Resistance of the Human β1-Adrenergic Receptor to Agonist-mediated Down-regulation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human β1-adrenergic receptor (β1AR) to agonist over a 24-h period, we instead observed an increase of ∼30% in both β1AR binding activity and immune-detected receptors. In contrast, β2AR expressed in these cells exhibited a decrease of ≥50%. We determined that the basal turnover rates of the two subtypes were similar (t½ ∼ 7 h) and that agonist stimulation increased β2AR but not β1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated β2AR turnover. As β1AR mRNA levels increased in agonist-stimulated cells, β1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; β1AR with a β2AR C-tail underwent down-regulation, and β2AR with a β1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being β2AR > β1AR with β2AR C-tail > β2AR with a β1AR C-tail > β1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although β1AR internalization was increased to that of β2AR, down-regulation still did not occur. Instead, β1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and β2AR but not β1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human ␤ 1 -adrenergic receptor (␤ 1 AR) to agonist over a 24-h period, we instead observed an increase of ϳ30% in both ␤ 1 AR binding activity and immune-detected receptors. In contrast, ␤ 2 AR expressed in these cells exhibited a decrease of >50%. We determined that the basal turnover rates of the two subtypes were similar (t1 ⁄2 ϳ 7 h) and that agonist stimulation increased ␤ 2 AR but not ␤ 1 AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A 1 had no effect on basal turnover of either subtype but blocked agonist-stimulated ␤ 2 AR turnover. As ␤ 1 AR mRNA levels increased in agonist-stimulated cells, ␤ 1 AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; ␤ 1 AR with a ␤ 2 AR C-tail underwent down-regulation, and ␤ 2 AR with a ␤ 1 AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being ␤ 2 AR > ␤ 1 AR with ␤ 2 AR C-tail > ␤ 2 AR with a ␤ 1 AR C-tail > ␤ 1 AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although ␤ 1 AR internalization was increased to that of ␤ 2 AR, down-regulation still did not occur. Instead, ␤ 1 AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and ␤ 2 AR but not ␤ 1 AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.
The three ␤-adrenergic receptor (␤AR) 1 subtypes, ␤ 1 AR, ␤ 2 AR, and ␤ 3 AR, are members of the G protein-coupled recep-tor (GPCR) superfamily. Although all three subtypes respond to norepinephrine and epinephrine by activating adenylyl cyclase, they differ in their distribution, regulation, and interaction with other signaling pathways. This is evident in the heart where ␤ 1 AR is the predominant subtype followed by ␤ 2 AR and ␤ 3 AR, and each subtype appears to differ in signaling properties (1)(2)(3)(4). ␤ 2 AR and ␤ 3 AR couple to G s and G i whereas ␤ 1 AR only couples to G s . Overstimulation of ␤ 1 AR is pro-apoptotic whereas ␤ 2 AR stimulation is anti-apoptotic. During chronic heart failure, the compensating increase in sympathetic drive leads to high catecholamine levels and chronic ␤-adrenergic signaling, in particular the norepinephrine/␤ 1 AR component contributes to the progression of the disease (5). Because ␤ 1 AR is implicated in chronic heart failure, there is considerable interest in its regulation.
Persistent agonist stimulation also leads to receptor downregulation. Down-regulation is defined as a reduction in the total number of receptors and is usually determined by loss of binding sites and often attributed to receptor proteolysis. Although the down-regulation of ␤ 2 AR has been studied extensively, the mechanisms and pathways remain unresolved, which has led to the proposal of several models (6). A major question is whether receptor down-regulation occurs in the absence of internalization or is dependent on endocytosis and trafficking to lysosomes. Some studies show that internalization of ␤ 2 AR is necessary for its degradation. Following agonistmediated endocytosis via clathrin-coated pits, most of the receptors are recycled, but some enter the lysosomal pathway and are degraded (7,8). Other studies, however, indicate that internalization and down-regulation of ␤ 2 AR are independent of each other. Certain mutations of ␤ 2 AR impair one process but not the other (9 -12), and inhibition of endocytosis does not block the degradation of ␤ 2 AR in mouse L cells or A431 cells (13). Based on a kinetic analysis, a two-pathway model of ␤ 2 AR down-regulation has been proposed: a high-affinity, low-capacity, internalization-independent pathway and a low-affinity, high-capacity, internalization-dependent pathway (14). An additional mechanism observed in some cells is the cyclic AMPmediated reduction in steady-state ␤ 2 AR mRNA levels (15)(16)(17)(18). The decrease is because of destabilization of the transcripts by proteins that bind to specific sequences in the 3Ј-untranslated region. Although some of these differences may be cell-specific, the precise mechanism of ␤ 2 AR down-regulation is not fully understood.
Less is known about the down-regulation of ␤ 1 AR, but in general it is not as responsive to this type of regulation compared with ␤ 2 AR. In rat C6 glioma cells exposed to the agonist ISO, down-regulation of both subtypes occurs at similar rates (19,20), but when the cells are exposed to atypical agonists, only ␤ 2 AR levels are reduced (19). When rat H9c2 heart cells are treated with agonist, ␤ 2 AR but not ␤ 1 AR undergoes downregulation (21). Distinctions also are found in vivo. Downregulation is greater for ␤ 2 AR than ␤ 1 AR in fat cells isolated from dogs with chronically elevated plasma catecholamines (22) and in myocardial tissue from rats infused with norepinephrine (23). The opposite, however, was found in tissue from human failing hearts (24). The two subtypes have been directly compared by heterologously expressing each in the same cell line. ␤ 1 AR is more resistant to agonist-mediated downregulation than ␤ 2 AR in CHW (25)(26)(27) and HEK 293 cells (28) and in some studies, undergoes up-regulation in the latter cells (29,30).
We initiated the present studies to identify some of the mechanisms involved in the resistance to down-regulation of human ␤ 1 AR during persistent agonist stimulation. As ␤ 1 AR also is more resistant than ␤ 2 AR to agonist-mediated internalization (25,26,28,31,32), we determined whether the latter contributed to its resistance to down-regulation. In addition, we investigated whether the two subtypes differed in basal or agonist-mediated turnover and whether basal and agonist-mediated turnover involved different pathways. Finally, as the C-tails of GPCRs including the ␤ 2 AR have been identified as important determinates of sorting between recycling and degradation (33)(34)(35)(36), we examined the effects of exchanging the C-tails of the two subtypes on receptor regulation. Our results indicate that in BHK cells, ␤ 1 AR is resistant to agonist-mediated down-regulation and instead undergoes up-regulation as does its mRNA; increased ␤ 1 AR internalization does not result in down-regulation; basal turnover is similar for both subtypes and appears to be non-lysosomal whereas agonist-mediated turnover of ␤ 2 AR is lysosomal; and finally the C-tails are key determinants of down-regulation, the ␤ 1 AR C-tail conferring resistance and the ␤ 2 AR conferring C-tail susceptibility.
Plasmid Construction-The plasmids Zem228c-h␤ 1 AR and -h␤ 2 AR encoding the respective human receptors under control of the metallothionein promoter have been described (26), and the plasmids pcDNA3.1-h␤ 1 AR and -h␤ 2 AR were generated by the same procedure. The plasmid pcDNA3.1-HA-h␤ 1 AR encoding an N-terminal, HA epitope-tagged h␤ 1 AR (MYPYDVPDYAGAGϪ) was generated by PCR. The HA-tagged wild-type ␤ 2 AR and chimeric ␤AR constructs in pCMV5 (32) were kindly provided by H. Kurose (Kyushu University, Fukuoka, Japan). The latter encode the chimeric receptors (numbers refer to amino acid positions in wild-type ␤AR sequences), ␤ 1 Met 1 -Ser 380 /␤ 2 Pro 330 -Leu 413 (␤ 1 /␤ 2 ct-AR) and ␤ 2 Met 1 -Ser 329 /␤ 1 Pro 381 -Val 477 (␤ 2 /␤ 1 ct-AR) in which the entire C terminus of one subtype has been replaced with that of the other. The ␤ 1 /␤ 2 ct-AR cDNA contains the 3Ј-untranslated region of the ␤ 2 AR gene whereas the ␤ 2 /␤ 1 ct-AR cDNA lacks any 3Ј-untranslated region of the ␤ 1 AR gene. The HA-␤ 2 AR and HA-␤ 1 / ␤ 2 ct-AR cDNAs were excised and inserted into the EcoRI/XbaI site of pcDNA3.1(ϩ), and the HA-␤ 2 /␤ 1 ct-AR cDNA was into the EcoRI/ HindIII site of pcDNA3.1(Ϫ), respectively. The plasmid pcDNA3-arrestin-2 was obtained from J. Benovic (Thomas Jefferson University, Philadelphia, PA). We generated Zem229-Arr2 by excising the coding region of the bovine arrestin-2 cDNA with NotI and ApaI and inserting it into the corresponding sites created in the BamHI cloning site of Zem229, which has a dihydrofolate reductase selectable marker.
Cell Culture and Transfection-The BHK cell line (clone tk-ts13) was obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Clonal lines stably expressing ␤ 1 AR or ␤ 2 AR or co-expressing both arrestin-2 and either subtype were obtained by transfecting the cells with one of the Zem228c-h␤AR plasmids or co-transfecting with both Zem228c-h␤AR and Zem229-Arr2, selecting for resistance to G418 or both G418 and methotrexate, and by limiting dilution of the resistant cultures (26). Arrestin-2 expression was confirmed by Western blotting. To obtain cells expressing wild-type or chimeric HA-␤ARs, cells were transfected with one of the pcDNA plasmids using LipofectAMINE Plus according to the manufacturer's instructions and either used after 24 h or selected with G418. Although the resistant cultures were not cloned, ␤AR expression levels remained fairly constant during the experimental period. All stably transfected cells were maintained in the culture medium containing 0.2 mg/ml G418 and/or 2 M methotrexate.
␤AR Binding Assays-Control and treated cells were assayed for total and surface receptors as described previously (27,37). Briefly, cells grown in 6-well plates or 35-mm dishes were incubated with 10 M ISO at 37°C for increasing times up to 24 h, rinsed with ice-cold Ca 2ϩ -and Mg 2ϩ -free DPBS, and lysed in 1 mM Tris-HCl, 2 mM EDTA, 1 mM EGTA, pH 7.4, and protease inhibitors at 4°C. Portions of the lysates were assayed for protein and for total ␤AR activity with 250 pM 125 ICYP for 1 h at 37°C. Portions also were dissolved in SDS sample buffer and used for Western blotting as described below. To measure surface ␤AR, cells grown in 24-well plates were exposed at 37°C to 1 M ISO for increasing times up to 30 min or to 10 M ISO for up to 24 h. The plates were placed on a bed of ice, rapidly washed twice with ice-cold DPBS, and exposed to 5 nM [ 3 H]CGP-12177 at 4°C for 1 h. Finally, the cells were washed as above and assayed for 3 H and protein. For both assays, nonspecific binding was determined in the presence of 10 M propranolol.
␤AR mRNA Assay-Control and agonist-treated cells were washed twice, scraped into ice-cold DPBS, and centrifuged. The cell pellet was suspended in TRIzol reagent (5-10 ϫ 10 6 cells/ml), mixed well with 1 ⁄5 volume of chloroform, and centrifuged for 15 min at 12,000 ϫ g at 4°C. The clear upper layer containing RNA was removed, adjusted to 35% ethanol, and further purified using an RNeasy kit (Qiagen). First strand cDNA in 20 l was generated from 500 ng of total RNA with Random Decamers using a RETROscript kit and protocol (Ambion). Portions (0.2-0.4 l) of the cDNA were used to quantify the relative amounts of ␤AR mRNA by generating [␣-32 P]-labeled PCR products using a QuantumRNA 18 S Internal Standards kit and protocol (Ambion). For ␤ 1 AR and ␤ 1 /␤ 2 ct-AR cDNA amplification, the sense and antisense primers were 5Ј-CAAGTGCTGCGACTTCGTCACC-3Ј and 5Ј-GCCGAGGAAACGGCGCTC-3Ј, which generated a 163-bp PCR product. For ␤ 2 AR cDNA, the primers were 5Ј-ACAAAGCCCTCAAGACGT-TAGG-3Ј and 5Ј-CCTTCAAAGAAGACCTGCG-3Ј, which generated a 240-bp product. For ␤ 2 /␤ 1 ct-AR cDNA, the ␤ 2 AR sense primer was paired with a ␤ 1 AR antisense primer, 5Ј-AGCAGAGCAGTCCCTG-GAAG-3Ј, which generated a 219-bp product. The 18 S PCR primer pair in the kit was used in a ratio of 18 S PCR primer pairs:18 S PCR Competimer of 1:9 to generate an internal control PCR 324-bp product. Amplification was done in a PerkinElmer Life Sciences GeneAmp PCR system 9600 with denaturation at 94°C for 30 s (3 min for initial denaturation), annealing at 55°C for 30 s, elongation at 74°C for 45 s, 26 cycles, and final extension at 74°C for 5 min. Pretests showed a liner correlation between the amount of ␤AR mRNA and its final PCR products under these conditions. The final PCR fragments were resolved on a 6% Tris/borate/EDTA gel, which was dried and exposed to a Bio-Rad storage phosphor screen-BI. The screen was scanned, and the labeled bands were detected and quantified using a Bio-Rad GS525 molecular imager system and multi-analysis/PC software. The abundance of each ␤AR mRNA was calculated relative to that of the control 18 S rRNA band from the same reaction.
␤AR Turnover Assay-Basal and agonist-mediated ␤AR turnover were determined by following the loss of surface-biotinylated receptors. Briefly, cells grown in 35-mm dishes were rinsed with ice-cold Buffer B (40 mM NaHCO 3 , 100 mM NaCl, pH 8.6), incubated with 1 mM sulfo-NHS-LC-biotin in Buffer B at 4°C for 30 min, and rinsed with ice-cold DPBS, 20 mM glutamine (27). The cells then were warmed to 37°C in fresh medium in the absence or presence of 10 M ISO and collected at different times as described above for the total ␤AR binding assay. Portions of the lysates were extracted in radioimmune precipitation assay buffer for 1 h at 4°C and centrifuged at 17,000 ϫ g for 20 min. ␤ARs were immunoprecipitated from the soluble extracts using protein A-Sepharose beads coated with anti-␤ 1 AR or -␤ 2 AR antibodies, eluted from the beads by heating in SDS sample buffer at 60°C for 15 min and subjected to SDS-PAGE and blotting with HRP-conjugated streptavidin as described below.
Whole Cell Phosphorylation of ␤ARs-BHK cells grown in 6-well plates were transfected with one of the pcDNA3 plasmids encoding wild-type or chimeric HA-␤ARs (1 g DNA/well; 3 wells per plasmid). After 24 h, one well of each set of three was assayed for total binding activity. The other two wells were washed with serum-and phosphatefree Dulbecco's modified Eagle's medium/Hepes, incubated in the same medium containing 200 Ci/ml of [ 32 P]orthophosphate for 2.5 h and then for 10 min Ϯ 1 M ISO. The cells were washed extensively with ice-cold Ca 2ϩ -and Mg 2ϩ -free DPBS and lysed in 0.7 ml of radioimmune precipitation assay buffer with protease and phosphatase inhibitors. The lysates were extracted and centrifuged as described above. The soluble extracts were pre-cleared by gently rotating with 30 l of protein A-Sepharose for 1 h. After removing the beads, the soluble samples were added to tubes containing 5 l of anti-HA-Sepharose and 25 l of protein A-Sepharose (added as carrier to have a visible pellet), and the tubes were rotated at 4°C overnight. The beads were washed five times with 300 l of radioimmune precipitation assay buffer, and bound proteins were eluted in 50 -60 l of sample buffer (based on lysate binding, adjusted to make ␤ARs/l the same in samples from control and stimulated cells) as described above. Portions of 40 l were separated by SDS-PAGE, and the gel was dried and exposed to a phosphor screen for 20 to 30 h. The screen was scanned and analyzed as described above. We confirmed that equal amounts of receptors were immunoprecipitated from control and stimulated cells by transferring proteins from the gel to a blot that was probed with rat anti-HA and HRPconjugated mouse anti-rat.
Western Blotting-Immunoblotting of ␤AR proteins was done essentially as described previously (27). Briefly, portions of cell lysates or immunoprecipitates dissolved in SDS sample buffer were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The latter were blocked with casein in Tris-buffered saline, blotted with anti-␤ 1 AR (1:10000), anti-␤ 2 AR (1:4000), or anti-arrestin (1:4000) antibodies, washed, and blotted with HRP-conjugated goat anti-rabbit or -mouse IgG or streptavidin (1:10000) as appropriate. Then the blots were visualized by enhanced chemiluminescence, and the images were captured on Eastman Kodak Co. Bio-Max MR or Lite 2 film and quantified using NIH Imaging software. ␤ 1 AR produces a linear response between 2.5 and 25 fmol (27). Similar results were observed with ␤ 2 AR (data not shown).
Data Analysis-Unless otherwise indicated, each experiment was repeated at least three times, and each data point within an experiment was done in triplicate. Data were fitted to curves by nonlinear regression analysis and analyzed for statistical significance by one-or twoway analysis of variance or two-tailed t test using Prism 3 (GraphPad Software).

RESULTS
Opposite Changes in Levels of ␤ 1 AR and ␤ 2 AR by Persistent Agonist Stimulation-To investigate the effects of persistent agonist stimulation on ␤ 1 AR and ␤ 2 AR, we exposed clonal lines of BHK cells stably expressing either subtype at ϳ 1 pmol/mg protein (BHK-h␤ 1 and -h␤ 2 cells) to 10 M ISO for up to 24 h. The cells then were lysed and assayed for total ␤AR binding activity using the hydrophobic radioligand 125 ICYP. The number of ␤ 2 AR binding sites decreased to 52% of control with a t1 ⁄2 of 2.6 h whereas that of ␤ 1 AR increased to 131% of control (Fig.  1A). Although there was an initial, modest reduction of ␤ 1 AR binding, overall ␤ 1 AR underwent up-regulation in contrast to the rapid down-regulation of ␤ 2 AR. To determine whether the changes in binding activity reflected corresponding changes in ␤AR proteins, we subjected the same samples to Western blotting with antibodies to ␤AR C-terminal sequences. With increasing time of agonist treatment, the levels of immune-detected ␤ 1 AR increased, and those of ␤ 2 AR decreased (Fig. 1, B and C), establishing that the respective up-and down-regulation of binding activities occurred at the receptor protein level. ␤ 2 AR binding activity initially decreased more rapid than the immune-detected receptor proteins (see Fig. 1 and Table I). A similar delay was observed in the down-regulation of HA-␤ 2 AR expressed in mouse L-cells (13). In general, our results are consistent with previous studies that ␤ 1 AR is more resistant to agonist-mediated down-regulation than ␤ 2 AR.
The changes in ␤ 1 AR and ␤ 2 AR binding activity after 24 h was dependent on agonist concentration with half-maximal effects occurring at ϳ1-3 nM ISO ( Fig. 2A). When we compared short term and prolonged agonist stimulation on ␤AR regulation, we found that a 15-min exposure had only a slight immediate effect on total ␤AR binding activity but led to changes 24 h later, especially for ␤ 1 AR (Fig. 2B). ␤ 1 AR levels after 15 min were 103 Ϯ 2% of control and increased to 126 Ϯ 3% after 24 h. ␤ 2 AR levels decreased to 92.8 Ϯ 9 and 79.6 Ϯ 1% of control after 15 min and 24 h, respectively. Thus, the up-regulation of ␤ 1 AR after 15 min of agonist stimulation followed by 24 h without agonist treatment was substantial when compared with the up-regulation obtained with constant agonist stimulation for 24 h. In contrast, the down-regulation of ␤ 2 AR over the same time period was small in comparison to the maximum caused by prolonged stimulation. As cAMP has been reported FIG. 1. Persistent agonist-mediated regulation of ␤AR subtypes expressed in BHK cells. BHK cells stably expressing ϳ1 pmol of ␤ 1 AR (q) or ␤ 2 AR (f) per mg of protein were exposed to 10 M ISO for the indicated times, washed, and lysed. The whole cell lysates were then assayed for the total number of ␤AR binding sites with 125 ICYP (A) and for receptor protein by Western blotting with anti-␤AR antibodies (B) and quantification of the immunoblots by densitometric analysis (C) as described under "Experimental Procedures." Data are the mean Ϯ S.E. of three-six independent experiments. to mediate down-regulation in some cells heterologously expressing ␤ARs (15, 27), we exposed the cells to a permeable cAMP derivative, (chlorophenylthio)-cAMP, for 24 h. The effect was an up-regulation of ␤ 1 AR (129 Ϯ 1% of control; p Ͻ 0.001, n ϭ 3) similar to that induced by agonist. There was no ␤ 2 AR down-regulation but instead a small, not quite significant upregulation (116 Ϯ 7.5% of control; p ϭ 0.07). Thus up-regulation of ␤ 1 AR levels may be mediated by agonist-generated cAMP whereas down-regulation of ␤ 2 AR may be because of agonist binding.
Agonist-mediated Up-regulation of ␤AR mRNA-To find out whether changes in ␤AR mRNA levels contributed to ␤AR regulation by agonist, BHK-h␤ 1 and -h␤ 2 cells were treated with 10 M ISO over a 24-h period, and ␤ 1 AR and ␤ 2 AR mRNA levels were quantified by reverse transcriptase-PCR. As shown in Fig. 3, ␤ 1 AR mRNA levels increased 3-fold within 1 h of ISO stimulation and then decreased but remained above control levels to the end of the 24-h treatment. ␤ 2 AR mRNA levels also increased but to a lesser degree. Similar results were obtained with a second clonal line expressing ␤ 1 AR or ␤ 2 AR and also co-expressing arrestin-2 (see below).
Prolonged ␤ 1 AR Regulation Unaffected by Increased Internalization-␤ 1 AR is more resistant to agonist-mediated internalization compared with ␤ 2 AR in CHW (25,26,31) and HEK 293 cells (28, 32) 2 As endocytosis of some GPCRs is necessary for their down-regulation (6), including ␤ 1 AR (27) and ␤ 2 AR (7, 8), we explored the possibility that subtype differences in internalization might account for differences in down-regulation in BHK cells. Exposing BHK-h␤ 1 and -h␤ 2 cells to ISO over a 30-min period resulted in time-dependent internalization of both subtypes with similar first order kinetics (t1 ⁄2 values of 3.2 and 2.9 min; see Table I). The maximal internalization of ␤ 1 AR, however, was only 16% in contrast to 38% of ␤ 2 AR (Fig. 4A). When the cells were continuously exposed to 10 M ISO for longer times, the proportion of ␤ 1 AR remaining on the cell surface actually began to increase, and the difference with ␤ 2 AR reached 6-fold at 24 h (Fig. 4B).
To investigate whether increasing ␤ 1 AR internalization would overcome its resistance to down-regulation, we co-expressed arrestin-2 with each ␤-subtype in BHK cells, which contain low levels of arrestin-3 and no arrestin-2 (38). Arrestins are necessary for endocytosis of GPCRs via clathrin-coated pits (39,40). We obtained clones that co-expressed ϳ3 pmol of arrestin-2 and ϳ1.3 pmol of either ␤ 1 AR or ␤ 2 AR per mg of protein (Arr2-BHK-h␤ 1 and -h␤ 2 cells) (Fig. 5A). The internalization of ␤ 1 AR was substantially increased to 52% and that of ␤ 2 AR was increased to 64% in these cells (Fig. 5B) with a reduction in the t1 ⁄2 values to 1.5 min (Table I). When we exposed Arr2-BHK-h␤ 1 and -h␤ 2 cells to persistent agonist treatment, ␤ 1 AR still underwent up-regulation even though 70% or more of the total ␤ 1 AR was internalized at any given time (Fig. 5C). The extent of ␤ 2 AR down-regulation was similar to that in cells not co-transfected with arrestin-2, but rate was significantly slowed (see Table I). In addition, because total ␤ 1 AR levels increased and ␤ 2 AR levels decreased, the relative amounts of internalized ␤ 1 AR were more than those of ␤ 2 AR, reaching almost 4-fold by 24 h. Thus despite the extensive intracellular accumulation of ␤ 1 AR, it remained resistant to agonist-mediated down-regulation.
Basal and Agonist-mediated Turnover of ␤ 1 AR and ␤ 2 AR-In the absence of agonist stimulation, receptors are TABLE I Kinetic parameters of acute and persistent agonist-mediated regulation of ␤ 1 AR and ␤ 2 AR expressed in BHK cells Clonal BHK and Arr2-BHK cells stably expressing ␤ 1 AR and ␤ 2 AR were exposed to ISO for up to 30 min and assayed for internalization of cell surface binding sites with [ 3 H]CGP-12177 or for up to 24 h, lysed, and assayed for total cellular binding sites with 125 ICYP or analyzed by Western blotting for amounts of immune-detected receptors as described under "Experimental Procedures." Cells also were biotinylated, incubated in the absence and presence of ISO for up to 24 h, and analyzed for amounts of biotinylated receptors remaining by immunoprecipitation and streptavidin overlay. Data were best fit by nonlinear regression analysis to one-phase exponential decay curves by Prism 3, and rates and extents of change are expressed as t1 ⁄2 and maximal % change. When the data could not be curve-fitted, the value at 24 h is given. All values are the mean Ϯ S.E. of three to six independent experiments.

FIG. 2. Effects of agonist concentration and treatment time on ␤AR levels in BHK cells.
A, BHK-h␤ 1 (q) and -h␤ 2 (f) cells were exposed to increasing concentrations of ISO for 24 h and assayed for total receptor binding sites with 125  maintained at a steady state level, the rate of receptor synthesis balanced by receptor turnover (14,41). We used a biotinylation procedure to identify any differences in either basal or agonist-mediated turnover of ␤ 1 AR and ␤ 2 AR. The cells were labeled with a non-permeable, non-cleavable biotin derivative, incubated up to 24 h in the presence or absence of agonist, and

FIG. 5. Effects of overexpression of arrestin-2 on regulation of ␤ARs by agonist in BHK cells. A, Western blot of arrestin expression
in BHK cells and the Arr2-BHK-h␤ 1 and -h␤ 2 cells. Increasing amounts of purified arrestin-2 or 20 g of cell protein were subjected to Western blotting using a monoclonal antibody against arrestins. B, Arr2-BHK-h␤ 1 (E) and -h␤ 2 (Ⅺ) cells were exposed to 1 M ISO for the indicated times, washed, and assayed for cell surface receptors using [ 3 H]CGP-12177 as described under "Experimental Procedures." Results represent the mean Ϯ S.E. of five-six independent experiments. C, Arr2-BHK-h␤ 1 (E, q) and -h␤ 2 (Ⅺ, f) cells were exposed to 10 M ISO for the indicated times, washed, and assayed for cell surface receptors (open symbols) or lysed and assayed for total receptors (solid symbols). Initially, 90% of the total receptors were on the cell surface. Data are expressed as % of initial total receptors and are the mean Ϯ S.D. from one of three similar experiments.

FIG. 3. Agonist-mediated regulation of ␤AR mRNA levels in BHK cells.
Cells stably expressing the indicated ␤ARs were exposed to 10 M ISO for up to 24 h. Total RNA was isolated from the cells, and ␤AR mRNA levels quantified using reverse transcriptase-PCR as described under "Experimental Procedures." The 32 P-labeled PCR-generated products were separated by gel electrophoresis and detected and quantified by a phosphorimaging system and imaging software. A, depicts a representative image of the 32 P-labeled PCR-generated products from ␤AR mRNAs in control and agonist-treated cells. solubilized. ␤ARs in the soluble extracts were immunoprecipitated with anti-␤AR antibodies, and biotinylated ␤ARs were detected by blotting with HRP-conjugated streptavidin. As shown in Fig. 6 and Table I, basal turnover of both ␤ 1 AR and ␤ 2 AR occurred with similar kinetics (t1 ⁄2 ϭ 6.5 Ϯ 0.7 and 6.9 Ϯ 1.8 h, respectively), and over 90% of the biotinylated ␤AR disappeared after 24 h. Whereas agonist stimulation increased ␤ 2 AR turnover significantly (t1 ⁄2 ϭ 3.0 Ϯ 0.2 h; p Ͻ 0.01), it had only a slight impact on ␤ 1 AR turnover (t1 ⁄2 of 5.0 Ϯ 0.5 h; p Ͼ 0.05). We also compared basal and agonist-mediated turnover in the Arr2-BHK-h␤ 1 and -h␤ 2 cells and found a similar pattern for the two subtypes (data not shown). Together these results are indicative that ␤ 1 AR is resistant to agonist-mediated degradation in BHK cells, even under conditions in which it undergoes extensive internalization.
To validate the biotinylation method, we used another approach to measure the basal turnover rate of each subtype. For ␤ 2 AR, we applied the methods and equations described by Williams et al. (14) to the down-regulation data and obtained a t1 ⁄2 of 7.2 h for basal turnover. As ␤ 1 AR did not down-regulate, we took advantage of the receptor being under control of the metallothionein promoter, exposed BHK-h␤ 1 cells to zinc sulfate to induce more receptors, and followed their turnover as described by Dunigan et al. (27). A t1 ⁄2 of 7.2 h for the turnover of zinc-induced ␤ 1 AR in BHK cells was obtained. These values are very close to the basal turnover rates obtained using biotinylation assay, indicating that biotinylation did not alter the turnover of either subtype.
Effects of Blocking Trafficking to Lysosomes on Basal and Agonist-mediated Turnover of ␤-Subtypes-Little is known about basal turnover of GPCRs, and although it is general accepted that agonist-mediated degradation occurs in lysosomes (6), non-lysosomal proteolysis of ␤ 2 AR has been described (13). To investigate the two possibilities, we used bafilomycin A 1 , which has been shown to block trafficking of ␤ 2 AR from endosomes to lysosomes (8). Biotin-labeled BHK-h␤ 1 and -h␤ 2 cells were incubated in the presence and absence of 1 M bafilomycin A 1 and/or 10 M ISO and were analyzed for the disappearance of biotinylated ␤AR after 4 h. Bafilomycin A 1 slightly increased the basal turnover of ␤ 1 AR and ␤ 2 AR but effectively blocked the agonist-mediated turnover of ␤ 2 AR (Fig.  7). Based on these results, it appears that the basal degradation of both subtypes may be non-lysosomal, whereas agonistmediated degradation of ␤ 2 AR is lysosomal.
Role of the C-tail of ␤AR in Agonist-mediated Regulation-As the C-tails of a number of GPCRs have been identified as structural determinants in targeting the receptors to lysosomes (33)(34)(35), we explored the effect of replacing the C-tail of one subtype with that of the other on agonist-mediated regulation. As the first step in the regulatory process is receptor phosphorylation, we 32 P-labeled BHK cells transiently expressing ␤ 1 AR, ␤ 2 AR, or the chimeras, ␤ 1 /␤ 2 ct-AR and ␤ 2 /␤ 1 ct-AR, stimulated the cells with agonist, and purified and analyzed the receptors for 32 P incorporation (Fig. 8). Whereas agonist stimulation increased the phosphorylation of ␤ 2 AR and ␤ 1 /␤ 2 ct-AR by 271 and 200% of control, respectively, it had no significant effect on the phosphorylation of ␤ 1 AR and ␤ 2 /␤ 1 ct-AR. We next compared the agonist-mediated internalization of the wild-type and chimeric receptors (Fig. 9A). ␤ 1 /␤ 2 ct-AR underwent more internalization than wild-type ␤ 1 AR, and ␤ 2 /␤ 1 ct-AR underwent less than wild-type ␤ 2 AR, which is in agreement with previous findings in HEK 293 cells (32). Having confirmed that the chimeras behaved similarly in BHK cells, we investigated the effects of persistent agonist stimulation on their regulation. As shown in Fig. 9B, ␤ 1 /␤ 2 ct-AR underwent down-regulated and thus was more similar to ␤ 2 AR, the source of its C-tail, than to ␤ 1 AR, which represented most of its structure. In an analogous manner, ␤ 2 /␤ 1 ct-AR exhibited up-regulation, the same as ␤ 1 AR from which it derived its C-tail. Western blotting confirmed the down-and up-regulation of the chimeras occurred at the protein level (Fig. 9C). Although the magnitude and rate of up-or down-regulation of each chimera were respectively less and slower than the corresponding wild-type ␤AR source of its C-tail, the results clearly demonstrated that the C-tails are important determinants not only of rapid agonist-mediated phosphorylation and internalization but also of persistent agonist-mediated up-or down-regulation of ␤ 1 AR and ␤ 2 AR.
It was noted that although the ␤ 1 /␤ 2 ct-AR expression vector contains the 3Ј-untranslated region of the ␤ 2 AR gene, the ␤ 2 / ␤ 1 ct-AR expression vector lacks the 3Ј-untranslated region of the ␤ 1 AR gene. Thus, the mRNA of the ␤ 2/1 ct-AR chimera does not have a 3Ј-untranslated region, which often contains specific sequences recognized by mRNA-binding proteins that modu- late mRNA stability. Therefore, we determined the mRNA levels of the chimeras. A 24-h exposure to 10 M ISO did not change the levels of ␤ 1 /␤ 2 ct-AR mRNA (98.5 Ϯ 1.2% of control) and slightly but significantly increased the levels of ␤ 2 /␤ 1 ct-AR mRNA (116 Ϯ 1.1% of control) (Fig. 3). Apparently, the upregulation of ␤AR mRNAs in BHK cells is not dependent on the 3Ј-untranslated region.
To further confirm the role of the C-tail on ␤AR regulation, we determined the turnover of the chimeric receptors. We labeled BHK cells stably expressing wild-type and chimeric ␤ARs with biotin and measured the loss of biotinylated ␤AR over a 4-h period. The basal turnover was similar for all the receptors except that ␤ 2 /␤ 1 ct-AR turned over more slowly (Fig.  9D). Agonist stimulation had no effect on ␤ 1 AR turnover but increased the turnover of the other receptors in the order of ␤ 2 /␤ 1 ct-AR Ͻ ␤ 1 /␤ 2 ct-AR Ͻ ␤ 2 AR. The same pattern emerged when we directly compared the percent of biotinylated receptors remaining after 4 h of agonist treatment. Based on these results, it appears that agonist-mediated ␤AR turnover is enhanced by the ␤ 2 AR C-tail and reduced by ␤ 1 AR C-tail. DISCUSSION In the present study, we have shown that in response to prolonged stimulation by agonist, ␤ 1 AR underwent up-regulation in contrast to the rapid down-regulation of ␤ 2 AR in BHK cells. The changes in receptor binding activity were accompanied by corresponding changes in immune-detected receptor proteins. Thus, up-and down-regulation occurred not by activating or inactivating pre-existing receptors but by varying the rates of receptor synthesis or turnover. To resolve how the two processes contribute to this subtype difference, we determined both the levels of ␤AR mRNA and the kinetics of ␤AR degra-dation. Our results showed that in agonist-treated cells, the mRNA levels of both subtypes were increased, being greater for ␤ 1 AR. Although agonist-induced decreases in levels of both human ␤ 1 AR and ␤ 2 AR mRNA have been observed in CHW cells (15,27), up-regulation of the mRNA levels of other GPCRs has been reported, an example being the dopamine D 2L receptor stably expressed in Chinese hamster ovary cells (42). The basal turnover rate as measured by the disappearance of biotin-labeled ␤AR was essentially the same for both subtypes. Although the degradation of ␤ 2 AR was significantly accelerated by agonist stimulation, ␤ 1 AR turnover was not. In cells treated with a permeable cAMP derivative, up-regulation of ␤ 1 AR was similar to that with agonist treatment whereas down-regulation of ␤ 2 AR was not observed. A 15-min pulse with agonist caused a substantial up-regulation of ␤ 1 AR in cells washed and incubated without agonist for 24 h. For ␤ 2 AR, the corresponding down-regulation was much less. This suggested that the brief stimulation of ␤ 1 AR produced a signal or activated a pathway in the cells that did not require the further presence of agonist. Based on these results, we propose that the up-regulation of ␤ 1 AR by prolonged agonist stimulation is because of a cAMP-mediated increase in receptor mRNA levels and the resistance to agonist-mediated degradation, and the down-regulation of ␤ 2 AR is because of the agonist-mediated increased rate of turnover.
We should note that the rates of basal turnover of both ␤-subtypes and of agonist-mediated turnover of ␤ 2 AR are relatively rapid. Many previous studies indicated that in the absence of agonist, ␤ARs turn over very slowly with t1 ⁄2 values of 30 -200 h (41). However, the t1 ⁄2 values for the basal turnover of endogenous ␤ 2 AR and ␤ 1 AR in rat C6 glioma cells are 6.4 and 9.4 h, respectively (43), and 11 h for endogenous ␤ 2 AR in mouse S49 lymphoma cells (44). Regarding the agonist-mediated turnover of ␤ 2 AR in BHK cells, the down-regulation of binding activity occurred with a t1 ⁄2 of 2.3 h, which is consistent with other reports on ␤ 2 AR. Examples are as follows: L cells, ϳ5 h; A431 cells, ϳ1 h (13); BEAS-2B cells, 3.3 h (14); and CHW cells, 2.6 -7.3 h (15,26,27). When making such comparisons, one must keep in mind that whereas basal turnover is independent of receptor density, agonist-mediated turnover is sensitive to receptor density (27).
We also established that bafilomycin A 1 , which disrupts trafficking of ␤ 2 AR from endosomes to lysosomes (8), blocked the agonist-mediated degradation of ␤ 2 AR but not the basal turnover of either subtype. Together with the similar basal turnover rates, it is reasonable to assume that both subtypes are turned over by the same process. Both human ␤ 1 AR and ␤ 2 AR have been located in microdomains of the plasma membrane known as caveolae (45), and similar results were obtained with BHK-h␤ 1 and -h␤ 2 cells. 3 Basal turnover of ␤ARs may involve a caveolae-associated process occurring either within the caveolae or after caveolae-mediated endocytosis (46). Although the mechanism(s) and pathway by which ␤ARs undergo basal turnover are not known, our results suggest that agonist-mediated turnover is a separate process and not just an increase in the rate of the basal process.
More than one mechanism has been proposed for the downregulation of ␤ 2 AR (6). Upon agonist stimulation, ␤ 2 AR is internalized through clathrin-coated pits and undergoes endosomal trafficking and sorting either to the recycling pathway or to the late endosome/lysosome pathway (7,8). Internalization is necessary for lysosomal degradation in this model. Other studies using mutagenesis or inhibitors of endocytosis showed that blocking of either internalization (10,11,13) or downregulation (9, 12, 47) of ␤ 2 AR does not prevent the other process from occurring. In this model distinct pathways are used for internalization and down-regulation of ␤ 2 AR, and degradation may be independent of internalization, as well as non-lysosomal. We found that in BHK cells, overexpression of arrestin-2 increased the extent of internalization but not down-regulation of ␤ 2 AR. This does not eliminate the possibility that ␤ 2 AR internalization is necessary for its down-regulation. In fact, the inhibition of agonist-stimulated turnover of ␤ 2 AR by bafilomycin A 1 is consistent with this possibility. Although ␤ 1 AR undergoes less agonist-mediated internalization than ␤ 2 AR, increasing ␤ 1 AR internalization by overexpressing arrestin-2 did not result in its down-regulation. ␤ 1 AR continued to undergo up-regulation in agonist-treated cells even though most of the receptors remained internalized. Thus, differences in subtype internalization did not contribute to differences in their down-regulation.
There is increasing evidence the C-tails of GPCRs are important determinants in regulating their endosomal trafficking (33)(34)(35)(36). For the C-tail of ␤ 2 AR, tyrosine 350 and 354 are required for receptor down-regulation (9), and the terminal four amino acid residues (DSLL) represent a PDZ domain binding motif that is involved in the sorting of ␤ 2 AR between recycling endosomes and lysosomes (34,36). ␤ 1 AR has no tyrosine residues in its C-tail but does terminate in a PDZ binding motif (ESKV) that is recognized by PSD-95 and MAGI-2, two postsynaptic scaffolding proteins that respectively inhibit and enhance receptor internalization (48,49). Mutation of the PDZ motif of mouse ␤ 1 AR enhances agonist-mediated endocytosis in mouse cardiac myocytes (50). When we used chimeric receptors in which the C-tails had been exchanged, we found that upon persistent agonist stimulation, ␤ 2 /␤ 1 ct-AR underwent up-regulation, and ␤ 1 /␤ 2 ct-AR underwent down-regulation, the order of down-regulation being ␤ 2 AR Ͼ ␤ 1 /␤ 2 ct-AR Ͼ Ͼ ␤ 2 /␤ 1 ct-AR Ͼ ␤ 1 AR. The same order was observed for agonist-mediated turnover of biotinylated receptors, as well as internalization of cell surface receptors. The C-tails also contribute to the differences in agonist-mediated desensitization of the two subtypes (51) and thus are involved in all the major mechanisms of ␤AR regulation. Furthermore, we demonstrated that agonist-stimulated phosphorylation of the receptors was determined by the C-tails as an increase occurred in ␤ 2 AR and ␤ 1 /␤ 2 ct-AR but not in ␤ 1 AR and ␤ 2 /␤ 1 ct-AR. The agonist-stimulated increase in phosphorylation of ␤ 1 AR also is much less than that of ␤ 2 AR in HEK 293 cells (52). These differences in subtype phosphorylation may account for the differences in other downstream regulatory events such as binding of arrestins, arrestinmediated desensitization and internalization, and possibly down-regulation.
As up-or down-regulation of the chimeras is not as effective as that of the wild-type receptors, other receptor domains may contribute to their regulation. One candidate is the proline-rich region in the third intracellular loop of ␤ 1 AR that is recognized by Src homology 3 domain-containing proteins of the endophilin family (53). Overexpression of endophilin (53) or deletion of the region (31) enhances ␤ 1 AR internalization. Another candidate is the N-terminal region that contains an allelic polymorphism at codon 49. When expressed in HEK 293 cells, the less frequent Gly-49 variant of the human ␤ 1 AR undergoes some agonist-mediated down-regulation whereas the more abundant Ser-49 variant is resistant and even exhibits up-regulation (29,30).
The cell type-specific effects on ␤ 1 AR regulation are very striking. In both BHK and HEK 293 cells, ␤ 1 AR is totally resistant to agonist-mediated down-regulation. In contrast, we have shown previously that ␤ 1 AR endogenously expressed in SK-N-MC cells (54) or stably expressed in CHW cells (27) undergoes agonist-mediated down-regulation. This apparent anomaly is most likely explained by the existence of sorting FIG. 9. Role of the ␤AR C-tail in the agonist-mediate regulation of receptors in BHK cells. A, stably transfected uncloned BHK cells expressing wild-type or chimeric ␤ARs were untreated or exposed to 1 M ISO for 30 min, washed, and assayed for cell surface binding using [ 3 H]CGP-12177. B, cells were treated with 10 M ISO for the indicated times, lysed, and assayed for total ␤AR using 125 ICYP. C, shown is a representative immunoblot of chimeric ␤ARs from control and agonist-treated cells. ␤ 1 /␤ 2 ct-AR and ␤ 2 /␤ 1 ct-AR were detected with antibodies against ␤ 2 AR and ␤ 1 AR C-tails, respectively. D, cells were biotinylated, incubated for 0 (control) or 4 h in the presence (solid bars) or absence (open bars) of 10 M ISO, and then analyzed for biotinylated ␤ARs as described in the legend to Fig. 6. Data represent the mean Ϯ S.E. of three-six independent experiments. *, p Ͻ 0.05; ***, p Ͻ 0.001 (two-way analysis of variance).
proteins that recognize specific motifs on receptors and control their endosomal trafficking. The PDZ binding motif in the C-tail of ␤ 2 AR interacts with PDZ domain-containing proteins of the Na ϩ /H ϩ -exchanger regulatory factor family (34). When the motif is mutated or phosphorylated by G protein-coupled receptor kinase 5, the interaction is disrupted, and receptor trafficking is shifted from recycling to degradation. More recently, a cytosolic protein named GASP (GPCR-associated sorting protein) has been identified that binds to the C-tails of a subset of GPCRs including ␤ 2 AR and facilitates receptor trafficking to lysosomes (55). Sorting nexin 1, a membrane-associated protein, also mediates GPCR sorting to lysosomes (56). Most likely, sorting proteins that recognize the C-tail of ␤ 1 AR exist, and their levels of expression vary among different cell types. Such variations have been observed for G protein-coupled receptor kinases and arrestins (38,57). In the absence of a ␤ 1 AR-selective sorting protein, the ␤ 1 -subtype may only be recycled. A further consideration is that differences in posttranslational receptor modifications such as phosphorylation (present study and Ref. 52) and ubiquitination (58) may contribute to cell type, as well as subtype, dissimilarities in receptor degradation.
Finally, the combination of limited internalization and upregulation of ␤ 1 AR in contrast to more extensive internalization, and down-regulation of ␤ 2 AR results in a large disparity between the subtypes in the number of cell surface receptors present after agonist stimulation for 24 h. Whereas ␤ 2 AR levels were less than 20% of control, ␤ 1 AR levels were more than control. As ␤ 1 AR has been found to undergo less agonist-mediated desensitization than ␤ 2 AR (26, 51), ␤ 1 AR may remain responsive to agonist stimulation for prolonged periods. This capacity may be useful in certain cell types but not in others such as cardiomyocytes where the apoptotic effects of ␤ 1 AR could be detrimental. Our identification of the receptor C-tail as an important structural element in determining subtype down-regulation may provide a basis for future studies and therapeutic strategies.