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J. Biol. Chem., Vol. 279, Issue 37, 38603-38607, September 10, 2004
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2-Adrenergic Receptor Routes Receptors to Degradation during Agonist Regulation*
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From the
Departments of Medicine, Pharmacology, and **Molecular Genetics and the ¶CardioPulmonary Research Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Received for publication, April 2, 2004 , and in revised form, June 22, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2-adrenergic receptors (2AR) of all species are N-linked glycosylated at amino terminus residues 
6 and 15. However, the human 2AR has a potential third N-glycosylation site at ECL2 residue 187. To determine whether this residue is glycosylated and to ascertain function, all possible single/multiple Asn 
Gln mutations were made in the human 2 AR at positions 6, 15, and 187 and were expressed in Chinese hamster fibroblast cells. Substitution of Asn-187 alone or with Asn-6 or Asn-15 decreased the apparent molecular mass of the receptor on SDS-PAGE in a manner consistent with Asn-187 glycosylation. All receptors bound the agonist isoproterenol and functionally coupled to adenylyl cyclase. However, receptors without 187 glycosylation failed to display long term agonist-promoted down-regulation. In contrast, loss of Asn-6/Asn-15 glycosylation did not alter down-regulation. Cell surface distribution and agonist-promoted internalization of receptors and recruitment of -arrestin 2 were unaffected by the loss of 187 glycosylation. Furthermore, acutely internalized wild-type and Gln-187 receptors were both localized by confocal microscopy to early endosomes. During prolonged agonist exposure, wild-type 2AR co-localized with lysosomes, consistent with trafficking to a degradation compartment. However, Gln-187 2AR failed to co-localize with lysosomes despite agonist treatments up to 18 h. Phylogenetic analysis revealed that this third glycosylation site is found in humans and other higher order primates but not in lower order primates such as the monkey. Nor is this third site found in rodents, which are frequently utilized as animal models. These data thus reveal a previously unrecognized 2AR regulatory motif that appeared late in primate evolution and serves to direct internalized receptors to lysosomal degradation during long term agonist exposure. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2-adrenergic receptor (2AR)1 undergoes a loss of cellular signaling during prolonged agonist exposure. This phenomenon, termed desensitization, serves to integrate 2AR function within the context of a cell that is receiving many signals (1). In pathologic conditions, this adaptive response can also contribute to the pathophysiology of the disease and can limit therapeutic effectiveness of agonists administered over prolonged periods of time (1). Early molecular events during short term agonist activation include phosphorylation of the 2AR by G-protein-coupled receptor kinases and protein kinase A, which act to rapidly regulate receptor function along a minute-by-minute time frame. With long term agonist exposure, an additional series of events leads to a net loss of cellular receptors, termed down-regulation, which markedly reduces the cellular response (such as cAMP production). The basis of receptor down-regulation includes degradation of the 2AR protein, which is linked to early desensitization events, because G-protein-coupled receptor kinase-mediated phosphorylation of 2AR leads to recruitment and binding of -arrestin, which, via its scaffolding functions, facilitates internalization (2). A sorting of internalized receptors can result in reinsertion into the cell membrane or movement of a receptor into a degradative pathway with an ultimate loss of the intact receptor (3). The structural features of the 2AR that appear to be involved in internalization, recycling, and/or down-regulation, include G-protein-coupled receptor kinase phosphorylation sites in the intracellular tail (4), the third intracellular loop protein kinase A phosphorylation site (5), the NPXXY motif of the seventh transmembrane spanning domain (6), and the PDZ-binding domain in the distal carboxyl-terminal tail of the receptor (7). Expression or trafficking of some G-protein-coupled receptors, such as the 2AR, gastrin-releasing peptide receptor, and the V2-vasopressin receptor is dependent on post-translational modifications including Nand O-linked glycosylation (8–10). All 2AR genes identified to date from multiple species have two sites for N-linked glycosylation within the amino terminus of the receptor at amino acid positions 6 and 15. Removal of these sites in the hamster 2AR by mutagenesis results in receptors that have an impaired capacity to insert into the membrane. Nevertheless, 50% of the total receptor compliment is ultimately expressed on the cell surface (8).
Interestingly, the human 2AR has an additional potential N-glycosylation site, localized to the second extracellular loop, at amino acid 187; this consensus sequence is not found in a wide variety of non-primate species reported to date (see Fig. 1). This prompted us to examine the role of Asn-187 of the human 2AR with the idea that this modification may represent a specific mechanism for receptor trafficking that occurred late in mammalian evolution and serves a unique function necessary for primate homeostasis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2AR cDNA was carried out so as to substitute Gln for Asn at amino acids 6, 15, and 187, both singly and in combination. This resulted in eight cDNAs encoding receptors with all possible glycosylation combinations, denoted as NNN (wild-type), QNN, NQN, NNQ, QQN, QNQ, NQQ, and QQQ. These cDNAs were subcloned into a pFLAG-CMV construct, which contained the 5'-FLAG epitope (DYKD-DDDK) in-frame with the 2AR. Chinese hamster fibroblasts (CHW-1102 cells) were stably transfected by a calcium precipitation technique as described, and selection was performed in 300 µg/ml G418. Cells were maintained in 100 units/ml penicillin and 100 µg/ml streptomycin in Dulbecco's modified Eagle's medium with 10% fetal calf serum in a 5% CO2 atmosphere at 37 °C. For -arrestin recruitment studies, human embryonic kidney-293 cells were transiently co-transfected by a calcium precipitation method with the 2AR construct of interest and a construct consisting of a -arrestin 2-green fluorescent protein fusion gene as described (11).
Radioligand Binding—Cells were washed twice with phosphate-buffered saline and detached by scraping in 5 mM Tris, 2 mM EDTA, pH 7.4, at 4 °C. This and subsequent buffers contained 5 µg/ml of the protease inhibitors leupeptin, soybean trypsin inhibitor, aprotinin, and benzamidine, and 0.1 mg/ml phenylmethylsulfonyl fluoride. Cell particulates were homogenized with a Polytron and centrifuged at 400 x g for 10 min, and the supernatant was pelleted by centrifugation at 30,000 x g for 10 min and resuspended in 75 mM Tris, 12 mM MgCl2, 2 mM EDTA, pH 7.4, at 37 °C. Competition and saturation radioligand binding studies with these membranes were carried out using 125I-cyanopindolol (125I-CYP) as described previously (12). To quantitate only the cell surface 2AR, radioligand binding was carried out using adhered intact cells in 12-well dishes with the hydrophilic radioligand 3H-CGP12177 (6 nM) in the absence or presence of 1.0 µM propranolol used to define nonspecific binding as described (13). Reactions were performed at 4 °C for 4 h. Then cells were washed three times in ice-cold phosphate-buffered saline, solubilized in 1% SDS in phosphate-buffered saline, and the contents were counted in a liquid scintillation counter. Additional experiments to ascertain internal versus cell surface 2AR utilized intact cell binding with 125I-CYP, in competition with 0.3 µM unlabeled CGP12177(identifies cell surface receptors) and 1.0 µM propranolol (identifies total cellular pool of receptors) as described else-where in detail (14). For the 125I-CYP binding experiments, reactions were terminated by a dilution in 5 mM Tris 2 mM EDTA buffer at 4 ° C, and bound radioligand was separated from free by vacuum filtration over glass fiber filters.
Adenylyl Cyclase Activities—Membranes were incubated with 30 mM Tris, pH 7.4, 2.0 mM MgCl2, 0.8 mM EDTA, 120 µM ATP, 60 µM GTP, 2.8 mM phosphoenolpyruvate, 2.2 µg of myokinase, 100 µM cAMP, and 1 µCi of [
-32P]ATP for 30 min at 37 °C as described (15). [32P]cAMP was separated from [-32P]ATP by chromatography over alumina columns. A[3H]cAMP standard included in the stop buffer accounted for individual column recovery. Activities were determined in the presence of vehicle (basal), 10 µM isoproterenol, or 100 µM forskolin.
Confocal Microscopy—To localize 2AR, -arrestin 2, and various intracellular organelles, confocal microscopy was performed with a Zeiss 510 laser scanning microscope and a 63/1.4 oil immersion lens (-arrestin 2-green fluorescent protein) or 63/1.2 water immersion lens (others). For 2AR localization, studies were performed on fixed cells. In a typical experiment, attached cells maintained in Dulbecco's modified Eagle's medium were treated with agonist for the indicated times at 37 °C in 5% CO2, washed three times with cold phosphate-buffered saline, and fixed with –20 °C methanol. 2AR receptors were identified with a mouse anti-FLAG-M2 primary antibody (Sigma) and the goat anti-mouse secondary antibody Alexa Fluor 488 (Molecular Probe). Lysosome compartments were stained with a rabbit anti-cathepsin-D primary antibody (Santa Cruz) (16), and the secondary antibody was goat anti-rabbit Alexa Fluor 543. The early endosome compartment was stained with a rabbit anti-EEA1 primary antibody (Affinity BioReagents) (17), with the secondary antibody being Alexa Fluor 543. Images were obtained with excitations of 488 or 543 nm and emission spectra of 505–530 or 560 nm, respectively. For -arrestin recruitment studies in live cells, cells transfected with 2AR and -arrestin 2-green fluorescent protein (11) were transferred onto collagen-coated glass bottom dishes. Forty-eight hours after transfection, cells were treated with the indicated agonist, and real time confocal microscopy was carried out using excitation at 488 nm and a 515–540 nm emission filter.
Other—For Western blots, cell membranes were solubilized in radio-immune precipitation assay buffer, and the proteins were fractionated on 10% SDS-PAGE and transferred to nitrocellulose membranes. They were blotted with primary antibody (mouse anti-FLAG-M2) at a titer of 1:750 and a secondary antibody (Amersham Biosciences) for chemiluminescence detection. For fractionation experiments, membranes were separated using a self-generating Percoll (Amersham Biosciences) gradient as described (18). Six 1.5-ml fractions were collected and probed by Western blot with the cathepsin-D and EEA1 antibodies or were used for radioligand binding studies to quantitate 2AR. Protein concentrations were determined by the copper bicinchoninic acid method (19). Radioligand binding and kinetic data were fit to standard models using the program Prism (GraphPad Software).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2ARs are shown in Fig. 2. As expected, an individual removal of position 6 or 15 glycosylation sites resulted in decreases in apparent molecular mass of the major high molecular mass form (from 60 to 56 kDa), and the loss of both sites reduced the molecular mass proportionately. Consistent with position 187 also being glycosylated, the NNQ receptor also had a lower apparent molecular mass like that of the QNN and NQN receptors. Removal of the position 187 site, in combination with those at 6 and/or 15, had the expected additive decrease in apparent molecular mass of the major receptor species as shown.
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2AR underwent a 34 ± 3.4% down-regulation. A similar extent of down-regulation was also observed for the single and doubly substituted QNN, NQN, and QQN receptors. In contrast, single removal of the 187-glycosylation site (NNQ receptor) resulted in a complete loss of agonist-promoted down-regulation, and in fact an increase in expression was noted (15 ± 6.7%, p s< 0.01 versus wild-type). This same phenotype was observed with all mutant 2ARs (NNQ, QNQ, NQQ, and QQQ) where the position 187 was mutated, regardless of the presence or absence of the other glycosylation sites (Fig. 3). The QQQ receptor displayed the greatest increase in expression during agonist exposure. This may be because of the fact that the QQN receptor has some-what less down-regulation than wild-type and thus less impediment to the up-regulation events imposed by QQQ, as compared with NNQ. Additional studies were also performed using the glycosylation inhibitor tunicamycin. Chinese hamster fibroblast cells expressing wild-type 2AR at 50% confluency were treated for 12 h with 0.25 µg/ml tunicamycin in the media and then for an additional 12 h with 10 µM isoproterenol. Tunicamycin-treated cells underwent only 46 ± 1.3% of the isoproterenol-promoted down-regulation observed with non-tunicamycin-treated cells. These results further support the notion that it is the loss of glycosylation imposed by the mutation at 187 that leads to the altered down-regulation phenotype of the NNQ receptor.
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2AR were predominantly expressed at the cell surface, as qualitatively shown by confocal microscopy (Fig. 4) and quantitatively determined by whole-cell radioligand binding with hydrophilic and hydrophobic ligands (95 ± 8.3 versus 90 ± 2.4% of the total receptor complements were at the cell surface for wild-type versus NNQ, respectively, n = 4). One of the earliest events in internalization of 2AR upon agonist binding is the recruitment of -arrestin from the cytosol to the cell-surface-expressed receptors. -arrestin-green fluorescent protein translocation was ascertained by confocal microscopy in live cells (Fig. 5). Agonist (10 µM isoproterenol) exposure resulted in a time-dependent recruitment of -arrestin in cells expressing either receptor. As shown, in wild-type (NNN)-expressing cells, the predominantly intracellular distribution of -arrestin becomes mostly cell surface by 5 min of exposure to isoproterenol. The translocation of NNQ was qualitatively indistinguishable from the wild-type 2AR. Consistent with these observations, agonist-promoted receptor internalization as determined by quantitative hydrophilic radioligand binding with intact cells revealed identical kinetics both in terms of the maximal response (45 ± 4.4 versus 49 ± 6.2% internalized) and the rate constant (k = 0.16 ± 0.029/min versus 0.12 ± 0.022/min, n = 4) for the NNN and NNQ receptors, respectively (Fig. 6). Taken together, these data indicate that the initial events in agonist-promoted down-regulation, which leads to the redistribution of cell surface 2AR to the cell interior, are not perturbed by the absence of glycosylation at Asn-187 of the receptor.
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2AR could be the result of an altered (enhanced) synthesis of new receptors during agonist exposure, or internalized receptors that are not targeted in a wild-type manner to the degradation pathway. New receptor synthesis in the presence of agonist was quantitated by radioligand binding after the exposure of the cells to an irreversible receptor-alkylating agent (Pindobind, Sigma). Cells were treated with 10 nM Pindobind for 2 h (which alkylated 70% of the receptors), washed, and placed back in the incubator. At various time points up to 12 h, cells were harvested, and 125I-CYP saturation binding was undertaken. Newly synthesized receptors appear as an increase in 125I-CYP binding over time. (Of note, pretreatment of cells with the protein synthesis inhibitor cycloheximide resulted in no increase in 125I-CYP binding, consistent with the technique identifying newly synthesized receptors.) The rate of new receptor synthesis in the presence of 10 µM isoproterenol, which is the condition relevant to the agonist-promoted up-regulation of NNQ, was not significantly different, and, if anything, the trend was toward being less for NNQ compared with NNN (k = 0.07 ± 0.02/min versus 0.12 ± 0.04/min, n = 4). It thus appears that the accumulation of NNQ receptors evoked by the agonist is not based on decreased receptor internalization or increased de novo receptor synthesis, suggesting that the NNQ receptor has altered entry into the degradation pathway(s) after internalization.
The fate of internalized receptors was assessed by confocal microscopy of fixed cells, with an emphasis on co-localization of NNN or NNQ with early endosomes (a short term event) and lysosomes (a long term event). Results of studies with the EEA1 antibody, which identifies early endosomes, are shown in Fig. 7. For both NNN and NNQ receptors, a 15-min exposure to isoproterenol resulted in internalization (Fig. 7, d and j) and co-localization with early endosomes (f and l). Similar findings were observed with incubations up to 4 h (data not shown). Taken together with the other short term agonist data shown in Figs. 5 and 6, it appears that NNQ internalizes and is localized to the early endosomes in a wild-type manner. However, as shown in Fig. 8, NNQ fails to co-localize with lysosomes during prolonged agonist incubation. For the NNN wild-type 2AR, one can clearly observe a loss of cell surface and total receptor expression (a versus d) and intracellular accumulation of receptor after 4 h of agonist exposure (d). The intracellular wild-type receptors co-localized with the lysosomal marker cathepsin-D (Fig. 8f). In contrast, overall expression of the NNQ receptor was maintained under the same agonist exposure conditions (Fig. 8, g versus j), and although internalized receptors were observed, they failed to co-localize with the lysosomal marker (l). Four hours of agonist exposure represents an optimal time point for co-localization of wild-type receptor with lysosomes, as longer exposures result in degradation and a decreased signal. Nevertheless, even with more prolonged agonist exposure (up to 18 h), we never observed the co-localization of NNQ with lysosomes (data not shown). To further support the confocal findings, cells expressing NNN or NNQ were treated with vehicle or isoproterenol for 4 h, whole-cell homogenates were prepared, and the proteins were fractionated on a Percoll gradient. Consistent with the findings of others (25), one of the six fractions (fraction 5) was enriched in lysosomes as determined by Western blots using the cathepsin-D antibody. 125I-CYP radioligand binding was performed on the proteins from this lysosomal fraction to ascertain whether agonist exposure increased receptors in this pool for NNN-expressing cells and had no effect on the pool of NNQ receptors. Although this fraction undoubtedly contains other compartments/proteins in addition to the specific lysosomes identified by confocal microscopy, we indeed observed increased agonist-promoted receptors in the fraction from NNN cells (from 123 ± 24 to 218 ± 42 fmol/mg, p = 0.03, n = 8), but not NNQ cells (from 65 ± 12 to 65 ± 17 fmol/mg). Although the magnitude of the agonist effect with the wild-type is modest in this assay, the lack of any change with the NNQ mutant is consistent with the confocal results.
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2ARs of other species that lack Asn-187 (e.g. hamster, guinea pig, rat 2AR) (20–24) nevertheless have been reported to undergo agonist-promoted down-regulation in vivo, although the phenomenon has been minimally explored in recombinant cells expressing non-human 2ARs (23). This may suggest that other sites/mechanisms are in place in these receptors that abrogate the necessity of Asn-187. As noted earlier, this site is absent in a wide variety of diverse species despite the high degree of surrounding sequence homology (Fig. 1). However, of the 11 2AR genomic sequences reported from primates, 5 have the Asn glycosylation site at the second intracellular loop residue, as is observed in the human receptor (Table II). These five Asn-187-containing primates are higher order primates (human, greater and lesser apes), whereas those without this glycosylation site are all lower order primates (monkey, lemur, tamarin, and galago). This suggests that perhaps Asn-187 provides a specific role for receptor regulation in the larger higher order primates. This mechanism may have evolved because of (i) pressure for this specific type of glycosylation-dependent trafficking or (ii) as a compensatory consequence to retain receptor down-regulation after the evolutionary loss of an alternate down-regulatory motif. 2AR sequence comparisons of primate species that carry Asn-187 with those that do not and comparisons with non-primates are hampered by the lack of full-length sequences in primates. Thus scenario (ii) above cannot be adequately explored at this time. Nevertheless, the current study has identified a previously unrecognized site that is essential for long term agonist-promoted down-regulation and has apparently appeared late in primate evolution.
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FOOTNOTES |
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|| Current address: Merck Research Laboratories, Rahway, NJ 07065.
To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 0564, Cincinnati, OH 45267-0564. Tel.: 513-558-0484; Fax: 513-558-0835; E-mail: stephen.liggett{at}uc.edu.
1 The abbreviations used are: 2AR, 2-adrenergic receptor; 125I-CYP,125I-cyanopindolol.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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