Adenosine Deaminase and A1 Adenosine Receptors Internalize Together following Agonist-induced Receptor Desensitization*

A1 adenosine receptors (A1Rs) and adenosine deaminase (ADA; EC 3.5.4.4) interact on the cell surface of DDT1MF-2 smooth muscle cells. The interaction facilitates ligand binding and signaling via A1R, but it is not known whether it has a role in homologous desensitization of A1Rs. Here we show that chronic exposure of DDT1MF-2 cells to the A1R agonist,N 6-(R)-(phenylisopropyl)adenosine (R-PIA), caused a rapid aggregation or clustering of A1 receptor molecules on the cell membrane, which was enhanced by pretreatment with ADA. Colocalization between A1R and ADA occurred in the R-PIA-induced clusters. Interestingly, colocalization between A1R and ADA also occurred in intracellular vesicles after internalization of both protein molecules in response to R-PIA. Agonist-induced aggregation of A1Rs was mediated by phosphorylation of A1Rs, which was enhanced and accelerated in the presence of ADA. Ligand-induced second-messenger desensitization of A1Rs was also accelerated in the presence of exogenous ADA, and it correlated well with receptor phosphorylation. However, although phosphorylation of A1R returned to its basal state within minutes, desensitization continued for hours. The loss of cell-surface binding sites (sequestration) induced by the agonist was time-dependent (t½= 10 ± 1 h) and was accelerated by ADA. All of these results strongly suggest that ADA plays a key role in the regulation of A1Rs by accelerating ligand-induced desensitization and internalization and provide evidence that the two cell surface proteins internalize via the same endocytic pathway.

Adenosine is an autacoid that exerts its physiological actions through specific cell surface receptors. Four adenosine receptors (A 1 , A 2A , A 2B , and A 3 ), belonging to the family of G protein-coupled receptors, have been cloned and pharmacologically characterized (1). Acting through different adenosine receptors, adenosine is a neuromodulator in both the central and peripheral nervous systems (2,3). Via A 1 adenosine receptors (A 1 Rs), 1 adenosine reduces heart rate (4), glomerular filtration rate, and renin release in the kidney (5); it induces bronchoconstriction (6,7) and inhibits lipolysis. A 1 Rs can be coupled to different pertussis toxin-sensitive G proteins (8 -10), which mediate inhibition of adenylate cyclase (11) and regulate Ca 2ϩ and K ϩ channels and inositol phosphate metabolism (1). A 1 R displays two different affinities for agonist, which have classically been attributed to a different coupling to heterotrimeric G proteins (12). According to this two-independent site model, coupled receptor-G protein complexes display high affinity for agonists (K d ϭ 0.1-0.2 nM), and uncoupled receptors display low affinity (1-2 nM) (12,13). The recently reported cluster-arranged cooperative model predicts that the high and low affinity sites are a consequence of the negative cooperativity of agonist binding and do not seem to be related to the content of free and G protein-coupled receptors (14). According to the cluster-arranged cooperative model, the agonist-induced conversion of the high to the low affinity state may partially explain the ligand-induced desensitization of A 1 R (14,15).
Receptors belonging to the G protein-coupled receptor family are desensitized and down-regulated in response to agonist stimulation. ␤ 2 -Adrenergic receptors (␤ 2 -ARs) constitute the best characterized system within the G protein-coupled receptor family. Agonist-induced ␤ 2 -AR desensitization is caused by a conformational change of the agonist-occupied receptor that facilitates receptor phosphorylation by second messenger-activated kinases or G protein-coupled receptor kinases (16,17). Following ␤ 2 -AR phosphorylation, ␤-arrestin binds to the phosphorylated receptor and uncouples the receptor from the heterotrimeric G proteins (18,19). ␤-Arrestin not only desensitizes the receptor but also functions as a clathrin adaptor, mediating receptor sequestration, i.e. receptor internalization toward intracellular compartments (20,21). Although sequestration is not required for phosphorylation and desensitization, it appears to be necessary for dephosphorylation and resensitization of ␤ 2 -ARs (22,23).
Like other G protein-coupled receptor members, A 1 R expression is regulated in response to agonist or antagonist stimulation. Desensitization of A 1 R has been described in intact animals and in cell cultures. Prolonged administration of A 1 R agonists to animals leads to functional desensitization of A 1 R in guinea pig heart (24), rat adipocytes (25), rat atrial muscle (26), and rat brain (27,28). The reduced functional response is due to a net loss of A 1 Rs or down-regulation, a decrease in the proportion of A 1 R displaying the high affinity state for agonists and a decrease in the content of G i proteins. Growth of rat adipocytes or smooth muscle DDT 1 MF-2 cells in the presence of N 6 -(R)-(phenylisopropyl)adenosine (R-PIA) leads to the desensitization and down-regulation of A 1 R (29) along with G i ␣-subunits (30,31). We have recently shown that treatment of DDT 1 MF-2 cells with the agonist R-PIA induces a rapid phosphorylation and clustering of A 1 Rs. The loss of binding sites on the cell membrane due to internalization of A 1 R is a slower event occurring within hours. Since activators of protein kinase A or C mimicked the effect of the ligand, Ser/Thr phosphorylation seems to be related to short term clustering and desensitization as well as to long term internalization of A 1 R (32).
A new perspective on the coupling of A 1 R to G proteins has recently been provided. Ecto-adenosine deaminase (ecto-ADA; EC 3.5.4.4), which is capable of degrading adenosine to inosine on the cell surface (for a review, see Ref. 33), was shown to modulate ligand binding and signaling through A 1 Rs on DDT 1 MF-2 cells (34). Irrespective of its catalytic activity, ADA seems to be necessary for a high affinity binding of agonists to A 1 R (35).
Here the effect of ADA on the ligand-mediated regulation of A 1 R in DDT 1 MF-2 cells has been studied. ADA affected both desensitization and internalization of A 1 Rs. Moreover, by means of immunocytochemistry, confocal microscopy, and flow cytometry using antibodies against A 1 R and ADA, we found that A 1 R and ADA colocalize and that, after receptor activation, they cluster on the cell surface and internalize via the same pathway. All of these results provide evidence for concerted modulation of ADA and A 1 R in response to receptor agonists. Antibodies and Fluorescent Probes-Antisera against purified calf ADA and against peptides of A 1 adenosine receptor (PC10 and PC20) were generated by immunization of female New Zealand White rabbits by the Biokit Company (Barcelona, Spain). Antibodies against A 1 Rs (PC11 and PC21) were purified from serum by affinity chromatography (peptide coupled to thiol-Sepharose 4B (Amersham Pharmacia Biotech)). Anti-ADA antibody was purified using ADA coupled to cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech). The specificity of antibodies for their respective antigens and extensive characterization of anti-ADA and anti-A 1 R antibodies (PC11 and PC21) have been previously reported (36,37). PC11 and PC21 antibodies do not recognize ADA from DDT 1 MF-2 cell extracts by immunoblotting, immunoprecipitation, or immunocytochemistry (34). PC21 and PC11 A 1 R antibodies were shown to be specific for cells expressing A 1 Rs (32).

Materials-[Adenine
Fluorescence probes were obtained by reacting (for 2 h at 20°C) 0.1 mg of fluorescein or rhodamine isothiocyanate with 1 mg of purified protein in coupling buffer (50 mM H 3 BO 3 , 200 mM NaCl, pH 9.2). Unbound fluorochrome was removed by rapid filtration through a Sephadex G-25 column (Amersham Pharmacia Biotech), and fractions were tested for protein content. Aliquots were stored at Ϫ80°C.
Cell Cultures and Protein Determination-DDT 1 MF-2 smooth muscle cells derived from a steroid-induced leiomyosarcoma of Syrian hamster vas deferens were obtained from the American Type Culture Collection. Cells were cultured (at 37°C, 5% CO 2 ) in Dulbecco's modified Eagle's medium (DMEM) (Whittaker, Walkersville, NY), 1% nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 5% (v/v) horse serum, and 5% (v/v) fetal calf serum (Life Technologies, Inc.). Desensitization and internalization assays were performed with cells growing in the absence or in the presence of 65 nM ADA and/or 50 nM R-PIA. At the indicated incubation times, cells were harvested and washed exhaustively in cold phosphate-buffered saline (PBS) before binding, immunocytochemistry, and intracellular calcium mobilization assays.
Protein content was measured by the BCA method (Pierce), as described by Sorensen and Brodbeck (38).
Radioligand Binding Assays-For binding assays, untreated or treated DDT 1 MF-2 cells (1 mg/ml) were resuspended in serum-free DMEM buffered with 20 mM HEPES, pH 7.4, and preincubated with 65 nM ADA for 30 min at 4°C. Radioligand binding was performed in the presence of 5 nM of [ 3 H]R-PIA for 4 h at 4°C. Nonspecific binding was performed in the presence of a 400-fold excess of R-PIA. Nonspecific binding with a 500-fold excess of the selective A 1 R antagonist 1,3dipropyl-8-cyclopentylxanthine resulted in similar displacement of [ 3 H]R-PIA binding. When equilibrium had been reached, incubates were filtered through glass fiber filters (GF/C filters, Whatman, Kent, United Kingdom) in a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD). Filters were washed in 5 ml of ice-cold PBS and transferred to vials containing 10 ml of scintillation solution EcoscintH (National Diagnostics, Atlanta, GA). After overnight shaking, vials were counted using a Packard 1600 TRI-CARB scintillation counter with 50% efficiency. All points represent the mean of 3-5 independent experiments performed in triplicate.
Immunostaining and Immunofluorescence Assays-For immunofluorescence staining, treated and untreated cells were washed in PBS and fixed in 4% paraformaldehyde in PBS, pH 7.4, for 15 min at room temperature. After two washes in PBS containing 20 mM glycine (buffer A) and in buffer A containing 1% bovine serum albumin (buffer B), cells were incubated with the rhodamine-conjugated anti-A 1 R antibody (PC21-TRITC, 75 g/ml) and the fluorescein-conjugated anti-ADA antibody (anti-ADA-FITC, 75 g/ml) or, alternatively, with the fluorescein-conjugated anti-A 1 R antibody (PC21-FITC, 75 g/ml) for 1 h at 37°C. Three washes in buffer B were performed before mounting the samples in immunofluorescence medium (ICN Biomedical Inc., Costa Mesa, CA).
Internalization was analyzed by immunocytochemistry and confocal microscopy. To follow dynamic internalization, unfixed cells growing on glass coverslips were incubated (37°C, 20 min) with 65 nM bovine serum albumin-FITC, with PC21-FITC (10 g/ml), and with 65 nM ADA-FITC and/or PC21-TRITC (10 g/ml) and then treated with 50 nM R-PIA at 37°C for 24 -72 h. Cells were washed twice in PBS, fixed in 4% paraformaldehyde in PBS for 15 min, and again washed twice in PBS 20 mM glycine. Glass coverslips were mounted as described above. Cells were examined with a LEICA TCS confocal scanning laser microscope attached to an inverted Leitz DMIRBE microscope (Leica Lasertechnik GmbH, Heidelberg). Images shown are representative of at least three experiments.
Internalization measurements for A 1 Rs and cell surface ADA (ecto-ADA) were performed by immunostaining and flow cytometry analysis. Cells were grown in complete medium in the absence or presence of 50 nM R-PIA for 0 -48 h. In some experiments, ADA (65 nM) was prebound by incubation for 2 h at 4°C; unbound enzyme was eliminated by washing twice in PBS. Cells were fixed using 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed in buffer A and buffer B for 20 min before staining with anti-ADA-FITC (75 g/ml) or PC21-FITC (75 g/ml). Three washes in buffer B were performed before analyzing the mean fluorescence intensity from 5000 -10,000 cells on an EPICS Profile flow cytometer (Coulter, Hieleah, FL). Similar assays were performed for cells incubated (30 min to 48 h at 37°C) with 65 nM fluorescein-conjugated ADA (ADA-FITC) in the absence or presence of 50 nM R-PIA. In order to remove as much of the cell surface-bound ADA as possible, cells were washed once in PBS and once in PBS/HCl, pH 2, for 15 min according to the method of Yang et al. (39). Cells were fixed and processed for flow cytometry as described above. For establishing comparisons in internalization studies, the mean of fluorescence intensity taken as reference was that found in cells incubated with 65 nM ADA-FITC for 15 min at 37°C. This fluorescence intensity was similar to that found in cells incubated with 65 nM ADA-FITC at 4°C for 2 h, which was considered as the background signal. Higher intensities would reflect internalization of ADA-FITC.
Measurement of Intracellular Free Calcium-Cells were washed and resuspended (5 ϫ 10 6 cells/ml) in Hanks' balanced salt solution (HBSS containing 1.2 mM CaCl 2 and 20 mM HEPES, pH 7.4) and loaded with 5 M Fura-2/AM for 30 min at 25°C. Cells were washed twice and resuspended in HBSS (1 ϫ 10 6 cells/ml) for 20 min at room temperature. Cells were placed in a cuvette (10 6 cells/2 ml of HBSS, 25 nM ADA), and calcium peak induction was achieved by treatment with 50 nM R-PIA at 30°C. For short term desensitization assays (1-15 min), cells were loaded and washed before resuspension in prewarmed (37°C) serum-free DMEM (1 ϫ 10 6 cells/ml) and incubated with the indicated reagents. Cells were washed three times in HBSS buffer to remove reagents before resuspension in the same buffer and R-PIA induction of calcium mobilization as indicated above. After these three washes [Ca 2ϩ ] base-line levels were similar irrespective of the type of pretreatment. Therefore, differences in calcium mobilization were not a consequence of different calcium base-line levels in response to pretreatments. For long term desensitization (12,24, and 48 h), incubations were performed before loading with Fura-2/AM. Calcium concentration was determined in a dual wavelength Shimadzu RF-5000 spectrofluorophotometer by using the ratio of excitation wavelengths 334/366 nm with emission cut-off at 500 nm. Free calcium concentration was calculated as described previously (40).
Statistical Analysis-Time course curves were fitted to a singlephase exponential decay equation, in the case of [ 3 H]R-PIA binding. Statistical comparisons were made using the two-tailed Student's t test. Differences were considered significant when p was Ͻ0.05.

Effect of ADA on Cell Surface Distribution of A 1 Rs in the
Absence and in the Presence of Agonist-A 1 adenosine receptor (A 1 R) distribution on the surface of DDT 1 MF-2 cells was examined by immunocytochemistry and confocal analysis. Untreated and R-PIA-treated cells were fixed, incubated with fluorescein-conjugated rabbit anti-A 1 R antibody (PC21-FITC), and analyzed by confocal microscopy. The PC21 antibody is directed against the second extracellular loop of A 1 R and its specificity for cell surface-expressed A 1 R has been previously demonstrated (32). Receptor distribution in untreated cells revealed homogenous bright staining on the surface of DDT 1 MF-2 cells (Fig. 1A). No major differences in receptor distribution were observed in the presence of exogenous ADA (65 nM) for 15 min (Fig. 1B). A 5-min exposure of cells to the A 1 R agonist R-PIA (50 nM) induced the appearance of bright accumulations of antigen on the plasma membrane, suggesting aggregation or clustering of receptor molecules (Fig. 1C). Clustering of A 1 Rs was more evident after 15 min of R-PIA exposure, when brighter clusters were found (Fig. 1, compare C and E). Incubation of DDT 1 MF-2 cells with ADA and R-PIA changed the pattern of cell surface A 1 R distribution dramatically. In the presence of ADA, clusters of A 1 R were already evident at 2 min, and the label was found as intense bright punctate accumulations at 5 or 15 min of R-PIA treatment (Fig.  1, D and F). This clearly shows the involvement of ecto-ADA in the regulation of the R-PIA-induced aggregation of cell surface A 1 R molecules. All images shown in Fig. 1 represent reconstructions of multiple sections at different levels. When middle sections were analyzed, only the cell surface staining was observed, thus ruling out intracellular staining (data not shown; see Ref. 32).
To analyze the codistribution of A 1 R and ADA on the plasma membrane of DDT 1 MF-2 cells, ADA-treated cells were incubated with or without 50 nM R-PIA for 15 min. Cells were fixed, washed, and stained by double immunofluorescence using the fluorescein-conjugated anti-ADA antibody (anti-ADA-FITC) and the rhodamine-conjugated anti-A 1 R antibody (PC21-TRITC). Homogenous distribution of ADA and A 1 R on the cell surface was observed in untreated cells (Fig. 2, top). Superimposition of images by confocal analysis showed extensive colocalization (yellow) of both proteins on the plasma membrane. When cells were treated (15 min) with R-PIA, a redistribution of ADA and A 1 Rs, which is consistent with clustering, was observed. R-PIA induced aggregation of both proteins in clusters seen as punctate accumulations of fluorescence and where colocalization between A 1 R and ecto-ADA was very high (Fig. 2,  bottom). ADA Affects Ligand-induced Phosphorylation of A 1 Rs-In a previous study, we have shown that ligand-induced phosphorylation of A 1 R correlates with the clustering of receptors on the DDT 1 MF-2 cell surface (32). To determine whether the potentiation of the clustering by ADA could involve changes in receptor phosphorylation, we immunoprecipitated the receptor from cells metabolically labeled with [ 32 P]orthophosphate. Exogenous ADA by itself had only a slight effect on A 1 R phosphorylation. R-PIA (50 nM) induced, in the absence of ADA, a steady increase of A 1 R phosphorylation from basal levels to a 2.1-fold increase at 15 min. Interestingly, in the presence of exogenous ADA (65 nM), R-PIA induced a rapid increase in A 1 R phosphorylation with a maximum rise (3-fold increase) after 1 min; thereafter (5 and 15 min), the level of phosphorylation declined (Fig. 3). In the presence of ADA, receptor phosphoryl- Agonist-induced Desensitization of A 1 Rs in the Presence of ADA-Activation of A 1 Rs with adenosine or analogs leads to both Ins(1,4,5)P 3 formation and intracellular calcium mobilization in DDT 1 MF-2 cells (41,42). To correlate changes in agonist-induced A 1 R phosphorylation with possible changes in second messenger production, we analyzed the effect of ADA and/or R-PIA on agonist-induced mobilization of intracellular calcium. Cells were exposed to vehicle (control) or to 65 nM ADA and/or 50 nM R-PIA. After extensive washes, calcium mobilization was induced by 50 nM R-PIA. R-PIA caused a transient Ca 2ϩ peak in naive (vehicle-treated) cells (Fig. 4A). Desensitization of the [Ca 2ϩ ] response in R-PIA-or R-PIA/ADA-treated cells occurred rapidly (in minutes) and was sustained for at least 48 h (Fig. 4B). R-PIA pretreatment reduced the maximum intracellular calcium concentration by 2, 30, and 44% at 1, 5, and 15 min, respectively. This effect was, however, potentiated in the presence of ADA, since the maximum desensitization (60%) was achieved at 5 min and maintained for up to 48 h (Fig.  4, A and B). No significant differences in maximum effect were observed when R-PIA pretreatment was done in the presence or in the absence of ADA (Fig. 4B). It should be noted that ADA pretreatment in the absence of R-PIA led to a significant and sustained desensitization (25-45% reduction of the response).
Loss of A 1 Rs from the Cell Surface-Characterization of A 1 Rs by radioligand binding using the agonist [ 3 H]R-PIA indicated the presence of two affinity states of the receptors (K high ϭ 0.8 -2 nM and K low ϭ 10 -100 nM) in cell membrane preparations, whereas only the low affinity state was detected in intact cells (43). Ligand binding assays were performed in intact cells preincubated with vehicle (control) or with 50 nM R-PIA and/or 65 nM ADA. Cells were extensively washed and assayed for [ 3 H]R-PIA binding at 4°C. The results indicated a time-dependent loss of binding sites on the cell membrane of treated cells (Fig. 5). The loss of binding sites after incubation of cells with ADA in the absence of R-PIA was small. In contrast, preincubation of cells with 50 nM R-PIA with or without ADA resulted in a time-dependent reduction of [ 3 H]R-PIA binding sites. The presence of ADA accelerated the reduction of binding sites, from t1 ⁄2 ϭ 10 Ϯ 1 h to t1 ⁄2 ϭ 2.9 Ϯ 0.5 h. The maximum loss of cell surface binding sites (around 41%) induced by agonist was not modified by the presence of ADA (Fig. 5).
Disappearance of A 1 Rs from the cell surface of DDT 1 MF-2 cells in response to agonist has been described elsewhere (32). Interestingly, the effect of ADA was evident even when the protein was prebound to the cell surface and the unbound enzyme was washed away (Fig. 6). On the other hand, the disappearance of A 1 Rs from the cell surface was reversible. Thus, when chronically stimulated cells were further cultured for 24 h in the absence of R-PIA, the level of cell surface expression was recovered. These results indicate that, in the presence of ADA, R-PIA induces the sequestration of A 1 R in intracellular compartments (see below).
Internalization of A 1 Rs Together with ADA-In order to determine whether agonist-induced disappearance of binding sites affects the expression of cell surface ADA (ecto-ADA), fluorocytometry analysis was performed using nonpermeabi-

FIG. 2. Colocalization between A 1 R and ADA on the cell surface.
Cells were treated with 65 nM ADA in the absence (top) or presence (bottom) of 50 nM R-PIA for 15 min at 37°C. After treatment, cells were washed, fixed, and processed for immunostaining as described under "Experimental Procedures." Staining was performed with the fluoresceinconjugated anti-ADA antibody (anti-ADA-FITC, 75 g/ml) and the rhodamineconjugated anti-A 1 R antibody (PC21-TRITC, 75 g/ml). Cells were analyzed by double immunofluorescence with a confocal microscope to detect ADA (green images) and A 1 R (red images). Superimposition of images reveals ADA/A 1 R colocalization in yellow. Scale bar, 10 m. lized cells. After pretreatment with medium (control) or 50 nM R-PIA, cells were fixed, and ecto-ADA was labeled using the anti-ADA-FITC antibody. A significant time-dependent decrease in fluorescence intensity was observed after 8 h of R-PIA treatment, and a maximum decrease (40%) was obtained at 24 h, which suggests A 1 R agonist-induced internalization of ecto-ADA (Fig. 7).
To follow ADA internalization, DDT 1 MF-2 cells were labeled with ADA-FITC, which binds to cell surface A 1 Rs (34). The labeling was performed in the presence or in the absence of 50 nM R-PIA. After extensive washes in acid buffer, cells fixed in paraformaldehyde were analyzed by flow cytometry. The acidic wash was necessary to remove ADA-FITC remaining on the cell surface; therefore, quantification of intracellular fluorescence is a direct measure of internalized ADA (see "Experimental Procedures"). A progressive time-dependent incorporation of fluorescence into the cells, in the period 2-48 h, was observed in the absence of R-PIA. Maximal incorporation of ADA-FITC, which occurred at 48 h, increased (37%) in the presence of R-PIA. The differences between control and R-PIA-treated cells were significant between 8 and 48 h. Internalization of ADA-FITC into the cells was also analyzed by immunofluorescence and confocal assays. Cells maintained in the presence of ADA-FITC for 24 -72 h showed accumulations of labeling in intracellular vesicles. R-PIA treatment for 24 -72 h resulted in a marked increase in intracellular labeling in brighter and larger intracellular vesicles (Fig. 8A). These variations were not due to fluid phase internalization, since R-PIA did not affect the extent of fluorescein-conjugated bovine serum albumin internalization (Fig. 8B).
As shown in Figs. 5 and 9A and as described elsewhere (32), R-PIA induces the disappearance of cell surface A 1 Rs even in the absence of exogenous ADA. Since cell surface ADA and A 1 R colocalize, these results indicate that R-PIA might induce internalization of A 1 R together with ADA. We tested this hypothesis by comparing the distribution of A 1 Rs and ADA in double immunofluorescence staining experiments analyzed by confocal microscopy. Double immunofluorescence staining with the rhodamine-conjugated rabbit anti-A 1 R (PC21-TRITC) and fluorescein-conjugated ADA (ADA-FITC) was performed in control and R-PIA-treated cells. Incubation of cells with ADA-FITC and PC21-TRITC for 24 h showed staining for ADA and A 1 R in intracellular vesicles (Fig. 9B, top). Superimposition of images revealed a high degree of colocalization between the two proteins (Fig. 9B, yellow). After 24 h of chronic exposure to 50 nM R-PIA, perinuclear staining and a marked decrease in the number of intracellular vesicles containing ADA and A 1 Rs, accompanied by a drastic increase in size and brightness, were observed (Fig. 9B, bottom). Colocalization of both proteins (yellow) was confirmed in numerous cells analyzed at various planes, indicating that A 1 Rs and ADA internalize together via the same pathway of endocytosis. DISCUSSION This study provides new insights into ligand-mediated mechanisms involved in the regulation and trafficking of A 1 adenosine receptors. Several studies have demonstrated agonist desensitization by means of a reduced inhibition of adenylate cyclase associated with a net loss of binding sites and G i proteins (29,30,31). However, little was known about the biochemical mechanisms involved in desensitization and downregulation or the intracellular pathways involved in the trafficking of A 1 Rs. Another interesting question concerns the role of ecto-ADA in the desensitization of A 1 R due to the fact that A 1 R and ADA interact on the cell surface (34). The high expression (100,000 receptors/cell) of A 1 Rs (43), capable of interacting with ADA (34) makes DDT 1 MF-2 cells a suitable model to study the role of ADA in the regulation of A 1 R expression.
By means of immunofluorescence staining using antibodies against A 1 R and ADA, a nearly homogenous distribution of A 1 R and ADA over the plasma membrane of DDT 1 MF-2 cells was observed. Confocal analysis demonstrated a high degree of colocalization between ADA and A 1 R, which was maintained in the cell surface clusters formed after R-PIA treatment. The appearance of clusters was accelerated when cells were incubated simultaneously with R-PIA and ADA. This suggests an ADA-mediated regulation of ligand-induced redistribution of A 1 Rs. Prolonged agonist stimulation induced the disappearance of membrane A 1 R binding sites on DDT 1 MF-2 cell surface, as previously shown (29). This phenomenon was also accelerated in the presence of ADA, although the maximum effect was the same as that achieved by R-PIA alone.
Ecto-ADA also underwent R-PIA-induced internalization. Internalized ADA appeared in relatively small intracellular vesicles, where it colocalized with A 1 R. This may be an agonistindependent process representing the natural recycling of the ectoenzyme, or it may be due to the presence of endogenous adenosine. This latter possibility seems unlikely, since no internalization of A 1 R occurred in the absence of R-PIA. Although ADA internalization may be agonist-independent, long term agonist treatment increased the degree of ADA internalization. Double immunofluorescence assays analyzed by confocal microscopy showed colocalization of ADA and A 1 Rs in the same intracellular vesicles. All these findings suggest that ADA and A 1 Rs participate in the regulation of each other and that this mutual regulation includes internalization via the same endocytic pathway. To our knowledge, this is the first report describing the trafficking of a purine ectoenzyme and demonstrating a common internalization pathway for A 1 R and the ectoprotein (ecto-ADA) interacting with it.
During the agonist-mediated redistribution (clustering) of A 1 Rs on the cell surface, a time-dependent phosphorylation of A 1 Rs occurs. Basal A 1 R phosphorylation increased rapidly between 5 and 15 min as a consequence of agonist stimulation. Agonist-induced A 1 R phosphorylation was accelerated and enhanced in the presence of exogenous ADA. Taking into account these data and the fact that ADA increased ligand-induced A 1 R aggregation, the involvement of phosphorylation in the clustering of A 1 receptors is highly probable. In fact, clustering and internalization of A 1 Rs were mimicked by activators of protein kinase A and protein kinase C (32), thus showing the need for Ser/Thr phosphorylation in these events. Simultaneous to ligand-induced receptor phosphorylation and aggregation, a decrease in ligand-induced second messenger response, i.e. a functional desensitization, was observed. Functional desensitization was a rapid time-dependent process that occurred within minutes but remained for hours. This contrasts with data found in Chinese hamster ovary cells transfected with the human A 1 R. Overexpressed A 1 Rs were neither phosphorylated nor desensitized after 10 min of R-PIA treatment. Interestingly, a stably expressed chimeric A 1 -A 3 adenosine receptor, in which the C-terminal domain of A 1 R distal to its predicted palmitoylation site was replaced by the corresponding region of the A 3 subtype, was able to undergo functional desensitization and agonist-stimulated phosphorylation (44). Differences in amino acid sequence of the C-terminal domain of A 1 R and A 3 R can account for the distinct desensitization pathways found in Chinese hamster ovary cells. However, the short term desensitization and phosphorylation reported here for A 1 R in DDT 1 MF-2 cells demonstrate that the cellular environment near the plasma membrane is important for receptor regulation. The regulation of A 1 R phosphorylation and desensitization by cell surface ADA is an example of the relevance of the membrane components for receptor regulation. To our knowledge, this is a novel finding, since no other receptor whose function is modulated by an ectoenzyme has been described.
Rapid ligand-induced desensitization of G protein-coupled receptors such as ␤ 2 -ARs (45), angiotensin II receptor (46), cholecystokinin receptor (47), ␦-opioid receptor (48), and neurokinin 1 receptor (49) correlates with receptor phosphorylation and uncoupling from G proteins. Several reports have provided evidence that receptor desensitization is short term and does not require endocytosis (45,50,51). An exception is the agonist-induced desensitization of the secretin receptor, which occurs in the absence of receptor phosphorylation and is basically induced by receptor internalization (52). ␤ 2 -AR (22,23) and neurokinin 1 receptor (49) resensitization seems to be a consequence of receptor dephosphorylation in endosomes and their recycling back to the plasma membrane. Our data indicate that although receptor phosphorylation returns to basal level, desensitization of A 1 R continued for hours (see Figs. 3 and 4). In a recent study, desensitization of the cholecystokinin receptor occurred in both rat pancreatic acinar cells and transfected Chinese hamster ovary cells, although receptor dephosphorylation was evident only in acinar cells. Moreover, cholecystokinin receptor in Chinese hamster ovary cells maintained its phosphorylation state throughout the time of internalization, whereas the acinar cell receptor was dephosphorylated to its basal state while remaining on the cell surface (47). All of these data suggest differences in regulation of G protein-coupled receptors, depending on the receptor subtype and the cell system in which the receptor is expressed.
The results provide new insights into the regulation and trafficking of A 1 Rs in response to agonist stimulation. Moreover, it is shown that ecto-ADA, besides its role in regulating the extracellular concentration of adenosine, modulates all the regulatory mechanisms involved in desensitization of A 1 R. It is remarkable that ADA and A 1 R colocalize on the cell surface and in intracellular vesicles after internalization. During the agonist-dependent internalization process, ADA and A 1 R follow a common endocytic pathway. This is probably the molecular mechanism underlying the recent discovery of increased plasma adenosine levels found after caffeine or sulfophenylteophylline consumption (53). Although it does not occur in DDT 1 MF-2 cells, there are cell types whose A 1 Rs internalize in response to xanthine antagonists. 2 The probable internalization of ADA and A 1 R from the cell surface in those antagonisttreated cells would lead to a decrease in adenosine deamination and, subsequently, to an increase in the extracellular levels of the nucleoside. As pointed out by Conlay et al. (53), alteration of adenosine levels by sudden changes in methylxanthine consumption could affect the physiology of many organ systems and provoke bronchospasm, alter blood pressure, change cardiac rhythms, or influence seizure thresholds. Confirmation that internalization of ecto-ADA is the cause of these alterations would be very valuable from a physiological and therapeutic point of view. After treatment, cells were fixed, washed, and analyzed by confocal microscopy. Images correspond to a middle section of representative cells. Scale bar, 10 m. B, DDT 1 MF-2 cells were grown in DMEM containing fluorescein-conjugated ADA (65 nM) and rhodamine-conjugated anti-A 1 R antibody (PC21-TRITC, 10 g/ml) in the absence (top) or in the presence (bottom) of 50 nM R-PIA for 24 h. After treatment, cells were fixed, washed, and analyzed by confocal microscopy to detect ADA (green images) and A 1 R (red images). Superimposition of images reveals the intracellular colocalization of ADA and A 1 R in yellow. Scale bar, 10 m.