Role for the Third Intracellular Loop in Cell Surface Stabilization of the alpha 2A-Adrenergic Receptor*

Stephen W. EdwardsDagger and Lee E. Limbird§

From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Previous studies have shown that alpha 2A-adrenergic receptor (alpha 2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant alpha 2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the Delta 3ialpha 2A-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and Delta 3i loop alpha 2A-ARs are extracted from polarized Madin-Darby canine kidney (MDCK) cells with 0.2% Triton X-100 and with a similar concentration/response profile, suggesting that Triton X-100-resistant interactions of the alpha 2A-AR with cytoskeletal proteins are not involved in receptor retention on the basolateral surface. The indistinguishable basolateral t1/2 for either the wild-type or nonsense 3i loop alpha 2A-AR suggests that the stabilizing properties of the alpha 2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of Delta 3i alpha 2A-AR cannot be attributed to alteration in agonist-elicited alpha 2A-AR redistribution, because alpha 2A-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the alpha 2A-AR on the basolateral surface of MDCKII cells involves multiple mechanisms, with the third intracellular loop playing a central role in regulating these processes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

The alpha 2-adrenergic receptor (alpha 2-AR)1 is a member of a large family of G protein-coupled receptors that are predicted to have seven transmembrane-spanning regions (1, 2). Three subtypes of alpha 2-ARs exist and couple to members of the Gi and Go class of G-proteins to mediate a variety of physiological responses (3, 4).

Receptor localization and stabilization on the cell surface of target cells are two critical contributors to the sensitivity and extent of signaling by G protein-coupled receptors. There is a growing body of evidence that discrete localization of G protein-coupled receptors may play a role in specificity of signaling by these receptors (5-8). A precedent already exists for the microcompartmentation of signaling molecules such as protein kinase C (9), cAMP-dependent protein kinase (10), Ca2+/calmodulin-dependent protein kinase II (11), kinases involved in the yeast mating response (12), and NO synthase (13, 14) by interaction of these effector molecules with signaling "scaffold" proteins.

In polarized cells, receptor localization is essential for vectorial information transfer, as occurs for alpha 2-AR regulation of Na+ and H2O transport in renal (15) and intestinal (16, 17) epithelial cells. Madin-Darby canine kidney (MDCKII) cells cultured in Transwell culture dishes have provided an excellent model system for polarized renal epithelial cells. The localization of the alpha 2A-AR subtype on the basolateral surface of these cells (18) recapitulates the basolateral localization of this receptor in vivo, based on physiological (19) and pharmacological (20) data.

Examination of molecular regions of the three alpha 2-AR subtypes in polarized MDCKII cells indicates that basolateral targeting of these receptors involves sequences in or near the membrane bilayer (18, 21, 22). In contrast, the large third intracellular loop of the receptor appears to play a role in stabilizing the alpha 2A-AR on the plasma membrane, because mutant alpha 2A-ARs that lack 119 amino acids from the large third intracellular loop (Delta 3i loop alpha 2A-AR) have a cell surface half-life (t1/2) of 4.5 h compared with a t1/2 of 10 h for the wild-type alpha 2A-AR (21). The present studies have explored the structural features of the alpha 2A-AR 3i loop responsible for stabilizing the alpha 2A-AR on the plasma membrane of MDCKII cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

Materials-- [gamma -32P]ATP (6000 Ci/mmol), [3H]yohimbine (70-90 Ci/mmol), p-[125I]iodoclonidine (2200 Ci/mmol), 35S-Express protein labeling mix (1200 Ci/mmol), [3H]methoxyinulin (Ci/mmol), and [alpha -35S]dATP (1389Ci/mmol) were purchased from NEN Life Science Products. Phentolamine was kindly provided by CIBA Pharmaceutical Co. 125I-Rau-AzPEC (17-hydroxyl-20-yohimban-16-(N-4-azido-3-[125I]iodophenyl)car-boxamide) was synthesized in our laboratory by the method of Lanier et al. (23). Biotin hydrazide, Sulfo-NHS-biotin, and streptavidin-agarose were purchased from Pierce. The protein A-purified 12CA5 monoclonal antibody was purchased from the Berkeley Antibody Co. Epinephrine was obtained from Sigma, and ascorbic acid was purchased from Fisher.

Cell Culture-- Madin-Darby canine kidney cells (MDCKII) were obtained from Enrique Rodriguez-Boulan (Cornell Univ. New York, NY) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 mg/ml streptomycin (referred to as complete Dulbecco's modified Eagle's medium) at 37 °C, 5% CO2. For polarity experiments, MDCK II cells were seeded at a density of 1 × 106 cells/24.5- mm polycarbonate membrane filter (Transwell chambers, 0.4-µm pore size, Costar, Cambridge, MA) and cultured for 5-8 days with a change of medium every 1-2 days. Before each experiment, the integrity of the monolayer was assessed by adding [3H]methoxyinulin to the apical medium and monitoring its leak after a 1-h incubation at 37 °C from the apical to the basolateral compartment by sampling and counting the basolateral medium in a scintillation counter (Packard Tricarb). Chambers with greater than 5% leak/h were not evaluated.

Construction of Mutant alpha 2A-AR Permanently Expressing Cell Lines-- Generation and characterization of Delta 3i loop alpha 2A-AR (Delta aa240-359) has been previously described(21, 24). Briefly, site-directed mutagenesis was used to create a novel NotI restriction enzyme site in the region of the alpha 2A-AR cDNA encoding the C-terminal end of the putative third intracellular (3i) loop. Cleavage of this mutant alpha 2A-AR cDNA with NotI restriction enzymes removes the DNA fragment encoding amino acids 240-359. This Delta 3i loop mutant receptor has 36 amino acids linking transmembrane domains 5 and 6 of the alpha 2A-AR as shown in Fig. 1A.

Oligo-directed mutagenesis in M13 phage was utilized to create incremental deletions of the predicted third intracellular loop of the alpha 2A-AR. Single oligos were designed against sequences flanking those encoding the amino acids selected for each deletion. The deletion mutations were confirmed by dideoxynucleotide sequencing and then subcloned into the pCMV4 mammalian expression vector. Deletions corresponding to DNA encoding the following amino acids were made in this manner: Delta aa252-267, Delta aa268-285, Delta aa286-303, Delta aa315-326, and Delta aa327-340. Fig. 1 provides a schematic diagram of the regions encoded by these deleted amino acids.

The nonsense loop was designed by taking advantage of the method used for making the original Delta aa240-359 (Delta 3i loop alpha 2A-AR). Because two NotI enzyme sites were used to remove the 3i loop, it was possible to subclone this segment of the gene back into the receptor in two orientations. The correct orientation produced a receptor that corresponded to the wild-type alpha 2A-AR sequence except for two point mutations at the site of the engineered NotI site (K359A, S360A). The opposite orientation also produces an open reading frame with a 3i loop of the same amino acid length but with very little sequence homology to the wild-type receptor (See Fig. 4). We refer to this mutant alpha 2A-AR as the nonsense 3i loop alpha 2A-AR.

All mutations were verified using dideoxy-DNA sequencing (Sequenase kit, U. S. Biochemical Corp.) of the single-stranded DNA utilizing T7 DNA polymerase with alpha -35S-dATP. Once verified, the mutant inserts were subcloned from M13 into the pCMV4-TAG-alpha 2A-AR expression vector (18) containing an N-terminal hemagglutinin epitope (YPYDVPDYA) to which antibodies are available commercially (Berkeley Antibody Co.). These plasmid constructs were verified by double-stranded DNA sequencing through the region of the mutation. COS M6 cells were transiently transfected with the plasmid DNA encoding the wild-type and mutant alpha 2A-ARs, and membranes from the transient transfectants were assayed for [3H]yohimbine binding before developing permanent MDCK cell lines expressing these mutant alpha 2A-AR. MDCK cell lines permanently expressing the wild-type or mutant alpha 2A-AR were created as described previously (18) (Table I).

                              
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Table I
Wild-type alpha 2A-AR and mutant alpha 2A-AR-expressing MDCK cell lines utilized in polarization and stability studies

Determination of the Half-life of Wild-type or Mutant alpha 2A-AR on the Basolateral Membrane-- To determine alpha 2A-AR half-life on the basolateral surface, a metabolic labeling strategy was employed. MDCK cells expressing wild-type or mutant alpha 2A-AR were incubated with [35S]Cys/Met ("pulse") and then incubated for various periods of time ("chase") before isolation of basolateral alpha 2A-AR using sequential biotinylation, extraction, immunoisolation, and streptavidin-agarose chromatography. The procedures utilized have been previously described (21) except for the modifications outlined as follows. Specifically, MDCK cells grown in Transwell culture were metabolically labeled with 1 µCi/µl 35S-Express protein labeling mix for 60 min in Cys/Met-free Dulbecco's modified Eagle's medium (18). After labeling, the cells were washed once with Dulbecco's phosphate-buffered saline (dPBS) and incubated for various periods of time (generally 0, 3, 6, and 18 h) at 37 °C, 5% CO2 in chase medium (complete Dulbecco's modified Eagle's medium supplemented with 1 mM cysteine and 1 mM methionine). At the conclusion of each chase period, alpha 2A-ARs residing on the basolateral surface of these cells were isolated by biotinylating the basolateral cell surface with biotin hydrazide or sulfo-NHS biotin and subjecting detergent extracts of membranes prepared from these cells to sequential immunoprecipitation with the 12CA5 anti-hemagglutinin epitope antibody followed by streptavidin-agarose chromatography (21). The streptavidin-agarose eluates were separated by SDS-polyacrylamide gel electrophoresis, and the amount of radiolabeled alpha 2A-AR remaining on the basolateral surface after various durations of chase was determined. The gels were exposed to film, and the gel area corresponding to the radiolabeled alpha 2A-AR was removed and counted (in 10 ml of scintillation Fluor) in a Packard beta counter. Similar-sized gel slices that did not correspond to any radiolabeled protein band were excised and counted to quantify the background 35S signal in each lane; these background counts were subtracted from the counts in the alpha 2A-AR slices to determine the specific alpha 2A-AR cpm at each time point.

Analysis of Ligand Binding to Adrenergic Receptors-- The density of alpha 2A-ARs in membrane preparations of MDCK cells was determined via saturation binding using the alpha 2A-AR antagonist [3H]yohimbine as the radioligand. Membranes were prepared by hypotonic lysis and were resuspended in membrane binding buffer (25 mM glycylglycine, 20 mM HEPES, 100 mM NaCl, and 5 mM EDTA, pH 8.0) such that the specific binding at 7.5 nM [3H]yohimbine in a 250-µl aliquot (12-400 µg of protein/ml) represented approximately 10,000 cpm of specific binding. The density of [3H]yohimbine binding sites was then assessed in a subsequent assay using increasing concentrations of [3H]yohimbine (0.5-15 nM) in a total volume of 250 µl. Binding was performed at 25 °C for 1.5 h and was terminated by the addition of 4.5 ml of ice-cold 25 mM glycylglycine, pH 7.6, followed by vacuum filtration through GF/B glass fiber filters and two washes with 4.5 ml of ice-cold buffer. Nonspecific binding was defined as that binding detectable in the presence of 10 µM phentolamine. The Bmax and KD for [3H]yohimbine binding to the alpha 2A-AR was determined by analysis of saturation binding data using GraphPad Prism software (GraphPad, San Diego, CA).

To assess the effects of agonist on alpha 2A-AR redistribution, MDCKII cells expressing either wild-type or Delta 3i loop alpha 2A-AR were treated for 24 h with 100 µM epinephrine and 100 µM ascorbic acid to prevent oxidation of the epinephrine, as described previously (25). Control cells were treated with 100 µM ascorbic acid alone. Receptor density was determined by [3H]yohimbine saturation binding.

Determining the Triton X-100 Extractability of Wild-type and Delta 3i Loop alpha 2A-AR-- MDCK cells permanently transfected with either wild-type or Delta 3i loop alpha 2A-AR were grown on Transwells for 7 days. Transepithelial leak of [3H]methoxyinulin was determined as described earlier for confirmation of functional polarization. To compare the Triton X-100 extractability of wild-type and Delta 3i loop alpha 2A-AR, the alpha 2A-ARs in intact cells were covalently modified via a radioiodinated photoaffinity label and then extracted with increasing concentrations of Triton X-100 (%v/v). Briefly, cells were washed with dPBS supplemented with 0.5 mM CaCl2 and 1.0 mM MgCl2 (dPBS-CM), and the Transwells were inverted and incubated 1 h at 22 °C in the dark with 150 µl of dPBS-CM containing 0.2 µCi/well (0.9 nM) 125I-Rau-Az-Pec. After 1 h, the wells were washed with dPBS containing 1 mM glutathione, suspended in a Rayonet UV photoilluminator, and photolysed for 3 min with 300-nm light. The alpha 2A-ARs expressed on the basolateral surface were identified by biotinylation with Sulfo-NHS-biotin in triethanolamine buffer for 2 20-min incubations as described above.

To determine the Triton X-100 extractability of alpha 2A-ARs in polarized cells, Transwells with the photolabeled alpha 2A-AR were washed with dPBS and extracted with increasing concentrations of Triton X-100 using a modification of a previously described protocol (26). The polycarbonate filters were first removed from the Transwell support and placed into a 12-well dish with each 24-mm well containing 190 µl of a Triton X-100 extraction buffer (15 mM Tris, pH 8, 120 mM NaCl, 25 mM KCl, 0.1 mM EGTA, 0.5 mM EDTA) with no Triton X-100. The cells on polycarbonate filters were rocked gently for 5 min at 4 °C. The filters were then transferred to a well with extraction buffer containing 0.05% Triton X-100 and rocked 5 min. This procedure was repeated with increasing concentrations of Triton X-100 (0.1, 0.2, 0.5, and 1.0%). After exposure to 1% Triton X-100, the residual cellular material, operationally defined as "Triton shells," was scraped into 200 µl of RIPA buffer. A set of control Transwells were subjected to the same procedure, except that each successive buffer contained no Triton X-100. The final RIPA extraction buffer from each well was transferred to a 0.6-ml Eppendorf tube, and the wells were washed with 200 µl of RIPA buffer. All extracts were brought up to a final volume of 500 µl with RIPA buffer, and biotinylated proteins were isolated using streptavidin-agarose chromatography. After an overnight incubation, the streptavidin beads were eluted with 1× SDS sample buffer at 90 °C for 30 min. This elution was repeated, and the combined eluates were loaded onto a 10% SDS-polyacrylamide gel. The dried gels were exposed to preflashed Kodak film for 3-5 days. The receptor was identified as a radioactive band migrating at a position characteristic of the alpha 2A-AR and whose photoaffinity-labeling was blocked in the presence of 10 µM phentolamine in separate control studies.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

No Small Region in the alpha 2A-AR Third Intracellular Loop Contains All of the Necessary Information for Stabilization of the Receptor on the Cell Surface-- We observed previously that deletion of 119 amino acids from the 3i loop of the alpha 2A-AR (amino acids 240-359) generates a structure (Delta 3i alpha 2A-AR) that has a basolateral t1/2 of ~4.5 h compared with 10-12 h for the wild-type alpha 2A-AR in polarized MDCKII cells. By analogy with the ability of a 21-amino acid insert into the short (D2S) dopamine receptor to create the long dopamine receptor isoform (D2L) and dramatically slow the rate of sequestration (27), the 3i loop of the alpha 2A-AR was examined to determine whether a single small amino acid sequence could account for the stabilization of the receptor.

Five ~20 amino acids deletions were made within the alpha 2A-AR 3i loop, as shown schematically in Fig. 1A. Demarcation of the regions selected for individual deletions was based on secondary structural predictions of Chou and Fasman analysis (52); for example, Delta aa286-303 and Delta aa315-326 are predicted by this analysis to form amphipathic alpha  helices. In addition, the Delta aa286-303 removes the LEESSSS sequence recognized for phosphorylation by G protein-coupled receptor kinases (28, 29). This was of interest because G protein-coupled receptor kinase-mediated phosphorylation of these receptors promotes association with arrestins that have been shown to act as adaptors and recruit some G protein-coupled receptors into clathrin-coated pits (30-32).


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Fig. 1.   No small region of the alpha 2A-AR third intracellular loop can account for the stabilizing properties of the entire 3i loop. A, a schematic diagram of the different deletions evaluated in this study. The Delta aa240-359 structure, termed Delta 3i loop alpha 2A-AR (21), and incremental deletions of the predicted third intracellular loop of the alpha 2A-AR (Delta aa252-267, Delta aa268-285, Delta aa286-303, Delta aa315-326, and Delta aa327-340) were prepared as described under "Experimental Procedures". B, the residence time of each alpha 2A-AR structure on the cell surface was determined by metabolically labeling MDCK cells expressing wild-type (WT) or mutant alpha 2A-AR (pulse) and then incubating those labeled cells for various periods of time (chase) before isolating basolateral alpha 2A-AR using sequential biotinylation, extraction, immunoisolation, and streptavidin-agarose chromatography, as described under "Experimental Procedures." The data shown are from one experiment representative of at least four separate experiments for wild-type, Delta aa286-303, and Delta 3i loop alpha 2A-AR. C, multiple incremental deletions of the alpha 2A-AR 3i loop were compared for the amount of receptor detected on the basolateral surface following a 1-h pulse (100%) versus a 1-h pulse followed by a 6-h chase. The results shown are the mean +S.E. of multiple individual experiments as follows: wild type (n = 9), Delta 3i loop (n = 7), Delta aa286-303 (n = 6), Delta aa327-340 (n = 4), Delta aa252-267 (n = 5), and Delta aa315-326 (n = 4).

The surface stability for each of the alpha 2A-AR structures examined (Fig. 1A) was determined by pulse/chase metabolic labeling strategies and isolation of alpha 2A-AR on the basolateral surface by sequential biotinylation and streptavidin-agarose isolation of detergent-solubilized receptor (see "Experimental Procedures"). As shown in Fig. 1B, Delta aa286-303 alpha 2A-AR does not mimic the accelerated turnover characteristic of the Delta 3i loop alpha 2A-AR. Similar studies were performed for the other deletions shown in Fig. 1A and are summarized in Fig. 1C. Although three of the deletions may affect alpha 2A-AR residence time on the cell surface somewhat, none of the smaller deletions within the 3i loop appear to mimic the accelerated turnover characteristic of the Delta 3i loop alpha 2A-AR. These data suggest that multiple noncontiguous sequences or bulk size of the 3i loop determine the residence time of alpha 2A-AR on the cell surface.

Direct Cytoskeletal Interactions Do Not Appear to Account for Stabilization of the alpha 2A-AR via Its 3i Loop-- One mechanism that might account for stabilization of the alpha 2A-AR on the basolateral surface of polarized renal epithelial cells could be direct interaction of the receptor with the underlying cytoskeleton. In this case, accelerated turnover of the Delta 3i loop alpha 2A-AR could result from loss of these direct cytoskeletal interactions. We utilized differential sensitivity to extraction by Triton X-100 as an indicator of direct and stable association with the cytoskeleton (33-35). This approach has been informative in revealing the association of the polytopic Na+-K+-ATPase (26, 36) and of the single transmembrane-spanning CD44 protein (37) with the cadherin-dependent ankyrin-fodrin matrix underlying the basolateral surface of polarized MDCK cells.

As shown in Fig. 2A, both the wild-type alpha 2A-AR and the Delta 3i alpha 2A-AR are released from polarized MDCKII cells when exposed to 0.2% Triton X-100 in the presence of 2 mM EDTA and 2 mM EGTA (0 mM [Ca2+]o and 0 mM [Mg2+]o). For comparison, proteins directly associated with the cytoskeleton, such as the Na+-K+-ATPase, are not completely extracted by 0.5% Triton X-100 under similar, but slightly more stringent, divalent cation-free extracellular conditions, whereas the alpha 2A-AR is completely extracted (26, 36),2 suggesting that the alpha 2A-AR does not interact directly or stably with the cytoskeleton. When Triton X-100 extractions were performed in the presence of 0.5 mM Ca2+ and 1 mM Mg2+, the amount of Triton X-100 required for extraction of >= 60% of the photoaffinity-labeled surface receptors was increased from 0.2% (Fig. 2A) to 0.5% (Fig. 2B) but with no difference in extraction efficiency between wild-type and mutant alpha 2A-AR. These data suggest that alpha 2A-AR stability on the basolateral surface is influenced by protein-protein interactions that involve a Ca2+ (likely cadherin)-organized substratum, but that these interactions cannot explain the difference in cell surface stability of the wild-type and Delta 3i loop structures, as they are extracted in a comparable manner even in the presence of Ca2+.


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Fig. 2.   Neither the alpha 2A-AR nor the Delta 3i loop alpha 2A-AR remains associated with Triton X-100-resistant structures in polarized MDCKII cells. Polarized MDCK cells grown for 1 week on Transwell filters were radiolabeled in intact cells using 0.2 µCi/well (0.9 nM) 125I-Rau-AzPEC, an alpha 2A-AR-selective photoaffinity label. Proteins present on the basolateral surface, including the alpha 2A-AR, were covalently modified with Sulfo-NHS-biotin as described under "Experimental Procedures." Wild-type or Delta 3i loop alpha 2A-AR were then extracted with increasing concentrations of Triton X-100 by rocking each Transwell in extraction buffers containing varying concentrations of Triton X-100 as indicated. All buffers contained either 2 mM EDTA, 2 mM EGTA (denoted [0 mM Ca2+]o and [0 mM Mg2+]o) (A) or 0.5 mM [Ca2+]o, 1.0 mM [Mg2+]o (B). After exposure to 1% Triton X-100, the residual cellular material, defined as Triton shells, was solubilized with RIPA buffer (Non Ext.). Biotinylated proteins were isolated using streptavidin-agarose chromatography. The alpha 2A-AR in the eluates was resolved on a 10% SDS-polyacrylamide gel. Control experiments indicated that the radioactive band shown corresponds to 125I-Rau-AzPec photoaffinity-labeled alpha 2A-AR, based on its relative migration on 10% gels and the blockade of its labeling by alpha 2A-AR antagonists. The results shown compare wild-type alpha 2A-AR (Tag3 clone at 25 pmol/mg of protein) and Delta 3i loop alpha 2A-AR (T3 at 3.4 pmol/mg of protein (A) or T66B at 2.8 pmol/mg of protein (B)). These data are representative of at least three separate experiments. This extraction profile is not dependent on receptor density because two cell lines with nearly 10-fold different levels of wild-type alpha 2A-AR expression (Tag3 clone at 25 pmol/mg of protein versus T24 clone at 3.4 pmol/mg of protein) were examined with the same results.

Specific Amino Acid Sequences Within the Third Intracellular Loop Do Not Appear to Be Required for Stabilization of the alpha 2A-AR on the Cell Surface-- If bulk size of the 3i loop is sufficient for stabilization of the alpha 2A-AR, then a loop containing the same number of amino acids as the wild-type alpha 2A-AR should manifest the same surface t1/2 regardless of the primary sequence within the 3i loop. To test this hypothesis, we constructed a receptor that contains a nonsense 3i loop corresponding to the exact number of amino acids as in the wild-type alpha 2A-AR 3i loop but with very little sequence homology. The sequences of the wild-type and nonsense alpha 2A-AR 3i loops are compared in Fig. 3. In four experiments using two different clonal cell lines expressing the alpha 2A-AR 3i nonsense loop, the half-life of this structure was indistinguishable from that characteristic of the wild-type receptor as shown in Fig. 3.


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Fig. 3.   Specific amino acid sequences within third intracellular loop do not appear to be required for stabilization of the alpha 2A-AR on the basolateral surface of polarized MDCKII cells. The nonsense 3i loop (NS) alpha 2A-AR was designed as described under "Experimental Procedures," and the residence time of this structure on the basolateral surface was compared with that of wild-type alpha 2A-AR. This mutant alpha 2A-AR contains a 3i loop of the same exact amino acid length but with very little sequence homology to the wild-type receptor; the calculated net charge of the wild-type alpha 2A-AR 3i loop is +1, whereas the calculated net charge of the nonsense loop is +11 because of a decreased number of acidic amino acids. The surface half-life of the wild-type and nonsense 3i loop alpha 2A-AR was determined as in Fig. 1B and described in detail under "Experimental Procedures."

These findings are consistent with a mechanism where the size of the 3i loop structure plays an important role in stability of the alpha 2A-AR on the cell surface. There are examples of membrane proteins localized in surface microdomains by virtue of so-called "corrals," often established by the cytoskeletal proteins underlying the cell surface (38-40). Consequently, alpha 2A-AR surface stability might arise by steric principles, dictated by the size of the 3i loop (Fig. 3). If corrals partitioned the alpha 2A-AR, and lack of corralling were responsible for accelerated turnover of the Delta  3i loop alpha 2A-AR, then we should expect a more rapid lateral diffusion coefficient and a significantly greater mobile fraction for the Delta 3i loop alpha 2A-AR. However, Uhlén et al. (41) have previously reported that the difference in surface half-life between alpha 2A-AR and Delta 3i loop alpha 2A-AR is not paralleled by a difference in lateral diffusion coefficients for each receptor structure, estimated at ~2.2 × 10-10 cm2/s using fluorescence recovery after photobleaching. Thus, the mechanistic significance of the retention of the alpha 2A-AR 3i nonsense loop on the basolateral surface for a duration comparable with the wild-type receptor is unexplained at present.

Sustained Agonist Exposure in MDCKII Cells Does Not Decrease Receptor Density for Either Wild-type or Delta 3i loop alpha 2A-AR-- One mechanism that might account for accelerated surface turnover of the Delta 3i loop alpha 2A-AR would be enhanced agonist-elicited redistribution and subsequent down-regulation of this mutant receptor compared with the wild-type receptor. Consequently, we examined the effect of prolonged agonist exposure on steady-state alpha 2A-AR density in MDCKII cells expressing wild-type or Delta 3i loop alpha 2A-AR. As shown in Fig. 4, treatment of MDCKII cells with 100 µM epinephrine for 24 h results in no detectable down-regulation of either the wild-type or the Delta 3i loop alpha 2A-AR. In fact, there is even a slight increase in receptor density following agonist incubation, perhaps because of ligand-dependent receptor stabilization (42).3 These findings are consistent with previous reports that the alpha 2A-AR subtype does not undergo agonist-induced down-regulation in MDCKII cells (43) and Chinese hamster fibroblast cells (28), although this subtype has been reported to down-regulate in Chinese hamster ovary cells (25, 44, 45). In addition, the alpha 2A-AR subtype, in contrast to the alpha 2B-AR subtype, does not manifest agonist-elicited redistribution (46),4 nor is there any evidence for intracellular localization of the Delta 3i loop alpha 2A-AR in MDCKII cells either by immunocytochemistry (48) or cell surface biotinylation.5 In contrast to our findings and the lack of effect of removal of the 3i loop on agonist-elicited alpha 2A-AR redistribution, several muscarinic receptor subtypes have been shown to undergo agonist-elicited sequestration and down-regulation in a manner influenced by the 3i loop; mutations within the 3i loop reduce sequestration (47, 49-51) and deletion of the 3i loop slows the rate of down-regulation for the m2 receptor (47).


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Fig. 4.   Sustained agonist exposure in MDCKII cells does not alter either wild-type (WT) or Delta 3i loop alpha 2A-AR density. MDCKII cells expressing either wild-type or Delta 3i loop alpha 2A-AR were treated for 24 h with 100 µM epinephrine (+100 µM ascorbic acid to prevent epinephrine oxidation). Control cells were treated with 100 µM ascorbic acid alone. Receptor density was determined by [3H]yohimbine binding as described under "Experimental Procedures." Results are the mean ±S.E. from six separate experiments. Results shown are from MDCKII cells grown to confluence on 60-mm cell culture dishes. However, analysis of MDCKII cells polarized in Transwells gave the same results. No decrease in receptor density was observed; in fact, an increase was noted by analogy with findings in other cultured cell systems3 suggesting that ligand occupancy stabilizes receptor density and affirming that epinephrine remains viable during the course of the incubation.


    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES

The present findings suggest that the retention of the alpha 2A-AR on the basolateral surface of polarized renal epithelial cells involves the 3i loop in its entirety, because smaller deletions within the 3i loop cannot mimic the accelerated turnover characteristic of the mutant Delta 3i loop alpha 2A-AR. Stabilization of the alpha 2A-AR on the basolateral surface does not appear to involve interaction with cytoskeletal proteins, because concentrations of Triton X-100 that do not destabilize direct membrane protein interaction with the cytoskeleton nonetheless extract the wild-type alpha 2A-AR and Delta  3i loop alpha 2A-AR with a similar concentration-response. Deletion of the 3i loop does not accelerate agonist-elicited redistribution and down-regulation of the alpha 2A-AR, suggesting that the 3i loop likely does not prevent alpha 2A-AR interactions with proteins involved in receptor internalization. A mutant alpha 2A-AR containing a 3i loop of similar length but disparate sequence from wild-type alpha 2A-AR (nonsense loop) has a surface half-life in polarized MDCKII cells indistinguishable from wild-type receptors, indicating that the specific amino acid sequences are not necessarily required for basolateral retention and suggesting that the bulk of the 3i loop may be sufficient to stabilize the alpha 2A-AR on the basolateral surface.

    ACKNOWLEDGEMENTS

We thank Jeffrey R. Keefer (Department of Pediatrics, Johns Hopkins University Medical Center, Baltimore, MD) for the design and creation of cDNA constructs with incremental deletions within the alpha 2A-AR 3i loop and for help in producing the MDCKII cell lines expressing these mutant alpha 2A-ARs. We also thank Carol Ann Bonner for assistance with transfection, screening, and maintenance of all cell lines used in these experiments and for synthesis of the 125I-Rau-AzPEC photoaffinity label. The precurser for the 125I-Rau-AzPEC photoaffinity label was a gift from Steve Lanier (Medical University of South Carolina, SC). We appreciate the advice from W. James Nelson (Stanford University, CA) especially regarding the Triton X-100 extraction experiments, and we are grateful to all members of the Limbird Lab for helpful discussions during these experiments.

    FOOTNOTES

* This work was funded by National Institutes of Health Grants DK43879 and HL25182 (to L. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by Pharmacological Sciences Training Grant GM07628 and Molecular Biophysics Training Grant GM08320.

§ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, MRBI 464, Nashville, TN 37232-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: lee.limbird{at}mcmail.vanderbilt.edu.

2 S. W. Edwards and W. J. Nelson, unpublished observations.

3 M. H. Wilson and L. E. Limbird, submitted for publication.

4 N. L. Schramm and L. E. Limbird, submitted for publication.

5 J. R. Keefer, S. W. Edwards, and L. E. Limbird, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: alpha 2A-AR, alpha 2A-adrenergic receptor; MDCKII, Madin-Darby canine kidney II; 3i loop, third intracellular loop; Delta 3i loop alpha 2A-AR, Delta aa240-359 alpha 2A-AR; 125I-Rau-AzPEC, 17-hydroxyl-20-yohimban-16-(N-4-azido-3-[125I]iodophenyl)carboxamide; dPBS, Dulbecco's modified phosphate-buffered saline; RIPA, radioimmune precipitation buffer.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
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