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CELLULAR AND MOLECULAR
2C-Adrenergic Receptors Exhibit Enhanced Surface Expression and Signaling upon Association with 
2-Adrenergic Receptors
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia
Received April 19, 2006; accepted June 2, 2006.
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Abstract |
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2C-adrenergic receptor (2CAR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of 2CAR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of 2CAR with more than 25 related GPCRs revealed that only coexpression with the 
2-adrenergic receptor (2AR) increased the surface localization of 2CAR in human embryonic kidney-293 cells. Coimmunoprecipitation of 2CAR with 2AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that 2CAR expressed alone was mainly intracellular, whereas 2CAR coexpressed with 2AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in 2CAR binding sites upon coexpression with 2AR, with no apparent change in affinity for 2AR ligands. Functional assays with the 2AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of 2AR with 2CAR enhanced 2CAR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced 2CAR internalization in response to 2AR agonists when 2CAR and 2AR were coexpressed. In addition, substantial cointernalization of 2CAR in response to AR agonists was observed when 2CAR was coexpressed with 2AR. These data reveal that 2CAR can interact with 2AR in cells in a manner that regulates 2CAR surface expression, internalization, and functionality.
1, 2, and 2) can be further divided into three subtypes each, and all of these subtypes are excellent targets for therapeutic pharmaceuticals. The specific roles of the various adrenergic receptor subtypes is becoming increasingly clear through studies on knock-out mice (Philipp and Hein, 2004
), and novel therapies making use of these insights await the development of more subtype-specific drugs. However, two of the adrenergic receptor subtypes, 2CAR and 1DAR, have proven extremely difficult to study in heterologous expression systems, because they do not traffic efficiently to the cell surface when expressed alone and are therefore largely non-functional (von Zastrow et al., 1993; Daunt et al., 1997; Chalothorn et al., 2002). Recently, it has been shown that 1DAR surface expression and functionality can be profoundly enhanced by coexpression with 1BAR or 2AR, presumably due to receptor heterodimerization (Uberti et al., 2003, 2005; Hague et al., 2004b).
The mechanisms underlying the 2CAR-trafficking defect remain enigmatic and are important to address because of the therapeutic importance of drugs targeting 2 receptors. It has been shown that 2CAR does traffic efficiently to the cell surface when expressed in several neuronally derived cell types, suggesting that the poor trafficking of 2CAR seen in other cell types is highly dependent on cellular context (Hurt et al., 2000). Other studies suggest that surface expression of 2CAR can be increased by exposure to cold temperatures, which may further contribute to tissue-specific regulation of 2CAR activity (Jeyaraj et al., 2001; Bailey et al., 2004). Studies on 2CAR knockout mice reveal a key role for this subtype in mediating spinal analgesia (Fairbanks et al., 2002) and in the regulation of epinephrine release (Hein et al., 1999; Brede et al., 2003), demonstrating that 2CAR is functional and relevant in vivo. Thus, it seems likely that efficient trafficking of 2CAR to the cell surface may require an associated partner that is expressed in a cell type-dependent manner. Such a partner could be a specialized chaperone protein, or it could be another receptor.
Classically, GPCRs have been thought to act as monomers. However, a growing body of literature suggests that dimerization is important for the function of many GPCRs. Interestingly, dimerization does not seem to be limited to homodimers, because heterodimerization of GPCRs has been shown to occur as well (Terrillon and Bouvier, 2004; Prinster et al., 2005). Depending on the number of GPCR heterodimers and their functional consequences, the physiological effects mediated by GPCRs may be much larger than could be ascribed to the approximately 750 GPCRs predicted to be contained in the human genome. The possibility of such an increase in receptor variation and a concomitant increase in potential drug targets makes investigation into the functions of GPCR heterodimers an important research direction. Heterodimerization has also been observed among adrenergic receptor subtypes, with various effects described on receptor trafficking and signaling, depending on the receptors involved (Lavoie et al., 2002; Stanasila et al., 2003; Xu et al., 2003; Breit et al., 2004; Hague et al., 2004a, 2006; Uberti et al., 2005). In this study, we investigated whether coexpression with other GPCRs might enhance the surface expression and functionality of 2CAR.
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Materials and Methods |
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1- and 2-Adrenergic receptor constructs were kindly provided by Robert Lefkowitz (Duke University Medical Center, Durham, NC). 1A-, 1B-, and 1D-Adrenergic receptor constructs were kindly provided by Ken Minneman (Emory University School of Medicine, Atlanta, GA). 2A-, 2B-, and 2C-Adrenergic receptor constructs were kindly provided by Lee Limbird (Vanderbilt University Medical Center, Nashville, TN). The 3-adrenergic receptor was kindly provided by Sheila Collins (CIIT Centers for Health, Research Triangle Park, NC). The serotonin 5HT1A receptor construct was kindly provided by John Raymond (Medical University of South Carolina, Charleston, SC). Angiotensin AT1 and AT2 receptor constructs, trace amine receptors constructs (1–5), P2Y2 receptor construct, NPY1 receptor construct, and thromboxane A2 receptor construct were purchased from the University of Missouri-Rolla cDNA Resource Center (Rolla, MO). Muscarinic m1–5 acetylcholine receptor constructs were kindly provided by Allan Levey (Emory University School of Medicine). The purinergic receptor P2Y1 construct was kindly provided by Ken Harden (University of North Carolina, Chapel Hill, NC). Opioid receptor constructs (µ, 
, and 
) were kindly provided by Ping-Yee Law (University of Minnesota Medical School, Minneapolis, MN). The histamine H3 receptor construct was kindly provided by Tim Lovenberg (The R. W. Johnson Pharmaceutical Research Institute, San Diego, CA).
The FLAG-tagged 2C-adrenergic receptor was generated from the HA-tagged 2CAR construct mentioned above. The 2CAR coding sequence was amplified via polymerase chain reaction using the primers 5'-GACTCTAGAGCGTCCCCAGCGCTG-3' (5' end, containing the XbaI restriction site) and 5'-GTCGGATCCTCACTGCCTGAAGCC-3' (3' end, containing the BamHI restriction site preceded by a stop codon). After polymerase chain reaction amplification, the receptor and plasmid pDoubleTrouble, containing N-terminal sequential hexahistidine and FLAG epitopes, were digested with XbaI and BamHI restriction enzymes and ligated with T4 DNA ligase, and the sequence was confirmed by DNA sequencing. All molecular biology reagents were obtained from Promega (Madison, WI).
Cell Culture and Transfection. All tissue culture media and related reagents were purchased from Invitrogen (Carlsbad, CA). HEK-293 cells were maintained in complete medium (Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and 1% penicillin/streptomycin) in a 37°C, 5% CO2 incubator. To express receptors, 2 µg of DNA from each construct was mixed with Lipofectamine 2000 (15 µl; Invitrogen) and added to 5 ml of complete medium in 10-cm tissue culture plates containing cells at 
80 to 90% confluence. After overnight incubation, complete medium was added to the culture dishes, and cells were trypsinized and replated on an appropriately sized dish.
For confocal microscopy, a transfection efficiency of >80% was achieved (by transfection) using the Nucleofector solution and following the protocol supplied by the manufacturer (Amaxa, Gaithersburg, MD). In brief, HEK-293 cells were trypsinized, collected by centrifugation, and resuspended in Nucleofector solution along with 1 µg of each cDNA. The suspension was then subjected to electroporation in the Nucleofector, complete medium was added, and cells were plated directly onto tissue culture-treated glass slides (BD Biosciences, Bedford, MA) and grown for 18 to 24 h.
Surface Expression Assay. HEK-293 cells stably transfected with 2CAR were transiently transfected with the appropriate epitope-tagged constructs and plated on poly-D-lysine-coated 35-mm dishes. Cells were washed, fixed, and rinsed. Cells were then incubated in blocking buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, and 5% w/v nonfat dry milk, pH 7.5) and incubated with horseradish peroxidase-conjugated anti-FLAG M2 (1:1000) or 12CA5 anti-HA (1:1000) monoclonal antibodies in blocking buffer. Cells were washed with blocking buffer and incubated with SuperSignal ELISA ECL reagent for 15 s before the chemiluminescence of the whole 35-mm plate, which corresponds to the amount of receptor on the cell surface, was quantified in a TD20/20 luminometer (Turner Designs, Sunnyvale, CA). For internalization assays, cells were stimulated with the appropriate agent in Dulbecco's modified Eagle's medium for 30 min at 37°C and then placed on ice and fixed before cell surface measurements were made.
Immunocytochemistry and Laser-Scanning Confocal Microscopy. The nucleofected cells were washed and fixed immediately, or to investigate internalization, cells were treated with brimonidine (UK 14,304; 10 µM) or isoproterenol (10 µM) for 30 min at 37°C and then placed on ice, washed, and fixed. The cells were then blocked and permeabilized by incubating in blocking buffer (1x phosphate-buffered saline, 2% bovine serum albumin, and 0.1% saponin, pH 7.4) and incubated with mouse anti-FLAG antibody (1: 1000; Sigma, St. Louis, MO) and rat anti-HA antibody (1:1000; Roche, Indianapolis, IN), washed, and incubated with anti-mouse-conjugated Alexa 488 and anti-rat-conjugated Alexa 546 (Molecular Probes, Eugene, OR). The slides were washed and dehydrated and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Cells were scanned with a LSM 510 laser scanning confocal microscope (Carl Zeiss GmbH, Heidelberg, Germany). For detecting Alexa 488, fluorescence was excited using an argon laser at a wavelength of 488 nm, and the absorbed wavelength was detected for 510 to 520 nm. For detecting Texas Red, rhodamine fluorescence was excited using a helium-neon laser at a wavelength of 522 nm.
Western Blotting. Samples in 1x sample buffer were centrifuged briefly before loading approximately 20 µl of the sample. The proteins were resolved by SDS-PAGE on a 4 to 20% Tris-glycine gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA). The membranes was incubated for 30 min in Tris-buffered saline with 0.1% Tween 20 plus 5% dry milk and then with the appropriate primary antibody for 1 h. The membranes were washed and incubated with a fluorescent-conjugated secondary antibody for 30 min followed by detection using the Odyssey imaging system (Li-Cor, Lincoln, NE).
Assays of ERK Activation. Cells grown on 12-well dishes were starved in serum-free Dulbecco's modified Eagle's medium overnight and exposed to vehicle in the presence or absence of 10 µM UK 14,304 for 5 min at 37°C, added directly to the starvation medium. At the end of the stimulation, the medium containing the agent was removed, and 60 µl of 1x sample buffer was added. Samples were sonicated, boiled for 5 min, and centrifuged briefly at 17,000g before loading 20 µl of each sample. The proteins were resolved by SDS-PAGE as described above, and the proteins were detected using monoclonal anti-phospho-p42/44 and rabbit anti-p42/44 antibodies to blot for phosphorylated and total mitogen-activated peptide, respectively. Fluorescent-conjugated secondary anti-mouse and anti-rabbit were then used for detection by scanning using the Odyssey imaging system, and band density was quantified using Odyssey imaging software (Li-Cor).
Coimmunoprecipitation. Membranes of cells transiently transfected were washed and collected in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) containing Complete protease inhibitor cocktail (Roche) and incubated for 60 min at 4°C with rotation. Unsolubilized membranes were pelleted, and the supernatant was incubated with anti-FLAG-conjugated agarose beads overnight at 4°C with rotation. The beads were washed in phosphate-buffered saline, and the protein was eluted from the beads in 1x sample buffer. Samples were analyzed by Western blotting as described above.
Radioligand Binding Assays. Cells were washed, collected, and centrifuged at 50,000g to collect the membranes, sonicated briefly, and resuspended in 3 ml of fresh binding buffer. The affinity of the receptors for [3H]dihydroalprenolol (DHA) (2AR antagonist) or [3H]rauwolscine (2CAR antagonist) was assessed in saturation binding assays using six concentrations of [3H]DHA or [3H]rauwolscine. The membrane preparation was incubated with [3H]DHA or [3H]rauwolscine for 30 min at 22°C. The reaction was stopped by filtration through Whatman GF/C glass fiber filters (Whatman Schleicher and Schuell, Keene, NH) on a Brandel cell harvester (Brandel Inc., Gaithersburg, MD). The amount of 3H ligand present was determined by liquid scintillation counting. Nonspecific binding was defined using 10 mM propranolol for 2AR or 10 mM norepinephrine or RX 821002 (2-methoxyidazoxan) for 2CAR. Nonlinear regression analyses of saturation binding assays and statistical comparisons were performed with Prism (GraphPad Software, Inc., San Diego, CA).
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Results |
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2CAR following Cotransfection with Other GPCRs. To investigate the effect of heterodimerization on 2CAR surface expression, 2CAR was coexpressed with a panel of 29 different GPCRs. The relative increase in FLAG-tagged 2CAR surface expression was investigated using an intact-cell ELISA assay that has been used previously to study other trafficking-defective GPCRs (Uberti et al., 2003, 2005; Hague et al., 2004a,b). Coexpression with most of the receptors examined had no detectable effect on the localization of 2CAR, but cotransfection with 2AR caused a marked increase (4-fold) in the amount of 2CAR at the cell surface (Fig. 1).
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2AR on 2CAR localization was confirmed via a second and independent technique, confocal microscopy. As described previously (von Zastrow et al., 1993; Daunt et al., 1997), 2CAR expressed alone in HEK-293 cells is largely intracellular (Fig. 2A). In contrast, 2AR expressed alone in our studies displayed strong surface localization in HEK-293 cells (Fig. 2B). Upon coexpression of 2AR with 2CAR, 2AR localization was unaltered by coexpression with 2CAR, but there was a striking increase in the surface localization of 2CAR, such that 2CAR colocalized well with 2AR at the plasma membrane (Fig. 2, C–E).
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One possible explanation for the ability of 2AR to alter the trafficking of 2CAR is an interaction between the two receptors. To explore this possibility, we investigated the ability of 2CAR to interact with 2AR by coimmunoprecipitation. Immunoreactivity for FLAG-2CAR was evident as a major band at 45 kDa and as a second band at approximately 100 kDa, which may represent receptor multimers not fully resolved on SDS-PAGE. Both 2CAR bands were efficiently immunoprecipitated with anti-FLAG antibodies (Fig. 3). The major band of HA-2AR immunoreactivity (52 kDa) was not immunoprecipitated by anti-FLAG antibodies when 2AR was expressed alone. However, HA-2AR was robustly coimmunoprecipitated with FLAG-2CAR when the two receptors were expressed together. These data reveal that 2CAR and 2AR can form a complex in a cellular environment.
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Binding Properties of 2CAR and 2AR. The effects of receptor coexpression on binding affinity and total receptor number for 2CAR and 2AR were assessed in saturation binding assays. Using the 2AR-specific ligand rauwolscine, we observed that the KD value was unchanged by coexpression with 2AR but that the Bmax value was increased by approximately 2-fold. Conversely, neither the KD nor Bmax values for [3H]DHA binding were altered when 2AR was coexpressed with 2CAR (Fig. 4; Table 1).
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Because agonist and antagonist binding might plausibly be affected differentially by receptor heterodimerization, we assessed the ability of agonists specific for 2CAR or 2AR to compete with their respective radioligands. However, competition binding assays revealed that the affinity values for UK 14,304 and norepinephrine binding to 2CAR were not significantly different when 2CAR was expressed alone versus coexpressed with 2AR (Fig. 5). The affinity values for epinephrine and isoproterenol binding to 2AR were also not changed when 2AR was coexpressed with 2CAR (Table 1).
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2CAR/2AR Coexpression on 2CAR Functionality. The 2AR subfamily is predominantly coupled to Gi/o and can strongly activate ERK1/2 (DeGraff et al., 1999). Assays of ERK1/2 phosphorylation are a sensitive, robust method for assessing the signaling activity of many GPCRs. Therefore, we explored the effect of 2AR coexpression on the functional properties of 2CAR using the phosphorylation of ERK1/2 as an endpoint. Cells transfected with 2CAR in the absence or presence of 2AR were incubated with UK 14,304. Increases in ERK1/2 phosphorylation were observed in both cases. However, the observed increase in ERK1/2 phosphorylation was much larger when 2CAR was coexpressed with 2AR than when 2CAR was expressed alone. The stimulatory effects of UK 14,304 in all cases were blocked by RX 821002, an 2AR antagonist (Fig. 6). In contrast to the large effect of 2AR coexpression on 2CAR signaling, 2AR-mediated stimulation of ERK1/2 phosphorylation by isoproterenol was not significantly altered by coexpression with 2CAR (data not shown).
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The predominantly intracellular localization of 2CAR in most cell types has been a confounding factor in previous studies aimed at assessing the capacity of 2CAR to undergo agonist-promoted endocytosis (Daunt et al., 1997; DeGraff et al., 1999; Olli-Lahdesmaki et al., 1999). However, the ability of 2AR to traffic 2CAR to the plasma membrane enabled us to more easily investigate 2CAR internalization following agonist stimulation. When 2CAR was expressed alone and stimulated with UK 14,304, the small population of 2CARs on the cell surface did not undergo any significant internalization, as assessed using the luminometer-based whole-cell ELISA assay. When 2CAR was coexpressed with 2AR, however, there was a striking 30% decrease in the amount of 2CAR on the cell surface following a 30-min treatment with UK 14,304. Furthermore, the 2AR-specific agonist isoproterenol also resulted in substantial endocytosis of 2CAR, suggesting cross-internalization between the two receptors (Fig. 7A).
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The effect of coexpression with 2CAR on 2AR internalization was also examined. As expected, a 30-min treatment with isoproterenol caused a robust 35% 2AR endocytosis, and this isoproterenol-induced internalization was not altered by coexpression of 2CAR. Unlike the apparent cross-internalization of 2CAR following isoproterenol stimulation of coexpressed 2AR, UK 14,304 stimulation of 2CAR was unable to promote internalization of coexpressed 2AR (Fig. 7B).
Agonist-induced receptor internalization was also studied via confocal microscopy. When 2CAR and 2AR were coexpressed and stimulated with isoproterenol, a loss of both receptors from the plasma membrane was observed, along with a concurrent accumulation of both receptors inside the cell (Fig. 8). In contrast, stimulation of the doubly transfected cells with UK 14,304 resulted in endocytosis of 2CAR but not 2AR (data not shown). Thus, the data from the confocal studies matched the results from the luminometer-based assay described above well, in that both techniques revealed cointernalization of the 2CAR/2AR complex upon treatment with AR agonists.
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Discussion |
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1DAR, 2CAR, and the olfactory receptors, are known to be inefficiently targeted to the cell surface when expressed heterologously in most cell types. Seminal studies demonstrating that coexpression with GABABR2 can facilitate GABABR1 trafficking to the cell surface suggested a key role for receptor heterodimerization in regulating the trafficking of certain GPCRs (Marshall et al., 1999). Likewise, associations of 1DAR and olfactory receptors with specific GPCR partners have been found to enhance the surface expression of these trafficking-defective receptors (Uberti et al., 2003, 2005; Hague et al., 2004a,b). The purpose of the studies reported here was to investigate whether the poor trafficking of 2CAR might also be enhanced by coexpression with an appropriate GPCR partner.
After examining 2CAR surface trafficking following coexpression with more than 25 different GPCRs, we observed that surface expression of 2CAR was markedly enhanced only by coexpression with 2AR. Confocal microscopy studies confirmed increased surface expression of 2CAR upon 2AR coexpression. We also observed that 2AR could be robustly coimmunoprecipitated with 2CAR. Thus, a reasonable interpretation of these data is that 2CAR surface expression is enhanced via association with 2AR, although it is not entirely clear whether the 2CAR/2AR interaction is direct (via heterodimerization) or indirect (via joint interaction with a scaffold protein). In any case, the effects of 2AR coexpression on 2CAR surface trafficking are analogous to previous observations that interactions with either 1BAR or 2AR enable 1DAR to localize normally to the plasma membrane (Uberti et al., 2003, 2005; Hague et al., 2004b). The effects of receptor coexpression on the trafficking of both 2CAR and 1DAR seem to be quite specific, because the vast majority of receptors examined had no significant effect on 2CAR or 1DAR surface expression. The interaction between GABABR1 and GABABR2 is also highly specific, as screens with several dozen other GPCRs revealed that only GAB-ABR2 is capable of efficiently promoting GABABR1 surface trafficking (Balasubramanian et al., 2004).
Certain GPCR heterodimers exhibit altered pharmacology relative to the individual receptors expressed alone. For example, heterodimers formed between opioid receptors (/ or µ/) possess ligand binding properties distinct from any of the three cloned opioid receptors expressed by themselves (Jordan and Devi, 1999; George et al., 2000). In our studies, coexpressed 2CAR and 2AR did not seem to display altered affinities for any of the agonists or antagonists examined, suggesting that the conformation of the binding pockets for both receptors remained unaltered, as has been observed for other GPCR heterodimer combinations (Pfeiffer et al., 2002; Uberti et al., 2003). An increased Bmax for [3H]rauwolscine binding was observed in saturation binding assays, where 2CAR levels were increased by almost 2-fold when coexpressed with 2AR, and a similar increase was also observed for 2CAR immunoreactivity upon 2AR coexpression (data not shown). Increased receptor stability has been described for other trafficking-defective receptors upon coexpression with appropriate partners, such as 1DAR coexpressed with 1BAR (Uberti et al., 2003). The observed increases in 2CAR levels upon 2AR coexpression might be explained by reduced 2CAR retention in the endoplasmic reticulum, where accumulating 2CAR would be rapidly degraded. Thus, because association with 2AR enhances the proportion of 2CAR in the plasma membrane, it would reduce the amount of 2CAR subject to rapid degradation and result in a modest but consistent increase in 2CAR binding and immunoreactivity.
Receptor-receptor interactions are known to have strong effects on regulating signaling for certain GPCR combinations. In the case of trafficking-defective GPCRs, such as 2CAR, associations with other receptors and the resultant-enhanced surface expression would seem to be critical due to the requirement for membrane-impermeant agonists to gain access to the receptors. In the current studies, UK 14,304-stimulated ERK1/2 activation by 2CAR was found to be significantly increased upon coexpression with 2AR. The 2-specific nature of the ERK activation was shown by blocking 2CAR with the specific antagonist RX 821002. Furthermore, 2CAR stimulation of ERK phosphorylation, both in the absence and presence of 2AR coexpression, was fully blocked by pertussis toxin treatment (data not shown), suggesting predominant coupling of 2CAR to Gi/o, even after association with 2AR. Thus, because 2CAR ligand binding and G protein coupling specificity did not seem to be altered by coexpression with 2AR, the most plausible explanation for the enhanced signaling is that 2AR-induced trafficking of 2CAR allowed for additional functional 2CAR to be inserted into the plasma membrane.
The trafficking and functionality of 2CAR are known to be heavily dependent on cellular context as well as the temperature at which cells are grown. Whereas 2CAR is largely intracellular and nonfunctional in most heterologous cell types, it has been shown that 2CAR is much more efficiently trafficked to the plasma membrane when expressed in certain neuronally derived cell lines (Hurt et al., 2000). It is tempting to speculate that the relative expression level of endogenous 2AR in these cell lines may be a key factor determining the trafficking and functionality of transfected 2CAR, although of course, the relative expression levels of other proteins involved in regulating 2CAR trafficking may also be very important. In various cell lines where transfected 2CAR is poorly trafficked to the cell surface, it has been shown that lowering the temperature of the cells can promote 2CAR plasma membrane expression (Jeyaraj et al., 2001; Bailey et al., 2004). Because the retention of misfolded proteins by the endoplasmic reticulum/Golgi complex is known to be less efficient at lower temperatures (Morello et al., 2000), it seems likely that an impairment in the ability of cells to retain 2CAR accounts for the reported effect of temperature on 2CAR trafficking. Whereas such temperature-dependent regulation of 2CAR trafficking may occur in certain blood vessels in the distal limbs, temperatures low enough to help 2CAR overcome its trafficking defect are unlikely to be achieved in most native cell types in which 2CAR is expressed. Thus, it seems probable that 2CAR trafficking and functionality in vivo are dependent on cellular factors, such as associations with other receptors as reported here and/or interactions with accessory proteins that promote proper receptor trafficking.
The regulation of 2CAR by agonist-promoted internalization has been difficult to study because of the poor surface expression of the receptor, although some progress has been made using ELISA-based assays similar to those used in the present studies (Daunt et al., 1997; DeGraff et al., 1999; Olli-Lahdesmaki et al., 1999). Results from previous studies suggested that, in Madin-Darby canine kidney cells, 2CAR was weakly internalized in response to agonist (Daunt et al., 1997), whereas in COS-1 cells, 2CAR internalization was not observed unless arrestin-3 was overexpressed (DeGraff et al., 1999). Because 2AR cotransfection robustly increased 2CAR surface expression in our studies, we took advantage of the opportunity to characterize the internalization properties of 2CAR in response to agonist. Furthermore, because 2CAR and 2AR associate in cells, we also assessed the consequences of this interaction for receptor endocytosis. We found that 2CAR was significantly internalized in response to UK 14,304, only when 2CAR was coexpressed with 2AR. We also observed a marked internalization of 2CAR in response to isoproterenol, indicating that 2CAR undergoes cointernalization with 2AR upon 2AR agonist stimulation. These findings were confirmed by confocal microscopy studies, which showed colocalization of 2CAR and 2AR in intracellular punctate regions following stimulation with isoproterenol. Interestingly, as with the luminometer assays, internalization of 2AR did not seem to be affected by UK 14,304 treatment, which may indicate that recruitment of arrestin to the 2CAR/2AR complex is dependent on whether the 2CAR component or 2AR component is stimulated by agonist. The isoproterenol-stimulated internalization of 2CAR observed here suggests a mechanism that may underlie various forms of cross-talk that have been reported between 2ARs and 2ARs (Maggi et al., 1980; Northam and Mobley, 1985; Nakamura et al., 1991; Atkinson and Minneman, 1992; Birnbaum et al., 1995). It is known that 2CAR and 2AR are coexpressed in many of the same tissues, including distinct structures within the brain, adrenal glands, and kidney (Rainbow et al., 1984; Rosin et al., 1996; Lee et al., 1998; Uhlen et al., 1998; Brede et al., 2003; Cesetti et al., 2003; Wallace et al., 2004). Further investigations into the consequences of 2CAR/2AR associations in native tissues, e.g., studies on knockout mice, may shed additional light on the physiological importance of the interaction between these receptors in vivo.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GPCR, G protein-coupled receptor; AR, adrenergic receptor; GABABR, GABAB receptor; DHA, dihydroalprenolol; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; RX 821002, 2-methoxyidazoxan; UK 14,304, brimonidine.
1 Current affiliation: Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes-Barre, Pennsylvania. 
Address correspondence to: Dr. Randy A. Hall, Department of Pharmacology, Emory University School of Medicine, 5113 Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322. E-mail: rhall{at}pharm.emory.edu
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