internalization of H2R-GFP continued in the same duration (Fig. 5C).

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Fig. 5. Visualization of antagonist-induced recycling of internalized H2R-GFP in HEK-293 cells. The three-dimensional distribution of internalized H2R-GFP was determined in a living cell using confocal time-lapse imaging in sequential z-axis planes. The images were recorded at 1-µm z-intervals, starting at the bottom of the cell, and 10 selected planes were represented. A: there was no agonist-induced internalization after treatment with 100 µM famotidine plus 100 µM histamine. B: addition of 100 µM famotidine after 80-min pretreatment with 100 µM histamine induced recycling of internalized H2R-GFP. The indicated times are shown; exposure to famotidine and exposure to histamine are indicated by brackets. C: H2R-GFP internalization continued in the presence of 100 µM histamine without the addition of famotidine. Typical findings are represented. Bars = 10 µm.
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Long exposure to antagonist (inverse agonist) induces upregulation of H2R-GFP.
To visualize the antagonist-induced upregulation of H2R-GFP, we used both confocal and evanescent wave microscopy to follow changes in GFP fluorescence in H2R-GFP-transfected living cells upon continuous treatment with 10 µM famotidine or 100 µM cimetidine for 24 h. When HEK-293 cells expressing H2R-GFP were treated with these H2R antagonists, there was a marked increase in GFP fluorescence in the plasma membrane compared with control levels (Fig. 6A). By evanescent wave microscopy, this increase in fluorescence was clearly detected adjacent to the basal plasma membrane attached to the glass-base dish. In the presence of 100 µM cimetidine, the diffuse fluorescence in the plasma membrane increased in a time-dependent manner (Fig. 6B).

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Fig. 6. Visualization of antagonist-induced upregulation of H2R-GFP in HEK-293 cells. Time-lapse images were recorded in a living cell. Typical findings are represented. A: persistent treatment with 10 µM famotidine for 24 h increased H2R-GFP fluorescence imaged by confocal microscopy, indicating upregulation. B: persistent treatment with 100 µM cimetidine for 24 h induced upregulation of H2R-GFP in the plasma membrane, as shown by evanescent wave microscope. Bars = 10 µm.
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Time course of H2R-GFP upregulation estimated by binding assay.
To characterize the time course of antagonist-induced upregulation, we also quantified H2R-GFP using the [3H]tiotidine binding assay. Upon continuous treatment with 100 µM cimetidine, [3H]tiotidine binding sites in HEK-293 cells expressing H2R-GFP significantly increased at 3 h, followed by a slower but persistent increase up to 24 h (Fig. 7A). To determine whether the antagonist-induced upregulation was reversible, HEK-293 cells expressing H2R-GFP continuously treated with 100 µM cimetidine for 24 h were rinsed to remove cimetidine. [3H]tiotidine binding, which was twofold higher with cimetidine treatment than in the control, gradually decreased to almost the control levels by 48 h after the removal of cimetidine (Fig. 7B).

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Fig. 7. Time course of H2R-GFP upregulation estimated by binding assay. A: antagonist-induced upregulation of H2R-GFP estimated by [3H]tiotidine binding [in disintegrations/min (dpm)]. The assay was performed with or without 100 µM cimetidine. Data represent means ± SE in triplicate assays. Similar results were obtained in 3 independent experiments. *P < 0.05 compared with control. B: recovery from upregulation of H2R-GFP after withdrawal of antagonist treatment. After 24-h pretreatment with 100 µM cimetidine, time-dependent recovery from upregulation was observed after the removal of cimetidine. Data shown as a percentage of the levels in cells with 100 µM cimetidine represent means ± SE in triplicate assays. Similar results were obtained in 3 independent experiments. *P < 0.05 compared with control.
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Analysis of mRNA levels and protein synthesis for upregulation of H2R-GFP.
To determine whether upregulation of H2R-GFP is mediated by an increase in mRNA levels, we performed real-time RT-PCR to quantify H2R mRNA over time. Treatment with famotidine or cimetidine had no detectable effects on the levels of H2R-GFP mRNA (Fig. 8A). To determine whether the upregulation is mediated at the translational level, we treated cells with CHX, an inhibitor of translation. Upregulation of H2R-GFP induced by 100 µM cimetidine for 24 h was dose dependently inhibited in the concurrent presence of CHX, although CHX did not alter the receptor amount without cimetidine (Fig. 8B). Thus antagonist-induced upregulation of H2R depends on the translation of H2R, whereas the receptor amount without ligand treatment (control experiment) is independent of protein translation. Furthermore, we asked whether maintenance of H2R upregulation depended on augmentation of H2R translation. Once the receptor was upregulated by the pretreatment with 100 µM cimetidine, the receptor amount was not affected by the addition of 60 µg/ml CHX in the presence of 100 µM cimetidine. In addition, after upregulation, the receptor number still decreased after the removal of cimetidine irrespective of the presence of CHX (Fig. 8C), indicating that CHX did not inhibit protein degradation. These findings suggested that the initial upregulation requires de novo protein synthesis, but this is not needed to maintain subsequent increases in receptor level.

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Fig. 8. Analysis of mRNA levels and protein synthesis during upregulation of H2R-GFP. A: quantitative RT-PCR of H2R-GFP mRNA during antagonist treatment. mRNA levels of H2R-GFP expressed in HEK-293 cells were quantified and normalized against -actin by real-time RT-PCR assay. The assay was performed with or without 10 µM famotidine or 100 µM cimetidine at 5 and 24 h. Data shown as a percentage of the levels in untreated cells represent means ± SE of 5 independent experiments. H2R-GFP mRNA levels did not increase significantly during antagonist treatment. B: dose-dependent inhibition by cycloheximide (CHX) of cimetidine-induced upregulation at 24 h. In each CHX treatment group, the assay was performed with or without 100 µM cimetidine. Data represent means ± SE in triplicate assays. Similar results were obtained in 3 independent experiments. *P < 0.05 compared with the values without cimetidine (control). C: treatment with CHX had no effect on either the upregulation of H2R-GFP or the recovery to normal levels after the removal of the antagonist. The receptor number upregulated by pretreatment with 100 µM cimetidine was not affected by the addition of 60 µg/ml CHX for 24 h, and recovery from upregulation after the removal of the antagonist was observed even in the presence of 60 µg/ml CHX. Data shown as a percentage of the levels in control cells represent means ± SE in triplicate assays. Similar results were obtained in 3 independent experiments. NS, not significant. *P < 0.05 compared with the values with cimetidine in each CHX treatment group.
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Degradative trafficking of H2R-GFP expressed in HEK-293 cells.
Because the antagonist-induced upregulation of H2R is suggested to depend on suppressing H2R degradation, we tried to confirm the degradative pathway of H2R-GFP expressed in HEK-293 cells. In many GPCRs, degradative trafficking has been observed before downregulation of the receptor with long exposure to agonist. Thus we examined whether H2R-GFP translocated to the lysosomal pathway with long exposure to H2R agonist. LAMP-1 is located in the limiting membranes of lysosomal pathway involving lysosomes and late endosomes (5). The application of 100 µM histamine for 6 h increased the colocalization between intracellular H2R-GFP and LAMP-1 (Fig. 9). This finding indicated that with agonist exposure the postendocytic pathway of H2R-GFP in HEK-293 cells was associated with the lysosomal pathway, resulting in receptor degradation.

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Fig. 9. Intracellular colocalization of H2R-GFP with a marker of degradative pathway by long exposure to agonist. HEK-293 cells expressing H2R-GFP were fixed, permeabilized, and immunolabeled for lysosomal-associated membrane protein (LAMP)-1. A and B: images for GFP (green; A) and Alexa 594-labeled LAMP-1 (red; B) fluorescence were obtained by confocal microscopy. Typical findings are represented. C: a small amount of H2R-GFP colocalized with LAMP-1 at 60 min in the presence of histamine. Long exposure to 100 µM histamine for 6 h increased colocalization between intracellular H2R-GFP and LAMP-1 (arrow). Bars = 10µm.
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DISCUSSION
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This study is the first to use living cells expressing functional GFP-tagged H2R to visualize intracellular H2R localization and sorting. The addition of GFP to the COOH-terminus of GPCRs has proven effective in monitoring the cellular distribution and regulation of these receptors (12). This study examined the mechanisms underlying the upregulation and intracellular trafficking of human H2R-GFP during treatment with an antagonist (inverse agonist). The main findings were that 1) agonist-dependent internalization involved initial accumulation in early endosomes, followed by trafficking to recycling endosomes; 2) short exposure to an antagonist (inverse agonist) induced recycling of the agonist-induced internalized receptor within 2 h; 3) long exposure to an antagonist (inverse agonist) for at least 3 h induced reversible upregulation of H2R, probably by suppressing H2R degradation; and 4) long exposure to an agonist enhanced the degradative trafficking of H2R-GFP.
Initially, we demonstrated that H2R was trafficked to the recycling endosome via early endosomes upon short exposure to agonist and then back to the plasma membrane upon agonist withdrawal. The trafficking pathway is similar to that used by transferrin and transferrin receptor, which have been well characterized. The time course of trafficking seems to vary among GPCRs. In the present study, H2R was internalized more slowly than the
2-adrenoceptor (13, 17, 44), angiotensin II type 1A receptor (10), or the serotonin type 2A receptor (2). This finding was consistent with the available data demonstrated in cell lines expressing H2R without a GFP tag, although the reasons for this difference in trafficking time among these receptors remains unclear.
Previous studies showed that longer exposure to a H2R antagonist induced upregulation of H2R (31, 37). In this study, we found this upregulation of H2R to be reversible upon withdrawal of the antagonist. In addition, we imaged the upregulation of H2R in the plasma membrane of living cells by confocal and evanescent wave microscopies, although the time-lapse imaging provides data that are only semiquantitative. Antagonist-induced upregulation of H2R was not due to any increase in the mRNA level but was initially inhibited by CHX, suggesting that de novo receptor protein synthesis was required for this initial step. These results are consistent with those in a previous report that demonstrated that upregulation of a
2-adrenoceptor constitutively active mutant induced by sustained treatment with an antagonist (inverse agonist) was not regulated at the mRNA level but was sensitive to inhibition of protein synthesis (14, 15). Of note, the elevated levels of H2R were not affected by the addition of CHX after cimetidine pretreatment, suggesting that the contribution of protein synthesis to maintaining elevated receptor levels is not considerable. These results indicate that translation is kept on a certain level and that receptor degradation and synthesis are in balance, irrespective of CHX treatment, and thus the equilibrium maintains the upregulation.
In the present study, we demonstrated two effects of antagonist (inverse agonist) on H2R regulation: 1) recycling to the plasma membrane without any detectable change in total receptor amount within 2 h and 2) upregulation after long exposure to antagonist. The direct link between antagonist-induced upregulation and recycling of H2R remains unclear, and no previous studies have discussed this link. However, in the case of downregulation induced by a persistent exposure to an agonist, receptor proteolysis via preceding endocytosis plays a major role (16, 39). Many GPCRs are regulated by endocytosis, in that short exposure to an agonist induces an increase in the perinuclear endosomal receptor pool due to receptor endocytosis before downregulation, probably by trafficking to lysosomes for degradation. In our expression system, we confirmed that this degradative pathway of H2R-GFP certainly exists during long exposure to 100 µM histamine because of colocalization with LAMP-1. In this study, antagonist-induced upregulation was observed at 3 h after treatment, whereas the decrease in the endosomal receptor was complete within 2 h. The decreased receptor pool in endosomes may lead to a subsequent reduction in receptor degradation. A part of the H2R upregulation upon long exposure to an antagonist needs de novo protein synthesis. The longer period for induction of H2R upregulation compared with other GPCRs probably corresponds to the time needed for redistribution of newly synthesized receptors. The upregulation could then be maintained by suppression of receptor degradation rather than augmentation of receptor synthesis. Taking these previous results and our current observations together, we propose a model for antagonist-induced upregulation of H2R (Fig. 10). In this model, the recycling endosome acts as an intracellular pool of membrane proteins rather than a mere point of recycling passage, which is supported by models proposed for other membrane receptors, channels, and transporters (27). Inverse agonist-induced redistribution of the H2R to the plasma membrane may reduce the normal degradation of the H2R, thereby shifting the synthesis-degradation balance and resulting in a net increase in H2R.

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Fig. 10. Model for antagonist (inverse agonist)-induced upregulation of H2R. In the resting state, ligand-independent endocytosis is in balance with receptor recycling back to the plasma membrane, as is de novo receptor synthesis and degradation via the endosome-lysosome pathway. In this state, the total number of receptors remains constant. A: short exposure to antagonist (inverse agonist) shifts the equilibrium between endocytosis and recycling predominantly to recycling. Consequently, the number of receptors localized in endosomes decreases. B: upon long exposure to antagonist (inverse agonist), the decrease in endosomal receptor pool may lead to a subsequent reduction in receptor degradation via trafficking to lysosomes. Therefore, de novo receptor synthesis predominates over degradation. In this state, the total number of receptors increases up to the altered point of equilibrium. EE, early endosome; RE, recycling endosome; LE, late endosome; N, nucleus; Lyso, lysosome.
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In conclusion, visualization of H2R in living cells revealed that a H2R antagonist, which acts as an inverse agonist, induced recycling of the agonist-induced internalized receptor from the endosomal pool to plasma membrane within 2 h. Persistent treatment with an antagonist induced upregulation of H2R-GFP after at least 3 h, especially in the plasma membrane. Upregulation of H2R-GFP is reversible after withdrawal of the antagonist. Treatment with an antagonist did not augment H2R mRNA, and subsequent inhibition of protein synthesis by CHX had no effect on H2R upregulation. The shift in equilibrium of receptor trafficking between endocytosis and recycling, which precedes H2R upregulation, occurs concurrently with suppression of receptor degradation and may explain the inverse agonist-induced upregulation of H2R.
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GRANTS
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This work was supported by a Center of Excellence grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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ACKNOWLEDGMENTS
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We are grateful to Dr. M. Futai (Osaka University, Osaka, Japan) for providing a genomic DNA clone of the human histamine H2 receptor.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Osawa, First Dept. of Medicine, Hamamatsu Univ. School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan (e-mail: sososawa{at}hama-med.ac.jp)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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