Abstract
The cellular localization, agonist-mediated internalization, and desensitization properties of the α1-adrenoceptor (α1-AR) subtypes conjugated with green fluorescent protein (α1-AR/GFP) were assessed using real-time imaging of living, transiently transfected human embryonic kidney (HEK) 293 cells. The α1B-AR/GFP fluorescence was detected predominantly on the cell surface. Stimulation of the α1B-AR with phenylephrine led to an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and promoted rapid α1B-AR/GFP internalization. Long-term exposure (15 h) to phenylephrine resulted in desensitization of the α1B-AR-mediated activation of ERK1/2 phosphorylation. α1A-AR/GFP fluorescence was detected not only on the cell surface but also intracellularly. The rate of internalization of the cell surface population α1A-AR/GFPs was slower than that seen for the α1B-AR. Agonist exposure also resulted in desensitization of the α1A-AR-mediated increase in ERK1/2 phosphorylation. The α1D-AR/GFP fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to phenylephrine. Phenylephrine treatment of α1D-AR/GFP expressing cells increased ERK1/2 phosphorylation. However, this increase was not significant. Cotransfection with β-arrestin 1 did not increase the rate or extent of agonist-stimulated α1A- or α1B-AR/GFP internalization. However, a dominant-negative form of the β-arrestin 1, β-arrestin 1 (319–418), blocked agonist-mediated internalization of both the α1A- and α1B-ARs. These data show that transfected α1-AR/GFP fusion proteins are functional, that there are differences in the cellular distribution and agonist-mediated internalization between the α1-ARs, and that agonist-mediated α1-AR internalization is dependent on arrestins and can be desensitized by long-term exposure to an agonist. These differences could contribute to the diversity in physiologic responses regulated by the α1-ARs.
The α1-ARs are members of the G-protein-coupled receptor (GPCR) family of receptors and are used by the sympathetic nervous system to regulate systemic arterial blood pressure and blood flow. The α1-ARs also play a major role in mediating growth responses in cardiac and vascular smooth muscle cells (for recent reviews on all aspects of the α1-ARs, see Garcı́a-Sáinz et al., 1999; Graham et al., 1996; Schwinn and Price, 1999; Zhong and Minneman, 1999; Piascik and Perez, 2001). Three genes encoding unique receptor subtypes, the α1A-, α1B-, and α1D-ARs, have been cloned and characterized. These subtypes use a variety of second messengers and G-proteins to modulate cellular processes. Alterations in normal α1-AR function may contribute to the pathophysiology of diseases such as hypertension, congestive heart failure, and benign prostatic hyperplasia.
GPCR signaling is also tightly regulated by a series of cellular proteins that promote receptor desensitization and internalization (Krupnick and Benovic, 1998; Lefkowitz, 1998). Agonist occupation promotes receptor phosphorylation by a series of GPCR kinases (Hausdorff et al., 1990; Inglese et al., 1993; Premont et al., 1995). The phosphorylated receptor exhibits high affinity for the arrestins, which, in turn, prevent further interaction between the receptor and G-proteins (Wilden et al., 1986; Benovic et al., 1987). There are currently four known members of the arrestin family: visual arrestin (arrestin 1), β-arrestin 1 (arrestin 2), β-arrestin 2 (arrestin 3), and cone arrestin (arrestin 4) (Ferguson et al., 1996; Krupnick and Benovic, 1998). The β-arrestins promote internalization by binding to both the receptor and clathrin, thus, directing the receptor to coated pits (von Zastrow and Kobilka, 1992; Krupnick et al., 1997a; Gagnon et al., 1998). Oakley et al. (2000) demonstrated recently that GPCRs have different affinities for the different arrestins. Class A GPCRs, which include the β2-AR, α1B-AR, and β-opioid receptor, have high affinity for β-arrestin 2, whereas class B GPCRs, such as the angiotensin II type 1A receptor, neurotensin receptor 1, and vasopressin V2 receptor, exhibit high affinity for both β-arrestin 1 and 2 isoforms.
With regard to the α1-AR subtypes, the desensitization, down-regulation, and internalization characteristics of the α1B-AR have been most extensively examined. For example, agonist-mediated phosphorylation and internalization of the α1B-AR have been demonstrated, and the domains of the receptor involved in internalization have been identified (Fonseca et al., 1995;Mhaouty-Kodja et al., 1999; Wang et al., 1997, 2000). We know much less regarding the molecular determinants of desensitization, down-regulation, and internalization for the α1A- and α1D-ARs.Vázquez-Prado et al. (2000) showed that the α1A-AR could undergo agonist-mediated phosphorylation, albeit not to the same extent as the α1B-AR. Yang and coworkers (1999) used fibroblasts stably transfected with each of the α1-ARs to show that the increase in inositol phosphates mediated by the α1A- and α1B-ARs could be desensitized, whereas the increase mediated by the α1D-AR was refractory to agonist-mediated desensitization. In contrast to this,Garcı́a-Sáinz et al. (2001) showed that the α1D-AR could be phosphorylated and desensitized.
In this report, we have examined subcellular distribution, agonist-mediated internalization and desensitization characteristics of green fluorescent protein (GFP)-tagged α1-ARs using real-time imaging in transiently transfected human embryonic kidney (HEK) 293 cells. We show that there are significant differences in these parameters that could account for differences in the cellular signaling properties of the α1-ARs.
Experimental Procedures
Materials.
α1-AR-green fluorescent protein (α1-AR/GFP) vectors were constructed by ligating the coding region of the human α1A-, α1B-, and α1D-AR into the EcoRI-KpnI site of the basic pEGFP-N3 protein fusion vector (BD Biosciences Clontech, Palo Alto, CA) as described previously (Hirasawa et al., 1997; Awaji et al., 1998). The generation of wild-type β-arrestin and a dominant-negative β-arrestin 1 (319–418) in pcDNA3 has been reported previously (Krupnick et al., 1997b). Rabbit polyclonal antibodies targeted against β-arrestin 1 were generated as described by Orsini and Benovic (1998).
Cell Culture and Transient Transfection.
HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic cocktail [10,000 units/ml penicillin G sodium, 10,000 mg/ml streptomycin sulfate, and 25 mg/ml amphotericin B in 0.85% saline (Invitrogen, Carlsbad, CA)]. The cells were grown in T75 flasks in a 37°C cell culture incubator with a humidified atmosphere of 95% air and 5% CO2 and were fed every 2 to 3 days. HEK cells used in immunocytochemistry protocols were grown on gelatin/laminin-treated coverslips in 35-mm tissue culture dishes (Corning Glassworks, Corning, NY), whereas cells for real-time studies were grown in culture dishes with a glass coverslip bottom (MatTek Co., Ashland, MA) that were also gelatin/laminin treated. Cells were grown to approximately 80% confluence and used for experimentation 3 days after being plated. HEK cells were transfected with cDNA encoding α1A-, α1B-, or α1D-AR/GFP fusion protein using calcium phosphate precipitation. In certain studies, the receptor/GFP constructs were cotransfected with a cDNA encoding wild-type β-arrestin 1 or β-arrestin 1 (319–418). β-Arrestin 1 overexpression was confirmed using specific antibodies in immunocytochemistry protocols as we have described previously (Hrometz et al., 1999; McCune et al., 2000).
Activation of ERK1/2 Phosphorylation and Agonist-Mediated Desensitization.
The coupling of the α1-AR-/GFP constructs to functional responses and agonist-mediated desensitization was assessed by measuring the phosphorylation of ERK1/2. Cells were challenged with 100 μM phenylephrine for a period of 2 h. After the appropriate time, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline for 10 min, and immunocytochemistry was performed as described previously by Hrometz et al. (1999) and McCune et al. (2000). In brief, cells were treated with mouse monoclonal IgG pERK (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:50 dilution and then incubated with Rhodamine Red-X-conjugated AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:100. The degree of ERK1/2 phosphorylation was assessed using laser scanning confocal microscopy as described below. Desensitization experiments were conducted on HEK 293 cells 72 h after transient transfection with cDNA encoding α1A-, α1B-, or α1D-AR-/GFP. Cells were treated with 100 μM phenylephrine for 15 h. Vehicle-treated cells served as controls. After the incubation, cells were washed three times (30-min intervals between each wash) with Dulbecco's modified Eagle's medium, after which cells were rechallenged with phenylephrine for 2 h, and the effect on ERK1/2 phosphorylation was assessed.
Laser Scanning Confocal Microscopy.
Transfected HEK 293 cells were imaged with a Spectra-Physics laser scanning confocal microscope attached to a TCS DM RXE microscope with a Plan-Apo 100x oil immersion objective lens (Leica, Wetzlar, Germany). The software used to collect the images was the Leica TCS NT version 1.6.587. The images were transferred to a computer for reduction and analysis with Adobe Photoshop version 4.0 (Adobe Systems, Mountain View, CA). The setting on the laser was constant for all experiments. However, both GFP and rhodamine signals were digitally enhanced by adjusting the photomultiplier tube (PMT). Initial adjustment of the PMT allowed us to minimize the background signal while maximizing the fluorescent signal(s) of interest. Because individual cells required a different PMT setting, the differences in intensity should not be construed as a measure of receptor expression levels.
Data and Image Analysis.
The rate and extent to which the α1-AR/GFP constructs were internalized after exposure to agonist were analyzed using the image analysis software NIH ImageJ 1.18x (http://rsb.info.nih.gov/ij/). The change in fluorescence intensity was measured in a rectangular area just below the cell surface before and during the internalization process. Data were normalized to a percentage of the fluorescence obtained before agonist treatment. The increase in fluorescence intensity above that observed in untreated cells is a measure of receptor internalization. A plot of the percentage increase in fluorescence intensity versus time after agonist treatment was then generated. The average phospho-ERK1/2 signal per determined area was quantitated using the same image analysis software. Only images acquired using exactly the same PMT settings were compared with each other. Treated cells were normalized to the control phospho-ERK1/2 activation signal. All data are reported as the mean ± S.E. Data were analyzed by analysis of variance followed by Student-Newman-Kuels analysis to determine where statistically significant differences existed. A P value of less than 0.05 was considered significant.
Results
Basal Cellular Localization.
HEK 293 cells were transiently transfected with expression plasmids encoding α1-AR/GFP fusion proteins and the living cells were visualized 72 h later. Transfection with a cDNA encoding the α1B-AR/GFP resulted in a specific fluorescence that was detected predominantly on the margin of the cell, indicative of a cell surface localization (Fig. 1). Although there was cell surface expression, the majority of the α1D-AR/GFP fluorescence was detected intracellularly in a perinuclear orientation (Fig. 1). Exhibiting localization properties of each of these subtypes, α1A-AR/GFP fluorescence was observed on the cell surface and in a perinuclear orientation (Fig. 1).
Functional Responses Mediated by the α1-AR/GFP Fusion Proteins.
To demonstrate that the expressed α1-AR/GFP fusion proteins were functional, transfected cells were stimulated with phenylephrine, and, after fixing of the cells, the effect on ERK1/2 phosphorylation was determined with a monoclonal antibody specific for phospho-ERK1/2. Treatment with phenylephrine resulted in a statistically significant increase in phospho-ERK1/2 immunoreactivity in cells transfected with either the α1A- or the α1B-AR/GFP constructs (Fig. 2, A, B, and D). This indicates that these GFP modified α1-ARs are functional when expressed in HEK 293 cells. Phenylephrine treatment of cells transfected with the α1D-AR also resulted in an increase in the level of ERK1/2 phosphorylation (Fig. 2, C and D). However, this increase was not significantly different compared with the untreated control (Fig. 2D). These findings could indicate that, although functional, the α1D-AR is poorly coupled to second messenger pathways such that only a modest increase in ERK1/2 phosphorylation could be observed.
Effect of Agonist Stimulation on Receptor Localization.
In addition to activating ERK1/2 phosphorylation, the ability of phenylephrine to promote changes in receptor localization was assessed in real-time. Addition of 100 μM phenylephrine to cells expressing the α1B-AR/GFP resulted in a rapid translocation of the receptor from the cell surface to intracellular compartments (Fig. 3). The α1-AR antagonist 1 μM prazosin blocked this internalization (data not shown).
The increase in intracellular fluorescence signal intensity, quantitated with image analysis software (as described underExperimental Procedures), was used to gain a measure of the rate of receptor internalization. A plot of the increase in intracellular fluorescence intensity versus time after phenylephrine administration is presented in Fig. 4A and shows that α1B-AR internalization occurred in a very rapid fashion.
Receptor activation with phenylephrine also promoted the internalization of the cell surface population of α1A-ARs (Figs. 3 and 4B). However, a significant increase in intracellular fluorescence was not detected until 50 min after agonist exposure. A plot of the increase in intracellular fluorescence intensity versus time revealed that the α1A-AR internalization occurred at a slower rate than that seen for the α1B-AR. Treatment of HEK cells expressing the α1D-AR/GFP fusion protein with phenylephrine did not cause a translocation of the cell surface population of α1D-ARs (Fig.5).
Agonist-Mediated Receptor Desensitization.
Transfected HEK 293 cells were incubated for 15 h with 100 μM phenylephrine, and the effect on α1-AR/GFP localization and the ability of the α1-ARs to stimulate ERK1/2 phosphorylation were assessed. Prolonged incubation with phenylephrine (followed by extensive washout) resulted in an internalization of α1A- and α1B-ARs (see GFP fluorescence in Fig. 2, A and B) in a fashion similar to that seen in untreated cells. Phenylephrine treatment for 15 h significantly decreased the ability of either the α1A- or α1B-AR to activate ERK1/2 phosphorylation in response to a second addition of phenylephrine (Fig. 2D). The long exposure to phenylephrine had no effect on the cellular localization of the α1D-AR. Long-term exposure to phenylephrine significantly reduced the level of phospho-ERK1/2 seen after rechallenge with agonist in α1D-AR expressing cells (Fig. 2D).
Effect of Arrestins on Agonist-Activated Receptor Internalization.
HEK cells were cotransfected with α1-AR/GFP constructs and an expression plasmid encoding β-arrestin 1. The overexpression of β-arrestin 1 was confirmed using immunocytochemical protocols with an antibody against β-arrestin 1 (Fig. 6). β-Arrestin 1 overexpression did not increase the rate or extent of α1A- or α1B-AR internalization after stimulation with phenylephrine (data not shown). In a similar fashion, cotransfection with β-arrestin 2 had no effect on agonist-mediated internalization (data not shown).
HEK 293 cells were cotransfected with a cDNA encoding the α1B-AR/GFP construct and a dominant-negative form of β-arrestin 1, β-arrestin 1 (319–418). β-Arrestin 1 (319–418) had no effect on basal α1B-AR cellular localization (Fig. 7A). However, the dominant-negative arrestin markedly decreased the ability of phenylephrine to promote α1B-AR internalization (Fig. 7A). Analysis of these data revealed that the dominant-negative arrestin significantly reduced the rate of increase in intracellular fluorescence intensity seen after the addition of phenylephrine to HEK 293 cells (Fig. 4A). Similar to effects seen with the α1B-AR, β-arrestin 1 (319–418) decreased the magnitude of the phenylephrine-induced α1A-AR internalization (Figs. 4B and 7B).
Wild-type β-arrestin 1 did not affect the basal cellular localization or the ability of phenylephrine to promote α1D-AR internalization (Fig.8A). Similarly, β-arrestin 1 (319–418) had no effect on the cellular localization of the α1D-AR/GFP (Fig. 8B).
Discussion
In this communication, we have examined the cellular localization, agonist-mediated internalization, and desensitization properties of α1-AR/GFP fusion proteins in transiently transfected HEK 293 cells. Previous studies with the α1B-AR/GFP construct demonstrated that it is fully functional and internalizes in the same manner as a non-GFP tagged α1B-AR construct (Awaji et al., 1998). In a similar fashion, previous studies showed that both the α1A- and α1B-ARs are coupled to the activation of ERK (for reviews, seeGarcı́a-Sáinz et al., 1999; Zhong and Minneman, 1999;Varma and Deng, 2000; Piascik and Perez 2001). In this report, we show that both the α1A- and α1B-ARs when coupled to GFP can promote an increase in ERK1/2 phosphorylation (Fig. 2). The phosphorylation of ERK1/2 is thought to mediate growth responses, at least in part. Demonstration of ERK1/2 phosphorylation in these studies is evidence that the α1-ARs are functional and retain their ability to activate cellular signaling when conjugated to the GFP.
In this report, we also noted that, although phenylephrine could increase the level of phospho-ERK1/2 in α1D-AR expressing cells, this increase was not statistically significant. This could indicate that the small population of cell surface α1D-ARs is not efficiently coupled to ERK1/2 phosphorylation. This result is consistent with the observations ofTheroux et al. (1996) who noted that the α1D-AR was the most poorly coupled of the α1-AR subtypes. In previous work with stably transfected fibroblasts, we showed that the α1D-AR was constitutively active with regard to ERK activation (McCune et al., 2000). We also noted that there was a high basal level of ERK activity in these cells and that phenylephrine could not promote a further enhancement of kinase activity (McCune et al., 2000). Thus, the inability to detect a significant increase in ERK1/2 phosphorylation in this present study could also be due to a constitutively active α1D-AR. Nonetheless, we must also accept the possibility that the α1D-AR/GFP construct may not be functionally active (however, see the additional discussion below).
Laser scanning confocal microscopy revealed that the α1B-AR was expressed predominantly on the cell surface (Fig. 1). Although there is some cell surface expression of the α1D-AR, the majority of this receptor is expressed in intracellular compartments. The α1A-AR has localization characteristics of both the α1B- and α1D-ARs, being expressed not only on the cellular surface but also intracellularly. Using immunocytochemistry with subtype selective antibodies, we previously observed a similar distribution pattern for the α1-ARs in stably transfected fibroblasts and vascular smooth muscle cells (Hrometz et al., 1999; McCune et al., 2000). However, studies of the cellular localization of the α1-ARs have been hampered by the low affinity of the commercially available antibodies. The present studies confirm and extend our initial findings with techniques that do not involve antibodies. Therefore, it seems likely that our observation of differential cellular localization of the α1-AR accurately portrays the distribution pattern in vascular smooth muscle cells that normally express all three α1-ARs. Indeed, an elegant series of studies using prazosin labeled with BODIPY-FL to image the α1-AR subtypes noted an intracellular expression of these receptors in cultured prostate smooth muscle cells and stably transfected fibroblasts (MacKenzie et al., 2000). These authors estimated that in smooth muscle cells, 40% of the total α1-AR population is expressed intracellularly.
Using real-time imaging of living cells, we observed differences in the agonist-mediated internalization properties of the α1-ARs (Figs. 3-5). In agreement with previous work (Fonseca et al., 1995; Wang et al., 1997, 2000), we observed that the α1B-AR undergoes rapid agonist-mediated internalization. However, there has been little investigation of the effect of agonist activation on the translocation of the other α1-AR subtypes. We noted that the α1A-AR also undergoes agonist-mediated internalization. Interestingly, this internalization occurs at a slower rate than for the α1B-AR (Fig. 4, compare A and B). We were unable to detect any agonist-mediated internalization of the α1D-AR (Fig. 5). We cannot discount the possibility that a small amount of receptor internalization did take place. However, this small increase in intracellular fluorescence could not be detected because of the considerable fluorescence produced by the intracellular population of α1D-ARs.
We then assessed the extent to which the receptors could be desensitized after prolonged exposure to phenylephrine. Transfected HEK 293 cells were incubated with phenylephrine for 15 h and then extensively washed. This long incubation period resulted in redistribution of each of the α1-AR/GFPs similar to that seen in nondesensitized cells (compare Fig. 2 with Figs. 1 and 3). After the washout period, the cells were rechallenged with phenylephrine. Using this protocol, we demonstrated that prolonged exposure to agonist desensitizes the ability of the α1A- and α1B-ARs to promote ERK1/2 phosphorylation (Fig. 2). Interestingly, after a 15-h exposure to phenylephrine in α1D-AR expressing cells, rechallenge with agonist could not promote any increase in the level of phospho-ERK1/2. Indeed, there was a statistically significant difference in the level of agonist-induced ERK1/2 phosphorylation in control HEK 293 cells and that seen after desensitization (Fig. 2). Thus even though phenylephrine could only promote a modest, nonsignificant increase in the level of phospho-ERK1/2 in control cells, this could be reduced by prolonged expose to agonist and supports the notion that the α1D-AR/GFP construct is functional.
Our results are consistent with other studies that have examined the phosphorylation and desensitization of the α1-ARs. Yang et al. (1999) noted that both the α1A- and α1B-ARs undergo agonist-mediated desensitization. However, these authors noted that the α1B-AR was desensitized by lower concentrations of agonist. Similarly, Vázquez-Prado et al. (2000)found that the α1B-AR underwent more extensive agonist-activated phosphorylation than did the α1A-AR. The more rapid rate of α1B-AR internalization noted here is also consistent with the observation that this receptor is more extensively phosphorylated and readily desensitized than the α1A-AR. Yang et al. (1999) also observed that functional responses mediated by the α1D-AR were not subject to desensitization. In contrast to the work of Yang et al. (1999), Garcı́a-Sáinz et al. (2001) noted that in stably transfected fibroblasts, the α1D-AR could be phosphorylated and desensitized. Therefore, a definitive answer regarding the desensitization characteristics of the α1D-AR requires additional studies.
Arrestins have been implicated in mediating the internalization of a variety of GPCRs. There has been little work performed to determine the role of arrestins in agonist-mediated α1-AR internalization. We were unable to observe any demonstrable effects of β-arrestin 1 overexpression to the degree to which agonist activation promotes the internalization of the α1A- or α1B-ARs. This probably reflects the fact that HEK 293 cells possess large amounts of β-arrestin 1 (Fig. 6). Therefore, overexpression of additional arrestin molecules would not be expected to have an effect on agonist-mediated receptor internalization. Similarly, overexpression of β-arrestin 2 had no effect on the degree of agonist-stimulated internalization of the α1A- or α1B-ARs (data not shown). A dominant-negative arrestin, β-arrestin 1 (319–418), completely blocked agonist-mediated internalization of both the α1A- and the α1B-ARs (Figs. 4 and 7). These data argue that agonist-activated internalization of the α1-AR subtypes is mediated by arrestins. Although the dominant-negative arrestin confirms the role of arrestins in α1-AR internalization, this reagent cannot be used to determine the specific role of β-arrestin 1 or 2 in the internalization process. This is because the dominant-negative arrestin binds to clathrin, thus preventing the binding of wild-type arrestin species. Therefore, the dominant-negative arrestin would be expected to block the actions of any wild-type arrestin.
The intracellular localization of α1D-AR was not affected by overexpression of either wild-type β-arrestin 1 or β-arrestin 1 (319–418), arguing that the intracellular distribution of the α1D-AR is not likely to be maintained by arrestin molecules (Fig. 8). The significance of the predominantly intracellular localization of the α1D-AR is not clear. We do not know which of the α1D-ARs, the small population of cell surface receptors or the large population of intracellularly expressed receptors, are signaling competent and responsible for the regulatory activity of this subtype. As noted above, data from several labs including ours show that the α1D-AR is constitutively active (Garcı́a-Sáinz and Torres-Padilla, 1999; McCune et al., 2000; Noguera et al., 1993). The observation of constitutive activity may shed some light on the relationship between cellular localization and functional responses. A constitutively active receptor assumes an activated conformation in the absence of agonist. The large degree of intracellular localization of the α1D-AR may be due to continuous internalization of the receptor due to its constitutively active nature.
The three α1-ARs are coexpressed on tissues and organs involved in cardiovascular regulation, yet these receptors modulate different physiological processes. We hypothesize that the observed differences in the cellular localization could contribute to the differences in the functional responses mediated by these receptors. We also propose that the α1B-AR most approximates a prototypic GPCR in terms of cellular localization, agonist-mediated internalization, desensitization, and coupling to cellular signaling. In contrast, we postulate that the α1D-AR is an atypical GPCR. Although the α1A-AR is expressed intracellularly, it seems to have signaling properties expected of a GPCR.
Acknowledgments
We thank Dr. J. L. Benovic for providing β-arrestin 1, β-arrestin 2, and dominant-negative β-arrestin 1 expression vectors as well as the antibodies to β-arrestin 1 and β-arrestin 2.
Footnotes
- Received October 10, 2001.
- Accepted January 28, 2002.
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This work was supported by National Institutes of Health Grant HL38120 (M.T.P.), American Health Association Grant-in-Aid (M.T.P.), a Predoctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (D.F.M.), and a Predoctoral Fellowship from the American Health Association (D.C.).
Abbreviations
- AR
- adrenoceptor
- ERK
- extracellular signal-regulated kinase
- GFP
- green fluorescent protein
- GPCR
- G-protein-coupled receptor
- HEK
- human embryonic kidney
- PMT
- photomultiplier tube
- BODIPY-FL
- 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester
- The American Society for Pharmacology and Experimental Therapeutics