Introduction

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Three subtypes of the ₁-adrenoceptor (AR), the _1A, _1B, and _1D, have thus far been identified, cloned, and characterized (Cotecchia et al., 1988; Schwinn et al., 1990; Lomasney et al., 1991; Perez et al., 1991). Our understanding of the complexity of the ₁-ARs and their interplay with other signaling systems has steadily increased. The ₁-ARs are major effectors used by the sympathetic nervous system to regulate systemic arterial blood pressure, blood flow, and cell growth. Previous work by us and others has shown that the mRNA encoding each of these receptors is widely distributed in the peripheral vasculature (Ping and Faber, 1993; Piascik et al., 1994, 1995; Eckhart et al., 1996; Guarino et al., 1996) and that the ₁-ARs are also expressed as protein in all of the peripheral arteries thus far examined (Piascik et al., 1997; Hrometz et al., 1999). Using both pharmacological approaches and antisense oligonucleotide technology, we have found evidence that the activation of smooth muscle contraction is caused by a single receptor in any given artery and that the ₁-AR subtype that mediates contraction varies throughout the vasculature (Piascik et al., 1995, 1997; Hrometz et al., 1999).

These findings have lead us to explore possible mechanisms to account for the observation that expression in blood vessels is not sufficient to link an ₁-AR to contraction. We have focused on the subcellular localization of the ₁-ARs as a factor that modifies the ability of these receptors to regulate cellular function. In models of cellular signaling, the heptahelical G protein-coupled receptors (GPCRs) are postulated to reside in the cell membrane where they transduce a variety of intracellular signals initiated by binding of neurotransmitters or exogenously administered pharmacologic agents. Recent evidence has indicated that the -ARs are not exclusively localized with the cell membrane. Using green fluorescent protein/₁-AR fusion proteins, Hirasawa et al. (1997) showed that the _1B-AR was expressed on the cell surface whereas the _1A-AR was expressed in intracellular compartments. In transfected fibroblasts, the _2C-AR was detected in intracellular compartments as well as on the cell surface, whereas the _2A-AR was found exclusively on the cell membrane (von Zastrow et al., 1993; Daunt et al., 1997).

Internalization of GPCR after activation by agonists is a well known phenomenon. Agonist-induced internalization of the GPCR regulates signal transduction by this class of transmembrane receptors. Like constitutively recycling receptors for transferrin and the low-density lipoprotein, GPCRs are internalized via clathrin-coated pits to endosomes (von Zastrow and Kobilka, 1992; reviewed by Krupnick and Benovic, 1998). In addition to activation of cellular signaling cascades, agonist occupancy of GPCR results in the recruitment of a GPCR kinase to the membrane, receptor phosphorylation, and promotion of arrestin binding to the receptor (Krupnick and Benovic, 1998). Arrestin binding is thought to uncouple the receptor from G proteins and initiate the process of internalization by directing the receptor to the clathrin-coated pits. This process has traditionally been thought of as a way of terminating cellular signaling. However, studies using transfected ₂-ARs or endogenous opioid receptors indicate that internalization of certain GPCR also activates cellular signaling. Daaka et al. (1998) and Ignatova et al. (1999) showed that receptor internalization was necessary to activate extracellular signal-regulated kinase (ERK). In these studies, expression of dominant negative mutants of -arrestin or dynamin prevented receptor internalization and receptor-stimulated ERK activity.

In this report we have examined the regulation of the cellular localization and signaling properties of the _1B- and _1D-ARs. We show that the _1B-AR is expressed primarily on the cell surface, is internalized by agonist occupation, and is coupled to inositol phosphate formation and ERK activity, consistent with traditional views of GPCR. The _1D-AR exhibits behavior atypical of a GPCR, and we postulate that this is due to the constitutive activity exhibited by this receptor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Cell Culture Conditions. Rat 1 fibroblasts stably transfected with cDNA for the human _1B-AR or _1D-AR (a gift from Glaxo Wellcome) were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Life Technologies), a 1% antibiotic/antimycotic mixture (Life Technologies), and 500 µg/ml geneticin (Life Technologies). Cells were maintained in cell culture flasks at 37°C in 5% CO₂, fed every 2 to 3 days with supplemented DMEM, and trypsinized every 5 to 7 days. After the fibroblasts reached confluency, they were plated out on sterile 20-mm × 20-mm glass coverslips and returned to the cell culture incubator for 72 h to allow attachment. Cells were deprived of serum overnight (18-24 h) before experimentation.

Immunocytochemistry and Laser Scanning Confocal Microscopy. Cells were fixed with 3.7% formaldehyde in PBS for 10 min, washed with PBS containing 0.05% BSA, and permeabilized with 0.1% Triton in PBS for 5 min. Cells were incubated in blocking solution containing 10% fetal bovine serum in PBS for 1 h at room temperature, then washed and incubated with primary antibody for 2 h. The primary antibodies (against the ₁-ARs, the transferrin receptor, and arrestin 2) are described in a section below. Fibroblasts were then incubated with either a fluorescein-conjugated (donkey anti-goat IgG, for ₁-AR visualization) or a rhodamine-conjugated (donkey anti-rabbit or -mouse IgG, for arrestin or transferrin receptor visualization) secondary antibody. Dual-label immunocytochemistry was used to simultaneously assess the localization of the ₁-ARs, arrestin, or transferrin. In certain experiments, the effect of phenylephrine or a series of antagonists on the cellular location of proteins of interest was assessed. We also examined _1B-AR immunoreactivity in COS 1 cells transiently transfected with the wild-type _1B-AR or a constitutively active mutant in which alanine 293 was mutated to lysine (A293K, Kjelsberg et al., 1992). Images were analyzed using a laser-scanning confocal microscope (Leica TCS, Exton, PA) with a 63× Water Immersion objective. Fluorescein isothiocyanate (FITC) fluorescence was excited by an argon laser at 488 nm and monitored at a wavelength no greater than 545 nm. Rhodamine fluorescence was excited using a 568-nm helium-neon laser and monitored at a wavelength no greater than 690 nm.

Quantitation of Intracellular Inositol Phosphates. Rat 1 fibroblasts plated on 60-mm culture plates were grown in DMEM (Bio-Whittaker, Walkersville, MD) supplemented with fetal bovine serum. On reaching 90% confluency, 3 µCi of ³H-myo-inositol (DuPont-NEN, Boston, MA) was added 24 h before use in experiments. Measurement of intracellular inositol phosphates was performed in serum-free DMEM after several changes of media. Assays were conducted in the presence of LiCl (10 mM) in a total assay volume of 3 ml. Cells were incubated at 37°C for 60 min in a 5% CO₂ atmosphere in the presence or absence of increasing concentrations of phenylephrine. The effect of 1 µM prazosin, an inverse agonist, which was added 24 h before experimentation was also determined. Incubations were terminated by removal of the media and addition of 1 ml of a 0.4 M perchloric acid solution. The cell lysates were scraped, collected, and neutralized by the addition of 0.5 ml of a 0.72 N KOH/0.6 M KHCO₃ solution. Soluble inositol phosphates in the cell lysates were isolated by passage through a Bio-Rad AG 1X-8 resin column that was buffered with a 0.1 M formic acid solution. After washing the column with 0.1 M formic acid, bound ³H-IPs were displaced from the column by eluting with a 0.1 M formic acid solution containing 1 M ammonium formate and the radioactivity was detected by liquid scintillation counting. Data were analyzed using a one-tailed t test analysis (GraphPad Prism).

Assays for ERK Activity. ERK activity was determined using in-gel kinase assays as we have described previously (Post et al., 1996). Briefly, fibroblasts were incubated with phenylephrine in the presence or absence of prazosin for the indicated times, washed with ice-cold PBS, and scraped into 1 ml of ice-cold buffered sucrose. The cell suspension was centrifuged (5 min, 1000g, 4°C), and the pellet was resuspended in 20 µl of lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 2.5 mM EDTA, 3 mM EGTA, 20 mM -glycerophosphate, 0.5% NP-40, 100 µM Na₃VO₄, 5 µM AEBSF (Calbiochem, La Jolla, CA), 1.5 nM aprotinin, 10 nM E-64, 10 nM leupeptin, pH 7.4). After incubation on ice for 30 min, the lysate was centrifuged (15 min, 15,000g, 4°C), the supernatant was collected, and protein content was determined. Protein was resolved on 10% SDS-polyacrylamide gels containing 0.5 mg/ml myelin basic protein (MBP). After electrophoresis, gels were first washed with 20% 2-propanol in 50 mM HEPES, pH 7.6 and then with 5 mM -mercaptoethanol in HEPES buffer. Proteins were denatured by washing gels in 6 M urea and then renatured in HEPES buffer containing 0.05% (v/v) Tween-20 (renaturation buffer) at 4°C. After overnight incubation in renaturation buffer at 4°C, gels were preincubated in 25 ml of cold kinase buffer (20 mM HEPES, 20 mM MgCl₂, 2 mM DTT, 5 mM -glycerophosphate, 0.1 mM Na₃VO₄) for 30 min. Phosphorylation of MBP was performed in situ by incubating gels in kinase buffer containing 20 µM ATP and 150 to 160 µCi [³²P]ATP for 90 to 120 min at 30°C. After extensive washing in 5% trichloroacetic acid/1% sodium pyrophosphate, gels were dried and exposed to film. ³²P incorporation into MBP was quantified by densitometry. The ERK activity reported in Table 1 is in integrated optical density units, whereas those presented in Fig. 4 are normalized to a percentage of the control level of ERK activity obtained in each cell line. Data were analyzed by a one-way ANOVA.

View this table: [in this window] [in a new window]   TABLE 1 Effect of prazosin on basal levels of inositol phosphate formation and ERK activity in fibroblasts expressing either the 1B- or 1D-AR Each point represents the mean ± S.E. for four (inositol phosphate) or five (ERK) independent experiments.

Antibodies and Reagents. The primary antibodies used were as follows: goat anti-human _1B IgG at 1:100 to 1:200, goat anti-rat _1D IgG at 1:25 to 1:50, (Santa Cruz Biotech, Santa Cruz, CA) and mouse anti-rat CD71, a marker for the transferrin receptor, at 1:100 (Research Diagnostics, Flanders, NJ). Arrestin antibodies were provided by Dr. Jeffrey Benovic (Thomas Jefferson University). All secondary antibodies were used at a dilution of 1:100, which included fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG, rhodamine-conjugated donkey anti-rabbit IgG, and rhodamine-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA). Prazosin and phenylephrine were purchased from Sigma (St. Louis, MO).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

In this report we have examined the regulation of the cellular distribution of the _1B- and _1D-ARs in Rat 1 fibroblasts stably transfected with either subtype. We have previously used antibodies to examine the distribution of the ₁-ARs in blood vessels, vascular smooth muscle cells, and stably transfected fibroblasts (Hrometz et al., 1999). In these studies we showed that the antibodies are specific and stain only those cells expressing the receptor against which the antibody was raised. Additionally, we show that preabsorption of the antibody with its immunizing peptide significantly reduces immunostaining.

In radioligand binding studies we determined that the _1B- and _1D-AR were expressed at similar levels in the Rat 1 fibroblast cell lines used in these studies (between 5.5 and 10 pmol/mg protein; data not shown). In _1B-AR-expressing fibroblasts, receptor immunoreactivity was detected on the margin of the cell, indicating that, as expected, this receptor was localized to the cell membrane (Fig. 1). In contrast, although there was _1D-AR immunoreactivity on the boundary of the cell, a significant degree of immunostaining was also detected in a perinuclear orientation (Fig. 1).

View larger version (75K): [in this window] [in a new window]   Fig. 1.   Immunolocalization of the 1B- and 1D-ARs in stably transfected fibroblasts. Immunocytochemistry was carried out as described in Materials and Methods.

Figure 2 shows the immunostaining pattern obtained in fibroblasts expressing the _1B-AR after treatment with the ₁-AR selective agonist phenylephrine. Agonist activation promotes a significant degree of _1B-AR internalization as evidenced by the enhanced FITC fluorescence detected in a perinuclear orientation (Fig. 2, panel 1). Internalization can be detected within 5 min of receptor activation (see Fig. 2, panel 4 for time course) and is blocked by prior treatment of the cells with prazosin. Isoproterenol activation of the ₂-AR promotes receptor internalization to endosomes (von Zastrow and Kobilka, 1992). We determined whether this is also true for the _1B-AR by performing dual-label immunofluorescence studies using an antibody against the transferrin receptor (CD71 in the figures), an endosomal marker. In these photomicrographs, the receptor is detected by a FITC-labeled secondary antibody, which emits a green color. The transferrin receptor is detected with a rhodamine-labeled secondary antibody that appears red (Fig. 2, panel 5). When these secondary antibodies are colocalized, the resulting fluorescent image appears yellow. Dual antibody labeling after activation of the _1B-AR with phenylephrine yields a perinuclear yellow fluorescence, indicating that the internalized _1B-AR is localized to endosomes (Fig. 2, panel 2). This colocalization and color change were prevented by pretreatment with prazosin.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Effect of 100 µM phenylephrine on the cellular localization of the 1B-AR. Also presented is the colocalization of the receptor with the transferrin receptor (CD 71 antibody, panel 2) and arrestin 2 (panel 3). The receptor was detected with a FITC-labeled secondary antibody. The transferrin receptor and arrestin 2 were detected with rhodamine-labeled secondary antibody. When the two proteins colocalize, the fluorescence is detected as yellow.

Arrestin binding to phosphorylated GPCR is thought to uncouple the receptor from its cognate G protein(s) and participate in the process of internalization by directing the receptor to clathrin-coated pits. Dual-label immunocytochemistry shows that in unstimulated Rat 1 fibroblasts, there is little colocalization of the _1B-AR with arrestin 2 (detected with a rhodamine-labeled secondary antibody, see Fig. 2, panels 3 and 5). Agonist mediated _1B-AR internalization promotes the association of the receptor with arrestin 2 (see Fig. 2, panel 3). Receptor internalization and arrestin colocalization were blocked by prior treatment with prazosin. Detection of agonist-stimulated internalization of the _1B-AR into endosomes by confocal microscopy has been reported previously in stably transfected HEK 293 cells (Fonseca et al., 1995) and cells expressing an _1B-AR/green fluorescent protein fusion protein (Awaji et al., 1998). Association of constitutively active _1B-AR mutants with arrestin 2 has also been demonstrated (Mhaouty-Kodja et al., 1999).

The functional response to _1B-AR activation was assessed by measuring inositol phosphate formation and ERK activity in stably transfected fibroblasts. Phenylephrine stimulated the formation of inositol phosphates and ERK activity in cells expressing the _1B-AR (see Figs. 3 and 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Phenylephrine stimulation of inositol phosphate formation in fibroblasts expressing the 1B- and 1D-ARs. Experiments were carried out as described in Materials and Methods. Each point represents the mean and S.E. of four independent experiments from each cell line.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Phenylephrine-mediated ERK activity in fibroblasts expressing the 1B- and 1D-ARs. Experiments were carried out as described in Materials and Methods. Data from each cell line is normalized to the percentage of the control optical density obtained from densitometric scans of the autoradiographs generated by the in-gel kinase assays. A representative autoradiograph is also presented. Each point represents the mean and S.E. of five independent experiments from each cell line.

Immunocytochemical analysis and cell signaling studies in Rat 1 fibroblasts expressing the _1D-AR revealed a distinctly different cellular localization pattern and functional responses than those observed for the _1B-AR. Although some _1D-AR immunoreactivity was noted on the boundary of the cell, a significant degree of immunostaining was detected in a perinuclear orientation (see Fig. 1). The _1D-AR colocalizes with the transferrin receptor, indicating that in unstimulated fibroblasts, the _1D-AR is localized to endosomes (see Fig. 5, panel 2). In unstimulated fibroblasts, the _1D-AR was also localized with arrestin 2 (see Fig. 5, panel 3). The control transferrin and arrestin immunoreactivities in _1D-AR-expressing fibroblasts were not different from those obtained in _1B-AR-expressing cells as shown in Fig. 2.

View larger version (84K): [in this window] [in a new window]   Fig. 5.   Cellular localization of the 1D-AR in stably transfected fibroblasts cultured in the absence and presence of prazosin. Also presented are dual-label immunocytochemistry with CD 71, an antibody against the transferrin receptor, and antibodies against arrestin 2. The receptor was detected with a FITC-labeled secondary antibody. The transferrin receptor and arrestin 2 were detected with rhodamine. When the two proteins colocalize, the fluorescence is detected as yellow.

Fibroblasts expressing the _1D-AR exhibited a high degree of basal inositol phosphate formation compared with _1B-AR-expressing cells (see Table 1). Phenylephrine was capable of inducing an additional dose-dependent increase in inositol phosphate formation in _1D-AR-expressing fibroblasts (Fig. 3). Basal ERK activity in _1D-AR-expressing fibroblasts was also greater than that seen in _1B-AR-expressing cells. However, we did not detect phenylephrine-induced increases in ERK activity above basal levels in _1D-AR-expressing cells (Fig. 4). Interestingly, basal ERK activity was similar in magnitude to serum-stimulated levels in _1D-AR-expressing cells. It may be that the high basal levels of kinase activity we observed precluded any additional agonist-induced increases in the _1D-AR-expressing fibroblasts.

In unstimulated cells, we noted that the _1D-AR is localized with arrestin and endosomes, the same cellular locale as the _1B-AR after agonist activation. In addition, fibroblasts expressing the _1D-AR exhibit a high level of basal phospholipase C and ERK activity compared with the _1B-AR. These data argue that the _1D-AR is constitutively active in Rat 1 fibroblasts. To more fully substantiate that the _1D-AR is constitutively active, we performed immunocytochemistry, inositol phosphate, and ERK assays after culturing _1B- or _1D-AR-expressing fibroblasts in the presence of prazosin for 24 h. Prazosin has recently been shown to have inverse agonist properties (Scheer et al., 1997). Treatment of _1B-AR expressing fibroblasts with prazosin had no effect on the cellular localization of the _1B-AR (data not shown). Likewise, prazosin did not decrease either basal inositol phosphate formation or ERK activity (see Table 1) in _1B-AR expressing cells. Treatment of _1D-AR-expressing fibroblasts with prazosin caused a significant degree of redistribution of the _1D-AR from a perinuclear orientation to the cell periphery (Fig. 5, panel 1). After prazosin treatment, the _1D-AR was no longer colocalized with either the transferrin receptor or arrestin 2 (see Fig. 5, panels 2 and 3). Treatment of fibroblasts expressing the _1D-AR with prazosin also decreased basal levels of inositol phosphate formation and ERK activity (see Table 1). The cellular localization of a constitutively active mutant (A293K, Kjelsberg et al., 1992) of the _1B-AR was assessed after transient transfection in COS 1 cells. Like the _1D-AR, we detect a significant degree of _1B-AR internalization in cells expressing the constitutively active mutant (Fig. 6). Therefore, receptor internalization in the absence of agonist is a property common to constitutively active ₁-ARs.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Immunolocation of the wild-type 1B-AR and a constitutively active mutant (A293K, Kjelsberg et al., 1992) of the 1B-AR in transiently transfected COS 1 cells. Immunocytochemistry and laser scanning confocal microscopy were carried out as described in Materials and Methods.

To determine whether the effects of prazosin on _1D-AR immunolocalization were unique to this compound, we examined the effect of a series of antagonists on _1D -AR expression. Rat 1 fibroblasts expressing the _1D-AR were cultured for 24 h in the presence of phentolamine, BMY 7378, 5-methylurapidil, or WB 4101 (₁-AR antagonists), the ₂-AR antagonist yohimbine, or the -AR antagonist propranolol (see Fig. 7). Yohimbine and propranolol had no effect on the cellular localization of the _1D-AR. Phentolamine and BMY 7378, which have been reported to have inverse agonist properties (Scheer et al., 1997; Garcia-Sainz and Torres-Padilla, 1999), induced redistribution of the _1D-AR from internal compartments to the cell membrane. Phentolamine was as effective as prazosin in promoting _1D-AR redistribution, whereas BMY 7378, WB 4101, and 5-methylurapidil were less effective than either phentolamine or prazosin at promoting _1D-AR redistribution. Therefore, the ability to promote _1D-AR redistribution is not unique to prazosin but is also manifested by other ligands that interact with this receptor. These data also support the notion that inverse agonists, like traditional agonists, differ in their intrinsic activity.

View larger version (84K): [in this window] [in a new window]   Fig. 7.   Effect of antagonist compounds (1 µM) on the cellular localization of the 1D-AR in stably transfected fibroblasts. Drug incubation, immunocytochemistry, and laser scanning confocal microscopy were performed as described in Materials and Methods.

Taken together, these data are strong evidence that the _1D-AR is constitutively active in fibroblasts. However, the data obtained in a stably transfected cell line may not accurately represent the localization and signaling characteristics of the _1D-AR in cells that natively express these receptors such as in vascular smooth muscle cells. Figure 8 shows immunoreactivity of femoral artery smooth muscle cells immunostained with _1B- and _1D-AR antibodies. Similar to the immunoreactivity seen in fibroblasts, the _1D-AR is localized in a perinuclear fashion in vascular smooth muscle cells. This similarity in immunolocalization suggests that the _1D-AR may be constitutively active in cells that natively express all three ₁-AR subtypes, an area of investigation that we are actively pursuing.

View larger version (110K): [in this window] [in a new window]   Fig. 8.   Immunolocalization of the 1B- and 1D-ARs in cultured femoral artery smooth muscle cells (FVSM) and fibroblasts expressing either receptor subtype. Immunocytochemistry was carried out as described in Materials and Methods.

The _1D-AR has been somewhat of an enigma. It is not as well studied as the other ₁-AR subtypes. Theroux et al. (1996) showed that this receptor was weakly coupled to second messenger pathways. Yang et al. (1997) were unable to detect a significant degree of expression of the _1D-AR in a variety of rat tissues. These findings are consistent with our observations that the receptor is predominantly expressed in intracellular compartments due to constitutive activity. As a result, the _1D-AR is not as responsive to agonist activation as the other subtypes of the ₁-AR. We noted that phenylephrine stimulated inositol phosphate accumulation but had no effect on ERK activity in _1D-AR-expressing cells. This suggests that the effect of constitutive activity on functional responses differs depending on the second messenger/kinase being studied.

Studies with either transfected HEK 293 or SK-N-MC cells failed to demonstrate any constitutive function of the _1D-AR (Theroux et al., 1996). The constitutive activity of the _1D-AR we observe may be related to the level of _1D-AR expression. In our studies, we note differences in cellular localization and signaling of the _1B- and the _1D-AR, even though these receptors are expressed at similar levels in the Rat 1 fibroblasts (5-10 pmol/mg protein), suggesting that the level of receptor expression is not sufficient to account for our results. Previous studies also using the stably transfected _1D-AR subtype in Rat 1 fibroblasts showed constitutive activation of the calcium response (Garcia-Sainz and Torres-Padilla, 1999), a function linked to the production of IP₃.

The _1D-AR also binds most agonists but not antagonists with higher affinity than the other two ₁-AR subtypes. This phenotype is a hallmark of constitutive activity based on the revised ternary complex model (Samama et al., 1993). Constitutive activity is also associated with constitutive phosphorylation and, therefore, desensitization. Current models of GPCR signaling indicate that the receptor is dephosphorylated and recycled back to the cell surface. The internalized pool of _1D-ARs may represent the equilibrium component of the recycling process due to its constitutive nature.

In summary, we have shown that there are significant differences in the cellular expression of the ₁-ARs. The _1B-AR exhibits characteristics of a typical GPCR as it is expressed on the cell surface, is subject to agonist-mediated internalization, and couples to second messenger/kinase activation. The results with the _1D-AR are not consistent with traditional models of GPCR. This receptor is expressed mainly in intracellular compartments. The unstimulated _1D-AR appears to be constitutively active and is subject to regulation by inverse agonists. In preliminary studies in vascular smooth muscle cells, we have observed that the _1D-AR is localized in a perinuclear orientation similar to that seen in fibroblasts. These data argue that the cellular localization we report in this paper is not an epiphenomenon of experiments in fibroblasts but rather is a characteristic of this receptor. We are currently investigating the possibility that the _1D-AR is constitutively active in cells natively expressing the receptor.