Agonist-regulated Interaction between α2-Adrenergic Receptors and Spinophilin
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m011679200
ISSN1083-351X
AutoresJeremy G. Richman, Ashley E. Brady, Qin Wang, Jennifer L. Hensel, Roger Colbran, Lee E. Limbird,
Tópico(s)Ion channel regulation and function
ResumoPreviously, we demonstrated that the third intracellular (3i) loop of the heptahelical α2A-adrenergic receptor (α2AAR) 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 α2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the α2AAR (Val217-Ala377), α2BAR (Lys210-Trp354), and α2CAR (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 α2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α2AR localization but also to agonist modulation of α2AR signaling. Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α2A-adrenergic receptor (α2AAR) 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 α2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the α2AAR (Val217-Ala377), α2BAR (Lys210-Trp354), and α2CAR (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 α2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α2AR localization but also to agonist modulation of α2AR signaling. α2 adrenergic receptor glutathioneS-transferase Madin-Darby canine kidney polyacrylamide gel electrophoresis protein-phosphatase 1 spinophilin (neurabin II) third intracellular loop mitogen-activated protein polyvinylidene difluoride hemagglutinin Dulbecco's modified Eagle's medium phosphate-buffered saline phenylmethylsulfonyl fluoride reduced glutathione n-dodecyl-β-d-maltoside cholesteryl hemi-succinate cyclohexylamino-1-propane sulfonic acid The three α2-adrenergic receptor (α2AR)1subtypes 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 (1Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (315) Google Scholar, 2Limbird L.E. FASEB. J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar, 3Jinsi A. Paradise J. Deth R.C. Eur. J. Pharmacol. 1996; 302: 183-190Crossref PubMed Scopus (30) Google Scholar, 4Richman J.G. Regan J.W. Am. J. Physiol. 1998; 43: C654-C662Crossref Google Scholar). In heterologous cell systems, these receptors also couple to the activation of a variety of signaling molecules, including Ras (5Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar, 6Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 7Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), p70S6 kinase (8Wilson M. Burt A.R. Milligan G. Anderson N.G. J. Biol. Chem. 1996; 271: 8537-8540Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), MAP kinase (9Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and phospholipase D (11MacNulty E.E. McClue S.J. Carr I.C. Jess T. Wakelam M.J. Milligan G. J. Biol. Chem. 1992; 267: 2149-2156Abstract Full Text PDF PubMed Google Scholar). Although all three α2ARs 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 α2AR redistribution have been noted in several experimental systems (12Von Zastrow M. Daunt D.A. Barsh G. Kobilka B.K. J. Biol. Chem. 1992; 268: 763-766Abstract Full Text PDF Google Scholar, 13Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 15Kurose H. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10093-10099Abstract Full Text PDF PubMed Google Scholar, 16Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 17Jones S. Leone S. Bylund D.B. J. Pharmacol. Exp. Ther. 1990; 254: 294-300PubMed Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar). The α2BAR subtype is readily internalized following agonist activation, whereas the α2AAR subtype typically is not (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar). The α2CAR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar). The α2AR subtypes also manifest different trafficking itineraries in polarized Madin-Darby canine kidney II (MDCKII) cells, even in the absence of agonist treatment. The α2AAR subtype is targeted directly to the basolateral surface (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar), whereas the α2BAR subtype is delivered randomly to both the apical and basolateral surfaces but is selectively retained on the basolateral surface (t12 = 10–12 h) in contrast to its rapid loss from the apical surface (t12 = 5–15 min) (20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These findings suggest that there is a molecular mechanism responsible for the selective retention of the α2BAR on the basolateral sub-domain of MDCK cells, probably a retention mechanism shared by the basolaterally targeted α2A- and α2CAR subtypes (20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although α2CARs, like α2AARs, 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 (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar, 20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar); the functional relevance of this intracellular α2CAR pool has yet to be clarified. Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the α2AR subtypes. For example, deletion of this loop in the α2AAR subtype (Δ3i α2AAR) results in accelerated basolateral receptor turnover (t12 ≅ 4.5 h) when compared with that for the wild-type receptor or with α2AAR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t12 of 10–12 h) (21Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Similarly, the Δ3i α2BAR is not enriched at the basolateral surface of MDCKII cells at steady state (22Saunders C. Limbird L.E. Mol. Pharmacol. 2000; 57: 44-52PubMed Google Scholar). Based on our findings that the α2BAR is rapidly removed from the apical surface following random delivery and that removal of the 3i loops of the α2A- and α2βAR subtypes accelerates surface turnover of these receptors, we hypothesize that α2ARs 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 (23Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 25Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (391) Google Scholar), and that this protein is enriched beneath the basolateral surface of polarized MDCK cells (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In addition, the multiple protein-interacting domains within spinophilin (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) 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 α2AR subtypes and, if so, if these interactions are regulated by agents that modify α2AR function. 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-β-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 α2AR 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 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% CO2as described previously (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar) 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 (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). 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. Rabbit anti-spinophilin antibodies were generated by injection of purified glutathioneS-transferase (GST) fusion proteins (fused to spinophilin amino acids 286–390) as described previously by MacMillan et al. (26MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). 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 0.1m 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 byA280. 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. Polarized MDCKII cells stably expressing the individual α2AR 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 α2AR 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. The residues corresponding to the 3i loops of the α2AAR (amino acids 217–377 (27Guyer C.A. Horstman D.A. Wilson A.L. Clark J.D. Cragoe E.J.J. Limbird L.E. J. Biol. Chem. 1990; 265: 17307-17317Abstract Full Text PDF PubMed Google Scholar)), the α2BAR (amino acids 210–354 (28Zeng D.W. Harrison J.K. D'Angelo D.D. Barber C.M. Tucker A.L. Lu Z.H. Lynch K.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3102-3106Crossref PubMed Scopus (143) Google Scholar)), and the α2CAR (amino acids 248–363 (29Lanier S.M. Downing S. Duzic E. Homcy C.J. J. Biol. Chem. 1991; 266: 10470-10478Abstract Full Text PDF PubMed Google Scholar)) 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 α2AAR 3i loop ((Met)4-α2A3i) 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 thePromega 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 proteins were generated with spinophilin amino acid regions 151–444 and 169–255 and expressed in DH5α. 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 mmisopropyl-β-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. Equimolar concentrations of GST-spinophilin fusion protein were incubated with 300,000 cpm (estimated to represent ∼40 pm) [35S]Met-labeled α2A, α2B, or α2C 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-spinophilinversus GST (controls) was determined by elution of the 3i loop into 1× Laemmli buffer (400 mm Tris, pH 6.8, 700 mm β-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. 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 α2A, α2B, and α2CARs (GenBank™ 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 (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar,20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Full-length spinophilin (GenBank™ 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 α2ARs (e.g. inhibition of adenylyl cyclase or stimulation of MAP kinase) typically is maximal following incubation with 100 μm epinephrine for 2–3 min (9Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 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 inn-dodecyl-β-d-maltoside (DβM)/cholesteryl hemi-succinate (CHS) extraction buffer (4 mg/ml DβM, 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 DβM/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 DβM/CHS buffer. HA-tagged α2AAR 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 DβM/CHS wash buffer (1 mg/ml DβM, 0.2 mg/ml CHS, 20% glycerol, 25 mmglycylglycine, 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 mcyclohexylamino-1-propane sulfonic acid (CAPS), pH 11, 10% methanol), and subjected to 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. Spinophilin is a ubiquitously expressed multidomain protein (25Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (391) Google Scholar) composed of an F-actin binding domain (amino acids 1–153), a PP1 binding/regulatory region (amino acids 427–470 (26MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 31Hsieh-Wilson L.C. Allen P.B. Watanabe T. Nairn A.C. Greengard P. Biochemistry. 1999; 38: 4365-4373Crossref PubMed Scopus (106) Google Scholar, 32Yan Z. Hsieh-Wilson L. Feng J. Tomizawa K. Allen P.B. Fienberg A.A. Nairn A.C. Greengard P. Nat. Neurosci. 1999; 2: 13-17Crossref PubMed Scopus (247) Google Scholar)), a single PDZ binding domain, and a C terminus that possesses a series of coiled-coil domains (see schematic in Fig. 2 A). Satoh et al. (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) showed that spinophilin was localized to the lateral sub-domain in polarized MDCK cells. As shown previously, the α2A-adrenergic receptor also is enriched on the lateral sub-domain of these cells (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) 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 α2AAR (Fig. 1). A rabbit polyclonal antibody was raised against amino acids 286–390 in spinophilin (26MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), a region that has virtually no sequence similarity to spinophilin's structural homolog, neurabin I (Fig.2 A). 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. (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The overlap of expression of the α2AAR 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 α2BAR subtype and spinophilin (data not shown). It should be noted, however, that some spinophilin also is detected intracellularly, including in a sub-apical compartment.Figure 1Laser-scanning confocal microscopy
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