Spinophilin Stabilizes Cell Surface Expression of α2B-Adrenergic Receptors
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304195200
ISSN1083-351X
AutoresAshley E. Brady, Qin Wang, Roger Colbran, Patrick B. Allen, Paul Greengard, Lee E. Limbird,
Tópico(s)Ion channel regulation and function
ResumoThe third intracellular (3i) loops of the α2A- and α2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the α2A, α2B, and α2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three α2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that α2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the α2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t ½ ≤ 60 min) apical versus slower (t ½ = 10–12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the α2-AR-interacting region (Sp151–483) by fusion with apically targeted p75NTR extended the half-life of α2B-AR at the apical surface to ∼3.6 h and eliminated the rapid phase (0–60 min) of α2B-AR turnover on that surface. Furthermore, we examined α2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp–/–) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-α2B-AR was accelerated in mouse embryo fibroblasts derived from Sp–/– mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of α2B-AR and presumably all three α2-AR subtypes at the surface of target cells and may act as a scaffold that could link α2-ARs to proteins interacting with spinophilin via other domains. The third intracellular (3i) loops of the α2A- and α2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the α2A, α2B, and α2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three α2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that α2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the α2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t ½ ≤ 60 min) apical versus slower (t ½ = 10–12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the α2-AR-interacting region (Sp151–483) by fusion with apically targeted p75NTR extended the half-life of α2B-AR at the apical surface to ∼3.6 h and eliminated the rapid phase (0–60 min) of α2B-AR turnover on that surface. Furthermore, we examined α2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp–/–) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-α2B-AR was accelerated in mouse embryo fibroblasts derived from Sp–/– mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of α2B-AR and presumably all three α2-AR subtypes at the surface of target cells and may act as a scaffold that could link α2-ARs to proteins interacting with spinophilin via other domains. α2-Adrenergic receptors (ARs) 1The abbreviations used are: AR, adrenergic receptor; 3i loop, third intracellular loop; BSA, bovine serum albumin; CHS, cholesteryl hemisuccinate; DβM, dodecyl-β-d-maltoside; DPBS/CM, Dulbecco's phosphate-buffered saline supplemented with 1 mm MgCl2 and 0.5 mm CaCl2; DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MEF, mouse embryo fibroblast; MDCKII, Madin Darby canine kidney II; p75NTR, p75 neurotrophin receptor; PP1, protein phosphatase 1; RIPA, radioimmune precipitation buffer; Sp, spinophilin; ELISA, enzyme-linked immunosorbent assay; MESNA, 2-mercaptoethanesulfonic acid. are members of the large superfamily of G protein-coupled receptors that contain seven putative transmembrane spanning regions. There are three α2-AR subtypes (α2A, α2B, and α2C), each of which is activated by the endogenous catecholamines, epinephrine and norepinephrine, and performs multiple physiological functions via pertussis toxin-sensitive Gi/Go proteins (1Limbird L.E. FASEB J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar). Cellular signaling pathways regulated by α2A-AR in native cells include inhibition of adenylyl cyclase, activation of receptor-operated K+ channels, inhibition of voltage-gated Ca2+ channels, and activation of the mitogen-activated protein kinase cascade (1Limbird L.E. FASEB J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar, 2Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (315) Google Scholar, 3Richman J.G. Regan J.W. Am. J. Physiol. 1998; 274: C654-C662Crossref PubMed Google Scholar). Many cells that express α2-ARs are polarized, including renal and intestinal epithelia, where the α2-AR serves to regulate sodium and water resorption (4Summers R.J. Fed. Proc. 1984; 43: 2917-2922PubMed Google Scholar, 5Clarke J.D. Cragoe Jr., E.J. Limbird L.E. Am. J. Physiol. 1990; 259: F977-F985PubMed Google Scholar), as well as neurons, where these receptors act to suppress neurotransmitter release (6Ruffolo Jr., R.R. Hieble J.P. Pharmacol. Ther. 1994; 61: 1-64Crossref PubMed Scopus (130) Google Scholar). The physiological functions mediated by α2-ARs in polarized cells are dependent upon precise localization of the receptor at the basolateral surface to gain access to neurally delivered and blood-delivered catecholamines. The α2-AR subtypes demonstrate unique targeting and retention profiles in polarized renal epithelial Madin-Darby canine kidney (MDCKII) cells in culture (7Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Previous work in our laboratory has shown that the α2A-AR subtype is directly targeted to the basolateral surface, where it exhibits a half-life of 10–12 h (8Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Direct and exclusive basolateral targeting of α2A-AR was found to be dependent upon several noncontiguous regions within or near the bilayer, whereas retention of the receptor at the basolateral surface appears dependent upon the third intracellular (3i) loop (8Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Deletion of the 3i loop results in accelerated surface turnover (t ½ = ∼4.5 h) of the α2A-AR at the basolateral surface (9Edwards S.W. Limbird L.E. J. Biol. Chem. 1999; 274: 16331-16336Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Unlike the α2A-AR, the α2B-AR subtype is randomly targeted to both the apical and basolateral subdomains and then selectively retained at the basolateral surface of polarized MDCKII cells, where the receptor has a half-life comparable with that of the α2A-AR subtype (t ½ = ∼10–12 h) (7Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Like for the α2A-AR, the 3i loop of the α2B-AR also is critical for basolateral surface stabilization of this subtype (10Saunders C. Limbird L.E. Mol. Pharmacol. 2000; 57: 44-52PubMed Google Scholar). In contrast to stable retention of the α2B-AR on the basolateral surface, the half-life on the apical surface is estimated to be dramatically shorter, on the order of minutes (7Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Taken together, these data suggest that the stabilization/retention of α2A- and α2B-AR at specific membrane domains is most likely mediated through interactions of the 3i loop with other proteins either within or underlying the basolateral membrane surface and not present (or expressed at much lower density) at the apical surface. The α2-AR 3i loop has been used as a ligand to identify potential interacting proteins and has led to the identification of two α2-AR-interacting molecules: 14-3-3ζ (11Prezeau L. Richman J.G. Edwards S.W. Limbird L.E. J. Biol. Chem. 1999; 274: 13462-13469Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and spinophilin (12Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 276: 15003-15008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Spinophilin is an 817-amino acid, ubiquitously expressed, multidomain-containing protein with an apparent molecular mass on SDS-PAGE of ∼130 kDa. It was originally identified both as a protein phosphatase 1 (PP1)-binding protein localized to dendritic spines, hence the name spinophilin (13Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (391) Google Scholar), as well as an F-actin-binding protein (14Satoh 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). Spinophilin (also known as neurabin II) is highly homologous to the brain-specific protein, neurabin I (14Satoh 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 to the domains described above, spinophilin contains a single PDZ (PSD-95, Discs large, ZO-1) domain and three coiled-coil domains at the C terminus, the latter of which mediate homo-multimerization in vitro (14Satoh 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) and may allow for the formation of multiprotein complexes in intact cells. Spinophilin previously was identified as a D2 dopamine receptor-interacting protein using the 3i loop of the D2 dopamine receptor as bait in a yeast two-hybrid screen (15Smith 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). The D2 dopamine receptor-binding domain in spinophilin (residues 151–444), located between the F-actin-binding domain and the PP1 regulatory domain, also interacts with all three of the α2-AR subtypes (12Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 276: 15003-15008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and will be referred to as the receptor-interacting domain. Because reports in the literature, as well as our own observations, indicate that spinophilin is specifically enriched at the basolateral surface of polarized epithelial cells (12Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 276: 15003-15008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Smith 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, 16Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (163) Google Scholar), we postulate that spinophilin may be involved in tethering and/or stabilizing the receptor at the cell surface via interactions with the α2-AR 3i loops. The present studies utilized two different biological systems to explore the role of spinophilin in α2-AR stabilization at the cell surface. First, the unique targeting profile of the α2B-AR subtype in polarized MDCKII cells (random delivery with rapid turnover at the apical surface) was exploited to determine whether redirection of the receptor-interacting domain of spinophilin to the apical surface of polarized MDCKII cells would result in enhanced apical retention of randomly delivered α2B-AR. Second, the role of spinophilin in α2-AR surface turnover was addressed by studying the internalization profile of the α2B-AR in mouse embryonic fibroblasts (MEFs) derived from wild type (Sp+/+) or spinophilin knock-out (Sp–/–) mice (17Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9287-9292Crossref PubMed Scopus (324) Google Scholar). The findings from both lines of investigation implicate spinophilin in the stabilization of the α2B-AR at the cell surface. Transwell culture chambers (0.4-μm pore size) were purchased from Costar (Cambridge, MA). Doxycycline hydrochloride was purchased from Sigma. NHS-SS-Biotin and Immunopure Immobilized Streptavidin were purchased from Pierce. Both [35S]EasyTag™ Express Protein Labeling mix (1200 Ci/mmol) and [3H]methoxy-inulin (126.5 mCi/g) were from PerkinElmer Life Sciences. Cysteine- and methionine-free DMEM was from Cellgro Mediatech. Dulbecco's modified Eagle's medium was prepared by the Cell Culture Core, a facility sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical Center. Fetal calf serum was purchased from Sigma. Mouse monoclonal 12CA5 antibody against the HA epitope was obtained from BABCo. Affinity matrix-coupled high affinity rat monoclonal anti-HA antibody (clone 3F10), rat monoclonal anti-HA antibody (clone 3F10), and mouse monoclonal HA.11 antibody (clone 16B12) were purchased from Roche Applied Science. Mouse monoclonal anti-c-Myc (clone 9E10) ascites was purchased from Covance Research Products Inc. (Denver, PA). Both the mouse anti-gp135 and mouse anti-EGFR were gifts from Peter J. Dempsey (Department of Pathology, University of Washington, Harborview Medical Center, Seattle, WA). Rabbit anti-spinophilin antibody raised against spinophilin amino acids 286–390 (18Macmillan 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) was purified in our lab (for details see Ref. 12Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 276: 15003-15008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Alexa Fluor 488-conjugated fluorescent goat anti-mouse, goat anti-rat, and goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). Cy3-conjugated donkey anti-mouse IgG was purchased from Jackson Immunochemicals. Sheep anti-mouse, donkey anti-rabbit, and goat anti-rat horseradish peroxidase-conjugated IgG were purchased from Amersham Biosciences. The rat p75NTR cDNA was a generous gift from Dr. Bruce Carter (Department of Biochemistry, Vanderbilt University). The retroviral vector pBabe-HA-α2B-AR was kindly provided by Drs. Dan Gil and John Donello (Allergan, Irvine, CA). MDCKII cells were plated at a density of 1.2 × 106 cells/100-mm polycarbonate membrane filter (Transwell culture chambers, 0.4-μm pore size) and cultured in DMEM supplemented with 10% fetal calf serum (Sigma) and 100 units/ml penicillin and 10 μg/ml streptomycin at 37 °C and 5% CO2 with medium changes every other day for 5–7 days. Cells grown under these conditions achieve a morphologically and functionally polarized phenotype, as described previously (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). Leak assays of [3H]methoxy-inulin were performed as described previously (20Saunders C. Keefer J.R. Bonner C.A. Limbird L.E. J. Biol. Chem. 1998; 273: 24196-24206Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) prior to each half-life experiment to verify that the MDCKII cells had developed tight junctions and that the apical and basolateral compartments were functionally separated. The cDNA encoding the pTRE-Myc-p75-Sp151–483 fusion protein was generated via overlapping PCR extension using Pfu Turbo DNA polymerase (Stratagene). The Myc tag was inserted 5′ to the coding start site of full-length rat p75NTR and 3′ of the N-terminal cleavable signal sequence. Four glycine residues were engineered via PCR onto the C terminus of p75NTR with the intention of permitting independent folding of the spinophilin subdomain and decreased steric hindrance for interacting with other potential binding partners. The pTRE cDNA backbone (Clontech) has a tetracycline-inducible promoter that is intended to confer regulated expression of the fusion construct by treatment with the synthetic tetracycline analog, doxycycline. Two fusion proteins were generated. Myc-p75-Sp151–483 includes the receptor-binding domain and the PP1 regulatory domain of the full-length spinophilin, whereas Myc-p75-Sp151–586 also contains the PDZ-binding domain (cf. schematic of spinophilin domain structure in Fig. 2B). The cDNAs were sequenced in their entirety via 33P-Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (U. S. Biochemical Corp.) to confirm that the sequences were correct. Permanent clonal cell lines were developed in MDCKII cells using FuGENE-6 (Roche Applied Science) transfection reagent, according to the manufacturer's protocol; 6 μg of linearized pTET-On plasmid (Clontech) was co-transfected with 6 μg of pTRE-Myc-p75-Spinophilin construct and 1 μg of a vector encoding a hygromycin resistance gene into MDCKII cells already stably expressing the HA-α2B-AR, generated as described previously (7Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Stably transfected cells were selected through growth in 400 μg/ml hygromycin and assayed for HA-α2B-AR expression via radioligand binding analysis using the antagonist [3H]rauwolscine (21Wilson A.L. Seibert K. Brandon S. Cragoe Jr., E.J. Limbird L.E. Mol. Pharmacol. 1991; 39: 481-486PubMed Google Scholar) and for Myc-p75-spinophilin expression and inducibility via Western analysis and immunofluorescence using anti-c-Myc antibody. Despite the use of an inducible expression system, Myc-p75-spinophilin expression occurred even in the absence of doxycycline. Nonetheless, the cells were treated overnight with 1 μg/ml doxycycline before the day of the experiment to assure maximal Myc-p75-spinophilin expression. Polarized MDCKII cells stably expressing HA-α2B-AR were grown on 12-mm Transwells for 5–7 days (as described above) and processed as described previously for immunolocalization of HA-α2B-AR and endogenous spinophilin (12Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 276: 15003-15008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and for detection of endogenous apical (gp135) and basolateral (EGFR) marker proteins (22Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (see Fig. 1). MDCKII cells expressing Myc-p75-Sp151–483 fusion protein in the HA-α2B-AR background were treated with 1 μg/ml doxycycline overnight to maximize the expression of Myc-p75-Sp151–483 prior to staining. All of the steps were performed essentially as described previously (22Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) except that a rat anti-HA (clone 3F10) antibody (Roche Applied Science) diluted 1:1000 in blocking buffer was used for the detection of HA-α2B-AR (see Fig. 2D). MDCKII cells lines stably expressing the HA-α2B-AR alone or Myc-p75-Sp151–483 or Myc-p75-Sp151–586 in the HA-α2B-AR background were grown to confluence in 100-mm tissue culture dishes. All of the dishes were treated with 1 μg/ml doxycycline for 16 h before harvesting the cells to maximize fusion protein expression. On the day of the assay, the dishes were washed twice with Dulbecco's phosphate-buffered saline supplemented with 1 mm MgCl2 and 0.5 mm CaCl2 (DPBS/CM) (4 °C) and scraped into 12 ml of lysis buffer (15 mm Tris-HCl, 5 mm EGTA, 5 mm EDTA, pH 7.6, with N-methyl-d-glucosamine) with protease inhibitors (1 μg/ml soybean trypsin inhibitor, 0.5 μg/ml leupeptin, 100 μm phenylmethylsulfonyl fluoride). The cells were passaged through a 20-gauge needle 10 times and then centrifuged for 20 min at 39,000 × g. The supernatant was aspirated, and the pellet was resuspended in 500 μl of radioimmune precipitation buffer (RIPA) (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) plus protease inhibitors (same as above) with five passages through a 20-gauge needle followed by 10 passages through a 25-gauge needle. The detergent-extracted membranes were cleared of RIPA-insoluble debris by centrifugation for 1 h at 100,000 × g. The supernatants were precleared for 30 min with rotation at 4 °C with protein G-agarose beads pre-equilibrated in RIPA buffer. Anti-c-Myc antibody (1:100 dilution) was added to each of the precleared samples and incubated overnight at 4 °C with rotation. The next day, 30 μl of a 1:1 slurry of protein G-agarose (pre-equilibrated in RIPA buffer containing 2.5 mg/ml bovine serum albumin (BSA)) was added to each tube and incubated for 2 h at 4 °C with rotation. The protein G-agarose was pelleted and washed four times with 1 ml of ice-cold RIPA buffer plus protease inhibitors. Immunoisolated protein from the protein G-agarose was eluted by two sequential incubations for 10 min each with 25 μl of 1× SDS sample buffer (50 mm Tris-HCl, pH 8.0, 2% SDS, 10% glycerol, 100 mm dithiothreitol, 0.1% bromphenol blue) at 70 °C. The eluates were pooled, and the entire sample was resolved by 10% SDS-PAGE and transferred to nitrocellulose for Western blot analysis as described previously (23Wilson M.H. Limbird L.E. Biochemistry. 2000; 39: 693-700Crossref PubMed Scopus (32) Google Scholar). Myc-p75-Sp151–483 or Myc-p75-Sp151–586 was detected by incubation with rabbit anti-Sp286–390 antibody (1: 1000) followed by donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000) and visualized by ECL (Amersham Biosciences). HA-α2B -Adrenergic Receptors—A metabolic radiolabeling strategy was used to determine receptor half-life of α2B-AR at the apical surface of polarized MDCKII cells because of the low steady state density of the α2B-AR on this surface (7Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Cells grown 5–7 days in 100-mm Transwell culture were treated overnight with 1 μg/ml doxycycline. The day of the assay the cells were washed once in DPBS/CM and then, simultaneous with performing a transepithelial [3H]methoxy-inulin leak assay (see above), incubated for 2 h at 37 °C in serum-free, cysteine/methionine-free DMEM. The cells were then pulsed for 45 min with 900 μl of 2 μCi/μl [35S]cysteine/methionine in cysteine/methionine-free DMEM at 37 °C and 5% CO2. "Chasing" of the metabolically labeled cells was achieved by adding serum-free DMEM containing 1 mm cysteine and 1 mm methionine (chase medium) (9 ml apical/9 ml basolateral) and returning the dishes to 37 °C for 40 min to allow delivery of all of the receptor labeled during the pulse phase to the cell surface. To begin the determination of the surface half-life of metabolically labeled receptors, the apical surface of polarized MDCKII cells was biotinylated as follows. Transwells were washed once with 4 °C DPBS/CM and transferred to the cold room on ice. After washing, the Transwells were equilibrated for 10 min in 4 °C TEA buffer (250 mm sucrose, 2 mm CaCl2, 2 mm MgCl2, 10 mm triethanolamine, pH 9.0) and then biotinylated for 20 min at 4 °C on the apical surface by incubating freshly made 1 mg/ml sulfo-NHS-SS-Biotin in TEA buffer in the apical chamber (an equal volume of TEA buffer without sulfo-NHS-SS-Biotin was also added to the basolateral chamber). The biotinylation step was repeated to assure quantitative labeling of the receptor. Washing the cells with 100 mm glycine in DPBS/CM for 10 min quenched the biotinylation reaction. After washing twice more with 4 °C DPBS/CM and once with 4 °C serum-free DMEM chase medium, the cells were transferred to 37 °C chase medium and returned to the 37 °C incubator for the indicated times. At varying time points, the amount of biotinylated HA-α2B-AR on the apical surface was quantified by sequential immunoisolation and streptavidin chromatography. Selected dishes were washed twice for 10 min at 4 °C with DPBS/CM, scraped into 12 ml of lysis buffer (15 mm Tris-HCl, 5 mm EGTA, 5 mm EDTA, pH 7.6, with N-methyl-d-glucosamine) containing protease inhibitors (1 μg/ml soybean trypsin inhibitor, 0.5 μg/ml leupeptin, 100 μm phenylmethylsulfonyl fluoride), triturated 10 times through a 20-gauge needle, and then centrifuged at 39,000 × g for 20 min. The pellet was resuspended in 1 ml of RIPA buffer plus protease inhibitors and incubated on ice 30 min before centrifugation for 1 h at 100,000 × g. Supernatants from this centrifugation (the solubilized preparation) were incubated with 25 μl of a 1:1 slurry of pre-equilibrated rat anti-HA affinity matrix overnight at 4 °C with rotation. The affinity matrix was pelleted and washed four times with 1 ml of ice-cold RIPA buffer plus protease inhibitors before elution of the immunoisolated HA-α2B-AR from the affinity matrix by incubation twice for 10 min with 100 μl of SDS sample buffer (1.6% SDS, 8.3% glycerol, 167 mm Tris, pH 8.0) at 70 °C. The eluates were pooled and brought to 1.5 ml with RIPA (containing no SDS) plus protease inhibitors. The sample was allowed to sit for 10 min at room temperature to equilibrate the component detergents. To isolate the apically biotinylated HA-α2B-AR from the entire immunoisolate, streptavidin chromatography was performed as follows. The samples were incubated with a 1:1 slurry of streptavidin-agarose (50 μl) pre-equilibrated in RIPA buffer for 2 h at 4 °C with rotation. Pelleted streptavidin-agarose was washed three times with 1 ml of ice-cold RIPA buffer containing protease inhibitors, and biotinylated HA-α2B-AR was eluted by incubation twice for 20 min with 100 μl of SDS sample buffer containing 50 mm dithiothreitol at 90 °C. The eluted samples were then incubated 40 min at 50 °C. N-Ethylmaleimide was added to a final concentration of 15 mm, and the samples were incubated for an additional 40 min at 50 °C. The rationale for the high dithiothreitol/N-ethylmaleimide treatment is to alkylate all sulfhydryl residues, thus better resolving the α2-AR preparation on SDS-PAGE (24Ross E.M. Wong S.K. Rubenstein R.C. Higashijima T. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 499-506Crossref PubMed Google Scholar). The samples were resolved overnight for a total of 160mAmp-hr on a 7.5–20% gradient SDS-polyacrylamide gel. The gels were treated with En[3H]ance Intensifying Solution (PerkinElmer Life Sciences) according to the manufacturer's protocols, dried, and exposed to BioMAX MR film. The bands on the film were quantitated using SCION image software, and/or bands were cut from the gel and counted directly in scintillation mixture. Equivalent findings were obtained from either quantitation procedure. Endogenous gp135—Because endogenous gp135 is expressed at a relatively high concentration on the apical surface of polarized MDCKII cells, the surface half-life of this protein was determined by surface biotinylation, extraction into RIPA at various time points, resolution by SDS-PAGE, and identification of biotinylated gp135 via Western blot analysis for gp135, using methods described previously (23Wilson M.H. Limbird L.E. Biochemistry. 2000; 39: 693-700Crossref PubMed Scopus (32) Google Scholar). A 13.5-day pregnant female mouse (Sp+/+ or Sp–/–) (17Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9287-9292Crossref PubMed Scopus (324) Google Scholar) was sacrificed, and the embryos were collected. The soft, dark colored tissues (i.e. heart, liver, and spleen) were dissected away from the embryo, and the head was removed. The remaining tissue was transferred to the barrel of a 5-ml syringe (five embryos/syringe) and passed through an 18-gauge needle into 3 ml of DPBS. The tissue was further dissociated by trituration five times, and the cell suspension was transferred to a 150-mm culture dish containing 25 ml of complete medium (DMEM with 10% fetal calf serum, 100 units/ml penicillin, and 10 μg/ml streptomycin supplemented with 2 mm glutamine). The cells were grown at 37 °C and 5% CO2 until the plates reached confluency, at which point the cells were split 1:5, expanded to confluency, and frozen at 2 × 106 cells/ml in freezing medium (50% fetal calf serum, 12% Me2SO in DMEM). Primary cultures of MEFs (Sp+/+ or Sp–/–) (17Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9287-9292Crossref PubMed Scopus (324) Google Scholar) were seeded at 1.4 × 106 cells/100-mm dish the day before transduction with 4 ml of one part retroviral supernatant containing HA-α2B-AR-encoding virions harvested from BOSC cells (25Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2301) Google Scholar) and one part complete DMEM containing a final concentration of 12 μg/ml polybrene. The viral application was repeated four times over the course of
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