Agonist-regulated Interaction between alpha 2-Adrenergic Receptors and Spinophilin*

Jeremy G. RichmanDagger §, Ashley E. BradyDagger , Qin WangDagger , Jennifer L. HenselDagger , Roger J. Colbran||, and Lee E. LimbirdDagger **

From the Departments of Dagger  Pharmacology and of || Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600

Received for publication, December 26, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha 2A-adrenergic receptor (alpha 2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether alpha 2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha 2AAR (Val217-Ala377), alpha 2BAR (Lys210-Trp354), and alpha 2CAR (Arg248-Val363) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because alpha 2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha 2AR localization but also to agonist modulation of alpha 2AR signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The three alpha 2-adrenergic receptor (alpha 2AR)1 subtypes are members of the type II, biogenic amine-binding, G protein-coupled receptor family. These receptor subtypes all couple via the Gi/Go family of GTP-binding proteins to the inhibition of adenylyl cyclase, inhibition of voltage-dependent calcium channels, potentiation of potassium currents via G protein-coupled, inwardly rectifying potassium channels, activation of phospholipase D, and activation of MAP kinase in native cells (1-4). In heterologous cell systems, these receptors also couple to the activation of a variety of signaling molecules, including Ras (5-7), p70S6 kinase (8), MAP kinase (9, 10), and phospholipase D (11).

Although all three alpha 2ARs appear to activate similar signaling pathways, differences in the cellular trafficking of these subtypes have been reported, both in naive cells and following agonist activation. Subtype-selective differences in agonist-elicited alpha 2AR redistribution have been noted in several experimental systems (12-18). The alpha 2BAR subtype is readily internalized following agonist activation, whereas the alpha 2AAR subtype typically is not (14, 18). The alpha 2CAR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14). The alpha 2AR subtypes also manifest different trafficking itineraries in polarized Madin-Darby canine kidney II (MDCKII) cells, even in the absence of agonist treatment. The alpha 2AAR subtype is targeted directly to the basolateral surface (19), whereas the alpha 2BAR subtype is delivered randomly to both the apical and basolateral surfaces but is selectively retained on the basolateral surface (t1/2 = 10-12 h) in contrast to its rapid loss from the apical surface (t1/2 = 5-15 min) (20). These findings suggest that there is a molecular mechanism responsible for the selective retention of the alpha 2BAR on the basolateral sub-domain of MDCK cells, probably a retention mechanism shared by the basolaterally targeted alpha 2A- and alpha 2CAR subtypes (20). Although alpha 2CARs, like alpha 2AARs, are directly targeted to and retained on the basolateral subdomain, a significant proportion of these receptors is identifiable in an intracellular pool at steady state (14, 18, 20); the functional relevance of this intracellular alpha 2CAR pool has yet to be clarified.

Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the alpha 2AR subtypes. For example, deletion of this loop in the alpha 2AAR subtype (Delta 3i alpha 2AAR) results in accelerated basolateral receptor turnover (t1/2 congruent  4.5 h) when compared with that for the wild-type receptor or with alpha 2AAR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1/2 of 10-12 h) (21). Similarly, the Delta 3i alpha 2BAR is not enriched at the basolateral surface of MDCKII cells at steady state (22).

Based on our findings that the alpha 2BAR is rapidly removed from the apical surface following random delivery and that removal of the 3i loops of the alpha 2A- and alpha 2beta AR subtypes accelerates surface turnover of these receptors, we hypothesize that alpha 2ARs interact, via their 3i loops, with protein(s) enriched beneath the basolateral surface of MDCKII cells to stabilize their steady-state localization. Consequently, we were particularly intrigued by recent findings that the 3i loop of another Gi/Go-coupled G protein-coupled receptor, the D2 dopamine receptor, interacts with spinophilin (23-25), and that this protein is enriched beneath the basolateral surface of polarized MDCK cells (24). In addition, the multiple protein-interacting domains within spinophilin (24) suggest that its interaction with the receptor may facilitate the formation of a signaling complex to modulate signaling or recruitment of other proteins to a functional microdomain. The present studies were undertaken to identify whether spinophilin interacts with the 3i loops of the alpha 2AR subtypes and, if so, if these interactions are regulated by agents that modify alpha 2AR function.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The pGEMEX-2 vector and TnT in vitro translation kit were from Promega (Madison, WI). The [35S]methionine (1000 Ci/mmol, at 10 mCi/ml) was purchased from PerkinElmer Life Sciences (Boston, MA). PVDF nylon membranes were from Millipore (Bedford, MA). The fast protein liquid chromatography and DEAE-Sephacel columns were from Amersham Pharmacia Biotech (Piscataway, NJ). Dodecyl-beta -maltoside and cholesteryl-hemisuccinate were purchased from Calbiochem (San Diego, CA) and Sigma Chemical Co. (St. Louis, MO), respectively. Antibodies against the HA epitope engineered into the alpha 2AR structures was obtained from BABCo (mouse) or from Roche Molecular Biochemicals (rat and mouse). Mouse anti-Myc antibodies were purchased from CLONTECH (Palo Alto, CA). Protein A-agarose was from Vector (Burlingame, CA). Centricon-10 concentrating filters were purchased from Amicon (Beverly, MA). Horseradish peroxidase-labeled anti-mouse and anti-rat antibodies were from Amersham Pharmacia Biotech. Horseradish peroxidase substrate for Western detection was Enhanced Chemiluminescence (ECL, Amersham Pharmacia Biotech). Cy3 and Alexa488 secondary antibodies were from Molecular Probes (Eugene, OR).

MDCKII Cell Culture and Polarization-- MDCKII cells were plated at confluence (~1-2.5 × 105 cells) and grown on 12-mm Transwell filters (0.4-µm pore size, Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma) and 100 units/ml penicillin and 10 µg/ml streptomycin at 37 °C/5% CO2 as described previously (19) except with daily media changes for 5-7 days. Under these conditions, cells form a monolayer and functionally polarize with distinct apical and basolateral surfaces separated by tight junctions. We routinely verify that tight junctions have formed and that the apical and basolateral compartments are functionally separated from one another using the nontransportable molecule [3H]methoxy-inulin (19). For these leak assays, 2 µCi of [3H]methoxy-inulin is added to the apical subcompartment and incubated for 1 h at 37 °C/5% CO2 followed by counting 100 µl of the medium in each of the apical and basolateral subcompartments. Leaks range from 5-10%, and we discard from study any culture wells of >10% leak.

Immunofluorescent Labeling and Confocal Microscopy

Antibody Purification-- Rabbit anti-spinophilin antibodies were generated by injection of purified glutathione S-transferase (GST) fusion proteins (fused to spinophilin amino acids 286-390) as described previously by MacMillan et al. (26). Antibodies were purified from serum by affinity chromatography. Affinity matrices were generated by mixing 2 ml of Affi-Gel-15 and 1 ml of Affi-Gel-10 (Bio-Rad) equilibrated in 0.1 M HEPES, pH 7.0, in a 10 ml of a Poly Prep chromatography column (Bio-Rad). Purified GST-Sp286-390 fusion protein (11.7 mg in 6.5 ml of PBS) was loaded onto the column and incubated with inversion for 4 h at 4 °C. The resin was washed with 1× PBS until free of unbound GST-Sp286-390, as determined by A280. Unbound sites on the Affi-Gel matrix were blocked by incubation with 1 M ethanolamine for 1 h at 4 °C with inversion. The column was equilibrated with 1× PBS (0.05% NaN3) and stored at 4 °C. A GST "subtraction column" was prepared in the same manner, except GST alone was coupled to the Affi-Gel 10/15 mixed matrix.

Serum (2 ml) was added to the GST-Sp286-390 affinity matrix and incubated with rotation for 2 h at room temperature. The column was washed three times with 1× PBS, once with 333 mM NaCl in 1× PBS, and then twice more with 1× PBS. Antibody was eluted twice with 2 ml of 100 mM glycine, pH 2.5, and collected into 200 µl of 1 M Tris-HCl, pH 9.0, to neutralize the sample. Eluted antibody was pooled, concentrated, and exchanged into 1× PBS using an Amicon Stirred Cell with a YM30 filter (Amicon). To remove antibody directed against the GST portion of the GST-spinophilin fusion protein, concentrated antibody was incubated with the GST subtraction column, prepared as described above, by rotation for 30 min at room temperature. The pass-through from this column was collected and concentrated using an Amicon Stirred Cell as described above, and utilized as the anti-Sp286-390 antibody. Antibody concentration was determined to be 1.44 mg/ml by protein assay (Bradford). Optimal working concentrations of antibody in Western and immunolocalization were derived empirically via Western blot analysis and immunofluorescence staining.

Fixation and Immunolabeling-- Polarized MDCKII cells stably expressing the individual alpha 2AR subtypes were grown on Transwells, as described above, and then rinsed once with PBS-CM (phosphate-buffered saline with 1 mM MgCl2 and 0.5 mM CaCl2) and fixed for 15 min with either 100% methanol (MeOH) at -20 °C or with 4% paraformaldehyde at room temperature (~22 °C) followed by quenching with two sequential 7.5-min incubations with 50 mM NH4Cl in PBS-CM. Spinophilin immunolocalization was best observed after MeOH fixation, whereas the alpha 2AR localization ("signal-to-background" ratio) was best visualized following paraformaldehyde fixation and quenching. For colocalization studies, we used MeOH for fixation of the polarized MDCKII cells.

After fixation, cells were rinsed two more times in PBS-CM, permeabilized in 0.2% Triton X-100 added to the cell surface of the excised Transwell for 20 min, and incubated in blocking buffer (0.1% Triton X-100 and 2% bovine serum albumin in PBS-CM) for 1 h. Primary antibody was added to the cell side of excised Transwells and incubated for either 1 h at room temperature or overnight (~15 h) at 4 °C. Mouse 12CA5 anti-HA antibodies were diluted at 1:250 (4 µg/ml), and rabbit anti-spinophilin 286-390 antibodies were used at a dilution of 1:100 (~10 µg/ml). MDCKII cells were washed three times for 15 min in PBS-CM at 22 °C before adding secondary antibodies. The secondary antibodies were Alexa488- or Cy3-conjugated anti-rabbit or anti-mouse antibodies, diluted 1:1000 (2 µg/ml) and were incubated with the cells for 1 h at room temperature. Cells were again rinsed three times for 15 min in PBS-CM and mounted cell-side-up onto a glass slide with Aqua-Polymount and sealed under a glass coverslip. Images were visualized on a Zeiss LSM 410, laser-scanning, confocal microscope in the Vanderbilt Cell Imaging Core Facility. Images were taken through a 40× oil objective at 1.5× magnification.

Generating [35S]Met-labeled alpha 2AR 3i Loops as Ligands

The residues corresponding to the 3i loops of the alpha 2AAR (amino acids 217-377 (27)), the alpha 2BAR (amino acids 210-354 (28)), and the alpha 2CAR (amino acids 248-363 (29)) were subcloned into the pGEMEX2 vector in-frame within the polylinker located downstream of the sequence encoding the methionine-rich viral coat protein Gene 10 (30). Alternatively, constructs were generated in which four methionines were inserted via polymerase chain reaction into the N-terminal region of the alpha 2AAR 3i loop ((Met)4-alpha 2A3i) and subcloned into the pGEMEX2 vector. All DNA constructs were verified by sequencing.

The Gen10-3i loop fusion proteins and (Met)4-3i loops were transcribed, translated, and [35S]Met-labeled using the Promega transcription and translation-coupled (TnT) rabbit reticulocyte lysate kit, as follows: 25 µl of TnT reticulocyte lysate was added to 1 µl of amino acid mix (1 mM, minus methionine), 2 µl of reaction buffer, 1 µl of TnT T7 RNA polymerase, 4 µl of [35S]methionine (1000 Ci/mmol, at 10 mCi/ml), and 1 µl of RNasin ribonuclease inhibitor (40 units/µl). Then, 1 µg of the appropriate plasmid DNA template was added, and the volume was adjusted to 50 µl with nuclease-free water. The mixture was incubated for 90 min at 30 °C. Following each synthesis, products were analyzed and quantitated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The band representing each probe was cut out of the dried gel and counted in scintillation mixture. GST-pull-down assays were performed such that each incubation contained an equivalent amount of [35S]Met-labeled 3i loop as radioligand.

GST-spinophilin Fusion Protein Generation

GST-spinophilin fusion proteins were generated with spinophilin amino acid regions 151-444 and 169-255 and expressed in DH5alpha . Bacteria were grown at 37 °C to an A600 of 0.6. GST or GST fusion protein expression was initiated with the addition of 1 mM isopropyl-beta -D-thiogalactopyranoside and allowed to proceed for 2-6 h at 37 °C. Bacteria were collected by centrifugation at 10,000 × g and then lysed in 50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 1 mg/ml lysozyme, 200 mM NaCl, 100 µM PMSF, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin (TT+ buffer) by one freeze-thaw cycle followed by probe sonication for three 30-s bursts on ice. GSH-agarose (1 ml of a 1:1 slurry equilibrated in TT+ buffer) was added to the supernatant of a 13,000 × g centrifugation and incubated for 1 h at 4 °C with inversion. This solution was transferred to a 0.8- × 4-cm Poly-Prep column (Bio-Rad) and washed with 12 ml of TT+ buffer, 3 ml of 333 mM NaCl in TT+ buffer, and then with 6 ml of TT+ buffer. GST or GST fusion protein was eluted from the GSH-agarose by adding 3 ml of 10 mM free acid GSH in TT+, pH 7.5. Eluted protein was concentrated and exchanged into PBS buffer using an Amicon Stirred Cell.

Binding of 3i Loops to GST-spinophilin

Equimolar concentrations of GST-spinophilin fusion protein were incubated with 300,000 cpm (estimated to represent ~40 pM) [35S]Met-labeled alpha 2A, alpha 2B, or alpha 2C 3i loop ligand (see above). GSH-agarose (1:1 slurry equilibrated with TT+ buffer) was then added to this incubation, rotated for 2 h at 4 °C, and the resin collected by centrifugation. The resin was then exposed to four 1-ml TT+ washes. Interaction with GST-spinophilin versus GST (controls) was determined by elution of the 3i loop into 1× Laemmli buffer (400 mM Tris, pH 6.8, 700 mM beta -mercaptoethanol, 1% SDS, 10% glycerol) and separation of the eluates by 12% SDS-PAGE. The degree of interaction was quantitated by cutting and counting the bands corresponding to 3i loop (determined via autoradiography) in scintillation mixture.

Detergent Extraction and Coimmunoprecipitation of Full-length HA-tagged alpha 2AR Subtypes with Full-length Myc-tagged Spinophilin

CosM6 cells were plated at 1.75 × 106 cells on 10-cm plates and maintained in DMEM supplemented with 10% fetal bovine serum and 100 units/ml penicillin and 10 µg/ml streptomycin at 37 °C/5% CO2. The following day, cells (at ~60-80% confluence) were transfected using FuGENE 6 reagent (Roche Molecular Biochemicals), according to the manufacturer's specifications, with an empirically optimized ratio of 3 µl FuGENE 6 reagent/1 µg of plasmid DNA. The alpha 2A, alpha 2B, and alpha 2CARs (GenBankTM accession numbers A38316, X74400, and X57659, respectively) were encoded in pCMV4 and tagged at their 5'-end after the start ATG codon with the sequence corresponding to the hemagglutinin tag (HA; YPYDVPDYA), as described previously (19, 20). Full-length spinophilin (GenBankTM accession AF016252) was expressed in pCMV4, and epitope-tagged with a Myc sequence inserted 5' after the start ATG codon (Myc; QKLISEEDLLRKR).

Medium was changed 24 h after the FuGENE transfection. Approximately 48 h after transfection, cells were rinsed in serum-free DMEM and incubated with 100 µM epinephrine, or not (control), for 3 min at 37 °C. Regulation of signaling pathways by alpha 2ARs (e.g. inhibition of adenylyl cyclase or stimulation of MAP kinase) typically is maximal following incubation with 100 µM epinephrine for 2-3 min (9). Cells were then rinsed twice in cold (4 °C) PBS-CM and extracted into lysis buffer (15 mM HEPES, 5 mM EDTA, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin, and 100 µM PMSF). Membranes were collected by centrifugation at 12,000 × g at 4 °C for 30 min and solubilized in n-dodecyl-beta -D-maltoside (Dbeta M)/cholesteryl hemi-succinate (CHS) extraction buffer (4 mg/ml Dbeta M, 0.8 mg/ml CHS, 20% glycerol, 25 mM glycylglycine, pH 7.6, 20 mM HEPES, pH 7.6, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin, and 100 µM PMSF) by five passes through a 25-gauge needle followed by ten passes in a glass/Teflon homogenizer. The supernatant of a 100,000 × g centrifugation for 1 h at 4 °C was defined as the Dbeta M/CHS-solubilized preparation. A 0.75-ml aliquot of this preparation was "precleared" by a 15-min incubation with 30 µl of protein A-agarose equilibrated with Dbeta M/CHS buffer. HA-tagged alpha 2AAR or Myc-tagged spinophilin was then immunoprecipitated following the addition of rat anti-HA monoclonal antibody or mouse anti-Myc monoclonal antibody, respectively, each at a 1:100 dilution, and incubation at 4 °C for 1 h. The immune complex was isolated by centrifugation following a 1-h adsorption to protein A-agarose; nonspecifically adsorbed proteins were removed by washing the protein A resin three times in Dbeta M/CHS wash buffer (1 mg/ml Dbeta M, 0.2 mg/ml CHS, 20% glycerol, 25 mM glycylglycine, pH 7.6, 20 mM HEPES, pH 7.6, 100 mM NaCl, 5 mM EGTA, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 units/ml aprotinin, and 100 µM PMSF) and centrifugation at 4 °C. Proteins were eluted with the addition of 1× Laemmli buffer and heating to 70 °C for 5 min. Eluates were separated via 10% SDS-PAGE, transferred to an Immobilon P membrane (PVDF; Millipore) with a constant current of 1 amp for 72 min in CAPS transfer buffer (1 M cyclohexylamino-1-propane sulfonic acid (CAPS), pH 11, 10% methanol), and subjected to Western blot analysis.

Western Blot Analysis

PVDF membranes were blocked for 15 min in Tris-buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1% Tween 20 (TBST) and 5% Carnation Instant powdered milk (w/v). The appropriate primary antibody was then added at a dilution of 1:1000 (Rat anti-HA) or 1:2000 (mouse anti-Myc monoclonal antibody) in blocking buffer and incubated at room temperature for 1.5-2 h. Blots were washed three times for 15 min with TBST and exposed to horseradish peroxidase-conjugated anti-rat or anti-mouse secondary antibodies, as appropriate, at a 1:2000 dilution in blocking buffer for 45 min at room temperature. Blots were washed again three times for 15 min in TBST, incubated with ECL Western blotting detection reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 1.5 min, and then exposed to x-ray film for variable times ranging from 5 s to 30 min.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Coimmunolocalization of Endogenous Spinophilin with the alpha 2AR Subtypes in MDCKII Cells-- Spinophilin is a ubiquitously expressed multidomain protein (25) composed of an F-actin binding domain (amino acids 1-153), a PP1 binding/regulatory region (amino acids 427-470 (26, 31, 32)), a single PDZ binding domain, and a C terminus that possesses a series of coiled-coil domains (see schematic in Fig. 2A). Satoh et al. (24) showed that spinophilin was localized to the lateral sub-domain in polarized MDCK cells. As shown previously, the alpha 2A-adrenergic receptor also is enriched on the lateral sub-domain of these cells (19, 20) and is revealed here using a Cy3 (red signal)-conjugated secondary antibody directed against the 12CA5 antibody that recognizes the N-terminal HA epitope in the alpha 2AAR (Fig. 1). A rabbit polyclonal antibody was raised against amino acids 286-390 in spinophilin (26), a region that has virtually no sequence similarity to spinophilin's structural homolog, neurabin I (Fig. 2A). As shown in Fig. 1, the affinity-purified polyclonal antibody against spinophilin, visualized here via Alexa488 (green signal)-conjugated secondary antibody, reveals considerable enrichment of endogenous spinophilin at the lateral surface of these polarized cells, corroborating initial reports of Satoh et al. (24). The overlap of expression of the alpha 2AAR and spinophilin in the lateral domain of MDCKII cells is demonstrated by the considerable amount of yellow signal present in the red/green overlay. Similar results were observed upon colocalization of the alpha 2BAR subtype and spinophilin (data not shown). It should be noted, however, that some spinophilin also is detected intracellularly, including in a sub-apical compartment.


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Fig. 1.   Laser-scanning confocal microscopy reveals colocalization of alpha 2AARs with endogenous spinophilin in polarized MDCKII epithelial cells. MDCKII cells stably expressing HA-tagged alpha 2AARs were grown and polarized on 12-mm Transwells for 6-8 days. The immunolocalization of the receptor and spinophilin was performed as described under "Experimental Procedures." Secondary antibodies were Cy3-conjugated donkey anti-mouse (red) and Alexa488-conjugated goat anti-rabbit (green). The presence of yellow in the overlay image indicates colocalization of the fluorescent signal. Images were taken through a 40× oil objective (NA = 1.4) via a Leica-TCS laser-scanning confocal microscope at 1.5× magnification in both the XY (top panels) and Z planes (lower panels corresponding to the blue line), as shown in the schematic diagram to the left of the confocal images.


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Fig. 2.   The 3i loops of all three of the alpha 2AR subtypes interact with amino acids 169-255 in spinophilin. A, schematic diagram of the predicted spinophilin domain structure and of GST fusion proteins that were used in pull-down assays. Neurabin is a closely related protein to spinophilin (51% identical and 74% functionally similar at the amino acid level) with its region of least homology being within the region from amino acids 151-444 of spinophilin, especially 286-390 (the region against which the anti-spinophilin antibody used in these studies was developed (25)). B, GST fusion protein pull-down assays were performed as described under "Experimental Procedures." The amount of 35S-labeled Gen10-3i loop fusion protein retained in the GSH-agarose-GST fusion protein pellet was visualized by autoradiography and quantitated via scintillation counting. C, the alpha 2AAR-binding domain of spinophilin can be further refined to Sp169-255; virtually no interaction with Neurabin 146-453 can be observed. For these assays, the radiolabeled probe was [35S]Met-(Met)4-alpha 2A 3i loop GST-Nb146-453. The "input" lane reflects 1/15 of the amount of [35S]Met-(Met)4-alpha 2A 3i loop added to each of the GST fusion protein binding incubations.

The 3i Loops of All Three alpha 2AR Subtypes Interact with Spinophilin-- Smith et al. (23) have demonstrated, via yeast two-hybrid screens and gel overlay strategies, that the 3i loops of the D2 dopamine receptor (short and long forms) interact with spinophilin in the region between the F-actin binding and PP1 domains (Fig. 2A). Consequently, we created GST fusion proteins of spinophilin bounded between amino acids 151 and 444 (GST-Sp151-444).

As shown in Fig. 2B, GST pull-down assays revealed that the 3i loop of each of the alpha 2AR subtypes is able to interact with GST-Sp151-444. Radiolabeled [35S]Met- Gen10 alpha 2AR 3i loops specifically interacted with GST-Sp151-444 but not with GST alone. To further refine the interacting regions of spinophilin, the 3i loop of the alpha 2AAR was incubated with a fusion protein of GST-spinophilin amino acids 169-255 (Fig. 2C). This region was selected because it represents the region of least homology with the spinophilin-related protein, neurabin I, a brain-specific protein (see Fig. 2A (33)). However, because there is a stretch of 14 amino acids identical between these protein family members within this region, it was of considerable importance to evaluate the ability of the corresponding region of neurabin I (Nb146-453) to interact with the alpha 2AAR 3i loop. As shown in Fig. 2C, little or no interaction was detected between the alpha 2AAR 3i loop and GST-neurabin 146-453 when compared with GST-spinophilin 151-444. Furthermore, binding to the 3i loop is demonstrated by the more restricted region of Sp169-255; in fact, binding of the alpha 2A-3i loop to this region is more readily detected to this region, than to GST-Sp151-444.

Interaction of the alpha 2AAR and Spinophilin within the Cell Is Regulated by Agonist-- It was of interest to determine whether or not these alpha 2AAR-3i loop-spinophilin interactions, detected in vitro via GST fusion protein assays, could be detected in the context of a living cell. For these studies, we transiently coexpressed cDNAs encoding full-length HA-tagged alpha 2AAR and Myc-tagged spinophilin in CosM6 cells. On the day of analysis, cells were incubated with or without 100 µM epinephrine prior to extraction of the cell membranes and solubilization with Dbeta M/CHS, a detergent that extracts receptor in a functional conformation (30). As shown in Fig. 3, immunoisolation of Myc-tagged spinophilin leads to the coisolation of the HA-alpha 2AAR in cells expressing both receptor and spinophilin (lanes 2 and 3; lane 1 is an extract from CosM6 cells expressing only the HA-tagged alpha 2AAR). HA-tagged alpha 2AAR also has been coimmunoprecipitated from cells transfected with only HA-alpha 2AAR using anti-spinophilin 286-390 antibodies, indicating that it does not require the overexpression of Myc-spinophilin to detect an interaction with alpha 2AARs (data not shown). Of particular interest, however, is the ability of the alpha 2AAR agonist, epinephrine, to increase the amount of alpha 2AAR seen in association with spinophilin, suggesting that regulated interactions with spinophilin could contribute both to receptor localization and coordination of signal transduction events mediated by alpha 2ARs.


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Fig. 3.   The HA-tagged alpha 2AAR coimmunoprecipitates with Myc-tagged spinophilin in an agonist-regulated fashion. A, CosM6 cells were transiently cotransfected with the HA-alpha 2AAR and Myc-spinophilin (lanes 2 and 3) or with the HA-alpha 2AAR alone (lane 1). Cells were treated with vehicle (-) or 100 µM Epi (+) as indicated. Lysates were prepared and precleared as described under "Experimental Procedures." Top, mouse anti-Myc antibody adsorbed to protein A-agarose was used to immunoprecipitate Myc-spinophilin. Resulting precipitates were separated via SDS-PAGE, transferred to a PVDF membrane, and blotted with rat anti-HA antibody. Bottom, the blot was stripped and reprobed with the rabbit anti-spinophilin amino acids 286-390 antibody. B, histogram representing the -fold increase over basal levels of coimmunoprecipitated receptor detected via Western blot following epinephrine treatment. *, two-tailed p < 0.005 determined using a paired t test comparing differences in band densities before and after epinephrine treatment (n = 4 independent experiments with single or duplicate immunoprecipitates).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our studies have demonstrated an interaction between the third intracellular loops of the alpha 2AR subtypes and spinophilin and significantly extend previous findings of in vitro studies demonstrating that the 3i loops of the D2 dopamine receptor short and long isoforms interact with spinophilin fusion proteins (23). Furthermore, our studies have refined this interaction to amino acids 169-255 of spinophilin (neurabin II), a region that shares little homology with what is otherwise a very homologous protein, neurabin I. Finally, our studies are the first to document G protein-coupled receptor-spinophilin interactions in the context of a native cell and to demonstrate that these interactions are fostered by agonist binding to the receptor.

A variety of novel interactions are being reported for G protein-coupled receptors via their C terminus (34-40) or 3i loops (30, 40, 41). In some cases, the interactions are fostered by agonist occupancy of the receptor such as for interaction of the beta 2AR with NHERF/EBP50 (35) or the somatostatin receptor with cortactin binding protein 1 (38). In some cases, interactions appear to affect receptor signaling (37, 39, 42), whereas in others, the interactions may be critical for receptor trafficking (43).

Interactions between alpha 2AARs and spinophilin in the cell should be considered in the context of interactions between the 3i loop of alpha 2ARs and other protein partners. Regions of the third intracellular loop of the alpha 2AAR have been shown to interact with 14-3-3zeta (30), beta -arrestin (41) and heterotrimeric G proteins (44). The alpha 2B- and alpha 2CAR 3i loops also have been demonstrated to interact with 14-3-3zeta (30). These interactions with 14-3-3zeta are competed for by a phosphorylated peptide of raf that blocks raf-14-3-3 interactions (45), suggesting that receptor activation of downstream signaling pathways, such as the raf-ras cascade, might disrupt pre-existing alpha 2AR-14-3-3 interactions, or vice versa. Interactions between alpha 2AAR and beta -arrestin are expected to occur following agonist-evoked G protein-coupled receptor kinase-mediated alpha 2AR phosphorylation (10). The 3i loop sequence employed for the alpha 2AAR in these studies includes regions that have been proposed to contribute to interactions with heterotrimeric G proteins (44), but these amphipathic helical sequences are not present in the amino acids encoded by the alpha 2BAR and alpha 2CAR 3i loop ligands. The ability of all three 3i loop ligands to interact with spinophilin comparably (e.g. Fig. 2) suggests that the alpha 2AR-spinophilin interactions can occur independent of interactions with G proteins. It also is probable that alpha 2AR-spinophilin interactions do not prevent interactions with G proteins, because agonist occupancy of the alpha 2AAR increases the amount of alpha 2AAR that coimmunoprecipitates with spinophilin. Because agonist occupancy of alpha 2AAR also favors receptor interactions with G proteins (46), it is likely that the alpha 2AAR can interact simultaneously with spinophilin and its cognate Gi protein. What remains to be established is whether this agonist-modulated interaction with spinophilin regulates acute or tonic receptor-mediated signaling, by analogy with findings for the D1 dopamine receptor (39, 47, 48) or mediates retention at the basolateral surface of polarized epithelial cells (Fig. 1), previously demonstrated to require the 3i loop of the AR subtypes alpha 2A (21) and alpha 2B (22).

    ACKNOWLEDGEMENTS

We are grateful to David M. Lovinger for his critical input and to Carol Ann Bonner for her superb technical support. We also are grateful for the exchange of reagents and data with Donelson Smith and Sharon Milgram, University of South Carolina and Chapel Hill, especially in early phases of these studies. We appreciate the shared enthusiasm of the other members of the Limbird laboratory.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK43879 (to L. E. L.) and NS37508 (to R. J. C.).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.

§ Funded by Postdoctoral Training Grant T32DK07563 during part of these studies.

Funded by an advanced pre-doctoral Fellowship in Pharmacology and Toxicology from the Pharmaceutical Research and Manufacturers Assn. Foundation. Confocal microscopy was performed in the Vanderbilt University Medical Center Cell Imaging Core Resource Facility (supported by NIH Grants CA68485 and DK20593).

** To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Rm. 464, Nashville, TN 37232-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: Lee. Limbird@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M011679200

    ABBREVIATIONS

The abbreviations used are: alpha 2AR, alpha 2 adrenergic receptor; GST, glutathione S-transferase; MDCK, Madin-Darby canine kidney; PAGE, polyacrylamide gel electrophoresis; PP1, protein-phosphatase 1; Sp, spinophilin (neurabin II); 3i loop, third intracellular loop; MAP, mitogen-activated protein; PVDF, polyvinylidene difluoride; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; GSH, reduced glutathione; Dbeta M, n-dodecyl-beta -D-maltoside; CHS, cholesteryl hemi-succinate; CAPS, cyclohexylamino-1-propane sulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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