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Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The localization of an epitope-tagged receptor for thyrotropin-releasing hormone (TRH) expressed in different cell contexts was studied with immunofluorescence microscopy. In pituitary lactotrophs, which normally express TRH receptors, and in AtT20 pituitary corticotrophs, TRH receptor immunoreactivity was primarily confined to the plasma membrane. In HEK 293 and COS7 cells, TRH receptors were predominantly intracellular. In transiently transfected COS7 cells, the TRH receptor colocalized with endoplasmic reticulum and Golgi markers. The pattern of TRH receptor immunofluorescence was the same over a wide range of receptor expression in transiently transfected COS7 cells, and all cell lines bound similar amounts of 3H- and rhodamine-labeled TRH analogs, suggesting that cell-specific differences in TRH receptor localization were not simply the result of overexpression. In all cell contexts, TRH receptors on the plasma membrane underwent extensive ligand-driven endocytosis. Inhibitors of glycosylation did not alter the subcellular distribution of receptors. In HEK 293 cells expressing the transfected TRH receptor, protein synthesis inhibitors caused translocation of intracellular receptors to the cell surface, as shown by a marked increase in cell surface immunofluorescence and [3H][N3-methyl-His2]TRH binding. These results demonstrate that the subcellular localization of the TRH receptor depends on the cell context in which it is expressed and that intracellular receptors are capable of translocation to the plasma membrane.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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G protein-coupled receptors
mediate the activity of a wide variety of hormones and
neurotransmitters. Ligands for most members of the heptahelical
receptor family are not membrane permeant, and receptors engaged in
signal transduction are localized on the plasma membrane. Relatively
few G protein-coupled receptors have been visualized with
immunofluorescent staining, and most of those have been shown to be
predominantly on the cell surface. Receptors localized primarily at the
plasma membrane include the 
2, mouse
2-10H, and 2A adrenergic receptors (1,
2), as well as receptors for gastrin-releasing peptide (3),
cholecystokinin (4, 5), and TRH (6). In contrast, the bulk of thrombin receptors in endothelial cells (7, 8) and mouse
2-adrenergic-4H receptors (1) are found intracellularly
in vesicular structures. It is not known whether the cellular
distribution of a G protein-coupled receptor is determined only by
intrinsic properties of the receptor or also influenced by the cellular
milieu.
The pituitary TRHR is a calcium mobilizing receptor coupled via Gq/11 to phospholipase C. Signal transduction by TRH and regulation of the TRHR have been extensively characterized (9-11). Recently, we localized the TRHR and a fluorescently labeled TRH ligand in pituitary cells (6). We stably expressed an epitope-tagged TRHR in the GH3 pituitary cell line and used immunofluorescence microscopy to show that the receptor is present on the plasma membrane before activation. The TRHR and labeled ligand undergo extensive temperature-dependent internalization after binding of an agonist. Fluorescently labeled TRH analog and transferrin colocalized in endocytic vesicles, and internalization was blocked by hypertonic sucrose, implying that the TRHR undergoes endocytosis via clathrin-coated pits.
TRH action has been studied in both pituitary lactotrophs and thyrotrophs, in which the receptor is expressed endogenously, and in heterologous cells after stable or transient transfection of wild-type or mutated TRHR cDNAs. Some aspects of TRH signaling, such as desensitization of the receptor and down-regulation of the receptor mRNA, have been reported to differ when the receptor is expressed in different cell types (9, 12-14). To determine whether differences in receptor localization could contribute to differences in TRHR regulation, we monitored the distribution of epitope-tagged TRHRs after transient or stable expression in several cell lines. The experiments also address the more general question of whether the subcellular localization of G protein-coupled receptors is cell type dependent. We report here that the distribution of the TRHR is strongly dependent on the cell in which it is expressed.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Rhod-TRH and the p-myc-FLAG-TRHR plasmid, which
encodes mouse TRHR with a myc epitope (GGEQKLISEEDLE)
inserted between residues Glu23 and Tyr24 in the amino-terminal portion
and a FLAG epitope (DYKDDDDK) after amino acid Asp369 in the
carboxyl-terminal tail in the pCDM8 vector, were prepared as previously
described (6). Plasmid encoding a 2-adrenergic receptor
bearing an amino-terminal FLAG epitope was a gift from Dr. Brian
Kobilka (Stanford University, Palo Alto, CA), and pRSV/Neo was from the
American Type Culture Collection (Rockville, MD). M2 mouse monoclonal
anti-FLAG antibody was from IBI (Rochester, NY). Rhodamine-labeled goat
anti-mouse IgG was from Hyclone (Logan, UT). [3H]MeTRH
(82.5 Ci/mmol) was from DuPont-New England Nuclear (Boston, MA).
N-Dodecylmaltoside was from Boehringer-Mannheim Biochemica (Mannheim, Germany). Monensin and TRH were from Calbiochem (La Jolla,
CA), and protease and glycosylation inhibitors and wheat germ
agglutinin-Sepharose from Sigma Chemical (St. Louis, MO). NBD hexanoic
ceramide and DiOC6 were from Molecular Probes (Eugene, OR).
Cell culture. Pituitary GHY cells, a subclone of the GH3 line that does not express TRHRs (15), were grown in Ham's F10 medium supplemented with 15% horse serum and 2.5% fetal bovine serum. HEK 293 cells, monkey COS7 cells, and mouse pituitary corticotroph AtT20 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cell lines were incubated at 37° in a humidified 5% CO2/95% air atmosphere.
Transfection. Stable lines of GHY and HEK 293 cells were established by cotransfecting cells with p-myc-FLAG-TRHR, which uses a CMV promoter, and pRSV/Neo with lipofection and selecting with G-418 (0.25 mg/ml for GHY and 0.60 mg/ml for HEK 293). Clones were screened for expression of the FLAG epitope-tagged receptor by [3H]MeTRH binding and immunofluorescent staining. Cells were transiently transfected with the p-myc-FLAG-TRHR plasmid by lipofection and DEAE-dextran methods.
Staining and fluorescence microscopy. Immunocytochemistry was performed as described previously (6). Briefly, cells plated on coverslips were treated as described in the text, fixed, and permeabilized with 0.2% Nonidet P-40. The cells were stained at room temperature for 60 min with 1 µg/ml of M2 antibody against the FLAG epitope, washed, and then stained for 20 min with a 1:100 dilution of rhodamine-labeled goat anti-mouse IgG. Stained coverslips were washed and mounted with polyvinyl alcohol on glass slides. No staining was observed when the same protocol was applied to cells that had been transfected with the wild-type receptor or to untransfected cells, and no staining was observed in cells expressing the epitope-tagged receptor after staining with an irrelevant monoclonal antibody at the same concentration. Cells that stained with antibody to the FLAG epitope did not stain well with monoclonal antibody against the myc epitope, suggesting that the extracellular myc epitope is sterically hindered, perhaps because of glycosylation, or that the anti-myc antibody binds less strongly under the conditions used. We were unable to compare the localization of the epitope-tagged versus the native TRHRs because no antibody to the native receptor is available, but the presence of epitope tags in two positions does not alter the subcellular localization of adrenergic receptors (1). Only the epitope-tagged TRHR was studied in all experiments unless stated otherwise.
Staining of live cells with Rhod-TRH was carried out as previously described (6). Cells were incubated with Rhod-TRH at ~170 nM in HEPES-buffered Hanks' balanced salt solution at either 0° or 37° and washed before visualization. As shown previously for GHY cells (6), only a dim and diffuse background fluorescence was obtained when receptor-negative GHY, HEK, or COS cells were stained with Rhod-TRH. Staining was reduced to this background level if cells were pretreated with TRH or incubated simultaneously with Rhod-TRH and an excess of TRH. Sequential staining of endoplasmic reticulum or Golgi apparatus and intracellular TRHR was achieved by staining live cells with DiOC6 (16) or NBD-C6-ceramide (17), followed by immunofluorescent staining of the epitope-tagged TRHR. Cells were incubated with 2.5 µg/ml DiOC6 in Hanks' balanced salt solution at room temperature for 10 sec or with 10 µM NBD-C6-ceramide in F10 medium containing 1 mg/ml bovine serum albumin at 37° for 10 min. Images were obtained using fluorescein filters. The cells were then immunofluorescently stained for the FLAG epitope on the microscopic stage using rhodamine-labeled second antibody, and images were obtained using rhodamine filters. Residual dye from live staining was minimal after the second staining procedure and did not appreciably bleed through the rhodamine filter. In all conventional fluorescence microscopy, a Nikon inverted fluorescence microscope equipped with a 150-W xenon lamp, a Nikon 40× objective, and selective filters was used. Digital images were taken with a Cohu CCD 4910 camera in conjunction with a Colorado video integrator unit. This unit has gain and integration controls that adjust the sensitivity of the system. Digital data were processed and stored with Metamorph software (Universal Imaging, Media, PA). Confocal microscopy was performed on an ACAS 570 (Meridian Instruments) using an argon laser with excitation at 514 nm and emission at 575 nm as previously reported (6). Cells shown are representative of the majority of cells observed in multiple experiments. When only one calibration bar is shown, it refers to all panels.Binding assays. [3H]MeTRH binding and internalization were analyzed as previously described (18). Cells were incubated with [3H]MeTRH in serum-free F10 medium at 37° or 0° for 1 hr, rinsed three times with 0.15 M NaCl, washed briefly with ice-cold acid/salt solution (0.5 M NaCl, 0.2 M acetic acid, pH 2.5), and then lysed with 0.1 N NaOH. Radioactivity removed with the acid/salt wash was presumed to be on the cell surface. Nonspecific binding, which was determined in parallel dishes containing a 1000-fold excess of unlabeled TRH, was < 5% of total and has been subtracted. TRHR was solubilized with either digitonin or N-dodecylmaltoside, as noted in the text, in Tris-Mg buffer (20 mM Tris, 2 mM MgCl2, pH 7.4) with protease inhibitors (0.3 mg/ml EDTA, 35 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin) at 0° for 30 min. Solubilized receptor was incubated at 4° overnight with either 10 nM [3H]MeTRH or, for nonspecific binding, 10 nM [3H]MeTRH plus 10 µM TRH. Bound [3H]MeTRH was assayed by filtration through Whatman GF/A filters that had been soaked in 0.3% polyethyleneimine (19). To confirm that tunicamycin effectively inhibited glycosylation, three 100-mm dishes of stably transfected GHY cells were incubated with and three dishes were incubated without 10 µg/ml tunicamycin for 18 hr; 5 nM [3H]MeTRH was added for the last hour to label receptors. Cells were washed and receptors were solubilized with 0.1% N-dodecylmaltoside. The solution containing solubilized receptors was incubated with 0.5 mg of wheat germ agglutinin-Sepharose for 2 hr at 4°, the reaction mixture was diluted in Tris-Mg buffer and centrifuged, and the resin was washed two additional times at 4°. The combined supernatant fractions were filtered through polyethyleneimine-soaked filters to determine the amount of [3H]MeTRH associated with unbound receptor, and the wheat germ agglutinin-Sepharose was counted to determine the amount of [3H]MeTRH bound to glycosylated receptor.
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Effect of cell type on receptor localization. To determine whether the distribution of the TRHR is affected by cell context, we expressed an epitope-tagged TRHR in several different cell lines and studied its localization by indirect immunofluorescence microscopy. The epitope-tagged receptor was stably transfected into the pituitary GHY line, which does not express an endogenous TRHR (15), and into HEK 293 cells. The receptor was transiently transfected into GHY cells, AtT20 pituitary corticotroph cells, HEK 293, and COS7 simian kidney cells. The Ca2+ response to 1 µM TRH was determined in Fura-2-loaded GHY and HEK 293 cells expressing similar densities of either the epitope-tagged or wild-type mouse TRHR and found to be of similar shape and amplitude (data not shown). Because the FLAG epitope was at the intracellular carboxyl terminus, cells were permeabilized before staining, allowing visualization of intracellular as well as cell surface TRHR.
The distribution of the TRHR depended on the cell type in which it was expressed (Fig. 1). In stably transfected GHY cells, fluorescence was confined to the plasma membrane, as previously reported for stably transfected GH3 cells, which express endogenous wild-type as well as epitope-tagged receptors (6); GHY is a subclone of GH3. Exposure of GHY cells to TRH at 37° caused a redistribution of the epitope-tagged TRHR to intracellular vesicles (Fig. 1).
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2-adrenergic
receptor in GH3 and COS7 cells using identical transient
transfection protocols. 2-Adrenergic receptor
immunofluorescence was mostly on the surface of GH3 cells
(Fig. 6). We observed a plasma membrane localization of
the 2-adrenergic receptor in approximately half of the
stained COS7 cells, such as those shown in the Fig. 6, and a reticular distribution in most of the remainder. These results are in general agreement with those of von Zastrow et al. (1), who found a plasma membrane distribution of the 2-adrenergic
receptor in 75% of transiently transfected COS7 cells. Localization of
the 2-adrenergic receptor was clearly different from
localization of the TRHR in the same cells in that we never observed a
COS7 cell with a predominantly plasma membrane localization of the TRHR.
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Receptor density in different cell lines. Despite the striking differences in the apparent density and cellular distribution of the epitope-tagged TRHR in the various cell types, the lines bound similar amounts of the potent TRH agonist [3H]MeTRH (Table 1). HEK 293 and COS7 cells are much larger than GHY cells (Fig. 4, calibration bars). The stably transfected HEK 293 cells expressed more [3H]MeTRH binding sites/cell than GHY cells but bound similar amounts per milligram of cell protein, whereas the transiently transfected COS7 cells bound approximately three times more per milligram of cell protein. The affinities of the epitope-tagged TRHRs for [3H]MeTRH were measured by Scatchard analysis. Measured Kd values were similar in different cell contexts: 1.4 nM (GHY), 4.7 nM (stably transfected HEK 293 cells), and 1.3 nM (transiently transfected COS7 cells). Values in the three transfected lines were all within a factor of 2 of the reported Kd value of the native TRHR in GH3 cells, 2-3 nM for [3H]MeTRH (10).
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Internalization in different cell lines. Endocytosis of receptor-bound [3H]MeTRH was measured biochemically by the formation of an acid/salt-resistant complex at 37°. [3H]MeTRH was internalized to a similar extent in all cell types (Table 1), and internalization was temperature dependent in all cell lines. The amounts [3H]MeTRH internalized after a 1-hr incubation at 4° were 37%, 25%, and 14% of the amounts internalized at 37° for GHY, HEK 293, and COS7 cells, respectively. The half-time for internalization of [3H]MeTRH was 3-5 min at 37° in both GHY and HEK 293 cells. Temperature-dependent internalization of Rhod-TRH was easily visible by 5 min and extensive at 10 min, as shown for HEK 293 cells in Fig. 3.
Properties of intracellular receptors. To determine whether the intracellular receptors in HEK 293 cells could bind ligand, we measured [3H]MeTRH binding after homogenization and detergent solubilization of receptors. Homogenization did not uncover new [3H]MeTRH binding sites, indicating that the intracellular receptors were inactive or oriented so they did not have access to [3H]MeTRH (Table 2). Furthermore, no additional [3H]MeTRH binding activity could be exposed after solubilization with detergents (Table 2). These data suggest that most of the intracellular receptors are not able to bind ligand.
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Effects of glycosylation inhibitors. One potential reason was much of the epitope-tagged TRHR protein failed to localize to the plasma membrane is that the receptor had not undergone normal glycosylation in the HEK 293 and COS7 cells. We tested this possibility indirectly by incubating with tunicamycin, a glycosylation inhibitor, GH cells that expressed epitope-tagged TRHRs (Fig. 8). Tunicamycin did not affect the normal plasma membrane distribution of receptors, suggesting that glycosylation is not necessary for transport of TRHRs to the plasma membrane. To confirm that tunicamycin effectively inhibited glycosylation, we measured the effect of tunicamycin on the ability of the TRHR to adhere to wheat germ agglutinin-Sepharose, which normally binds the N-glycosylated receptor (19), as described in Materials and Methods. Tunicamycin treatment reduced the amount of receptor-bound [3H]MeTRH absorbed to wheat germ agglutinin from 7400 ± 909 to 2600 ± 147 cpm (p = 0.01) and reduced the amount not absorbed by the lectin from 3705 ± 513 to 2522 ± 238 cpm (p = 0.10). Monensin (10 µM), which interferes with transfer through the Golgi stacks and also inhibits glycosylation, did not alter the apparent localization of the epitope-tagged TRHR (data not shown).
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Effects of protein synthesis inhibitors.
As shown in Fig.
9, cycloheximide caused a marked redistribution of
receptor immunofluorescence from the intracellular space to the plasma
membrane in transfected HEK 293 cells. This redistribution required
~24 hr and was observed at cycloheximide concentrations of 
2
µg/ml. Puromycin, which is another protein synthesis inhibitor, caused a similar redistribution of receptors to the plasma membrane. Incubation of pituitary cells with cycloheximide did not cause a
detectable redistribution of TRHRs (data not shown).
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Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The findings reported here show that the distribution of a single G protein-coupled receptor can differ dramatically in different cell types. The TRHR was confined to the plasma membrane in two pituitary lines, GHY and AtT20, but was predominantly intracellular in two nonpituitary lines, HEK 293 and COS7. Cell type-dependent differences in receptor localization have been demonstrated previously for thrombin receptors (7, 8). There is a sizable intracellular pool of thrombin receptors in human umbilical vein endothelial cells, which express an endogenous receptor, or in transfected Rat1 cells, and these vesicular receptors cycle to the membrane to replenish the plasma membrane receptors depleted by thrombin activation. In megakaryoblastic cells, however, there does not seem to be a significant intracellular pool of thrombin receptors. For most G protein-coupled receptors, subcellular localization has not been found to differ with cell context. Gastrin-releasing peptide, cholecystokinin receptors, and several classes of adrenergic receptors display the same subcellular distribution when expressed in a variety of cell contexts (1-5). It is not possible to predict which receptors will be localized aberrantly in a heterologous cell and which will not.
The molecular basis for the cell type dependence of receptor localization remains uncertain. Because the FLAG epitope is at the carboxyl terminus of the epitope-tagged TRHR, only completely synthesized receptors were visualized with immunocytochemistry. One possible explanation for the cell type dependence of receptor localization is that the levels of expression in COS7 or HEK 293 cells were so high that some aspect of post-translational processing or trafficking was saturated so receptors were retained in the endoplasmic reticulum; in this model, blocking synthesis of other proteins might facilitate transport of the long-lived TRHR to the plasma membrane. However, simple overexpression is unlikely to be the entire explanation for two reasons. First, cycloheximide was more effective than puromycin in allowing receptors to move to the plasma membrane, although the drugs were equally effective at blocking protein synthesis. Second, the pattern of localization was the same in COS7 cells expressing low as well as high levels of total receptor, based on fluorescence intensity.
The TRHR has two potential N-glycosylation sites at its amino terminus (20) and is normally glycosylated (19). It was recently reported that deletion of residues 2-22 of the TRHR, which removes the two amino-terminal glycosylation sites, does not prevent receptor expression or binding (21). In the current study, inhibition of glycosylation did not cause any detectable trapping of TRHRs intracellularly in pituitary cells. These results both indicate that glycosylation is not essential for plasma membrane localization. It is not known whether the TRHR is post-translationally modified in any other manner (e.g., by palmitoylation or phosphorylation) or whether such modifications influence receptor localization.
In addition to demonstrating cell type dependence of the localization
of the TRHR, our results support previous studies showing that
different G protein-coupled receptors can have different localizations
in the same cell. von Zastrow et al. (1) showed that
different subtypes of the 2-adrenergic receptor differed from one another in their subcellular distribution, as well as in their
response to agonist. The same differences were found when these
adrenergic receptors were transfected into either COS7 or HEK 293 cells. When transiently expressed in COS cells, the 2-adrenergic receptor was clearly localized on the cell
surface in half of the cells, but the TRHR was not predominantly plasma membrane in any cell. Because the TRHRs and 2-adrenergic
receptors were tagged with the same epitope and the intensity of the
immunofluorescence was similar in cells expressing the two types of
receptors, it is likely that the differences in localization result
from intrinsic differences in receptor structures. The importance of
receptor structure to subcellular localization has also been
highlighted through the study of naturally occurring mutations in a
number of membrane proteins. Certain mutations in rhodopsin (22) block normal trafficking to the plasma membrane and result in the
accumulation of receptor in the endoplasmic reticulum, accounting for
some hereditary forms of retinitis pigmentosa.
All of the available data for the TRHR indicate that receptors that do reach the plasma membrane are functional. [3H]MeTRH binds with comparable affinity to receptors expressed in a variety of cell types, and agonist binding drives a temperature-dependent internalization of both the ligand and the receptor in all cell lines (6, 9, 23, 24). Clathrin-dependent pathways are used in different cell types.1 Similar findings have been reported for the hCG/LH receptor, which internalizes identically in Leydig cells, where it is expressed naturally, and after transfection into L-cells (25). It is not possible, however, to generalize the conclusion that receptors will internalize identically in different cell contexts. The cholecystokinin receptor that occurs naturally in pancreatic acinar cells does not undergo agonist-dependent internalization, but the cholecystokinin receptor expressed at higher densities in CHO cells is internalized via a classic clathrin-dependent mechanism when activated (4, 5).
A number of aspects of TRHR expression and regulation have been reported to depend on the context in which the receptor is expressed (9, 12-14). The cell type dependence of receptor localization shown here may help explain some of these findings. In pituitary cells, the level of TRHR mRNA is regulated by steroid, thyroid, and peptide hormones, as well as by drugs that activate second messenger pathways, including cAMP analogs and phorbol esters (for a review, see Ref. 9). Both the rate of transcription of the TRHR gene and the stability of the TRHR message are controlled. Our results suggest that all of the receptor synthesized in pituitary cells will be transported to the plasma membrane; therefore, it is likely that any change in the level of the message and the rate of receptor synthesis will result in a change in the number of functional receptors. In contrast, only a fraction of TRHRs synthesized in transfected HEK 293 or COS7 cells will appear at the plasma membrane, and changes in receptor mRNA levels in these cells are likely to have little effect on the density of functional receptors at the plasma membrane. These differences may contribute to the cell type dependence of the effects of TRH, cAMP, and phorbol esters on TRHR mRNA. Any agent that affects the trafficking of receptors from the endoplasmic reticulum to the plasma membrane could also alter the number of functional TRHRs.
It is often convenient to express receptors in heterologous lines such as COS and HEK 293 that are easy to transfect and grow. The current results show that the possibility of aberrant receptor localization needs to be considered when interpreting data obtained in such systems. For example, it is difficult to determine experimentally whether a mutated receptor is not expressed or is expressed but does not bind ligand. Western blots would not be useful for distinguishing between these possibilities for TRHRs in cells like COS7 and HEK 293 because most receptors are not at the membrane. It is also well established that the repertoire of G proteins and effectors may differ with cell type and can influence signal transduction. Additional experimentation will be required to establish the molecular basis for the localization of G protein-coupled receptors.
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Footnotes |
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Received September 3, 1996; Accepted January 17, 1997
1 R. Yu and P. M. Hinkle, unpublished observations.
This work was supported in part by National Institutes of Health Grant DK19974 and Cancer Center Core Research Grant CA11098 and by a Pharmaceutical Manufacturers' Association Advanced Predoctoral Fellowship and Wilmot Fellowship (R.Y.).
Send reprint requests to: Dr. P. M. Hinkle, Box 711, Department of Pharmacology & Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. E-mail: hinklep{at}pharmacol.rochester.edu
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Abbreviations |
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MeTRH, [N3-methyl-His2]thyrotropin-releasing hormone; TRH, thyrotropin-releasing hormone; Rhod-TRH, rhodamine-labeled thyrotropin-releasing hormone analog; HEK, human embryonic kidney; TRHR, thyrotropin-releasing hormone receptor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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