The ζ Isoform of 14-3-3 Proteins Interacts with the Third Intracellular Loop of Different α2-Adrenergic Receptor Subtypes
1999; Elsevier BV; Volume: 274; Issue: 19 Linguagem: Inglês
10.1074/jbc.274.19.13462
ISSN1083-351X
AutoresLaurent Prézeau, Jeremy G. Richman, Stephen W. Edwards, Lee E. Limbird,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoThe α2-adrenergic receptors (α2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the α2AAR (Val217–Ala377), α2BAR (Lys210–Trp354), and α2CAR (Arg248–Val363) were used as ligands in gel overlay assays. A protein doublet of ∼30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (α2B ≥ α2C≫ α2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the ζ isoform of 14-3-3 proteins. [35S]Met-14-3-3ζ binds to all three native α2AR subtypes, assessed using a solid phase binding assay (α2A≥α2B> α2C), and this binding depends on the presence of the 3i loops. Attenuation of the α2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251–266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the α2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction. The α2-adrenergic receptors (α2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the α2AAR (Val217–Ala377), α2BAR (Lys210–Trp354), and α2CAR (Arg248–Val363) were used as ligands in gel overlay assays. A protein doublet of ∼30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (α2B ≥ α2C≫ α2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the ζ isoform of 14-3-3 proteins. [35S]Met-14-3-3ζ binds to all three native α2AR subtypes, assessed using a solid phase binding assay (α2A≥α2B> α2C), and this binding depends on the presence of the 3i loops. Attenuation of the α2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251–266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the α2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction. The three α2-adrenergic receptor (α2AR) 1The abbreviation used is: α2AR, α2 adrenergic receptor; GPCR, G protein-coupled receptor(s); MDCK, Madin-Darby canine kidney; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; eIF2Bα, eukaryotic initiation factor 2Bα; FPLC, fast pressure liquid chromatography; DβM, dodecyl-β-d-maltoside; CHS, cholesterol hemisuccinate.1The abbreviation used is: α2AR, α2 adrenergic receptor; GPCR, G protein-coupled receptor(s); MDCK, Madin-Darby canine kidney; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; eIF2Bα, eukaryotic initiation factor 2Bα; FPLC, fast pressure liquid chromatography; DβM, dodecyl-β-d-maltoside; CHS, cholesterol hemisuccinate. subtypes, encoded by distinct genes (1Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar), all couple via the Gi/Go family of GTP-binding proteins to inhibition of adenylyl cyclase, suppression of voltage-sensitive calcium channels, and activation of receptor-operated potassium channels (2Limbird L.E. FASEB J. 1988; 2: 2686-2695Crossref PubMed Scopus (275) Google Scholar). These receptors also couple to activation of Ras (3Alblas 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, 4Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (407) Google Scholar), the mitogen-activated protein kinase cascade (3Alblas 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, 5Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. 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Chem. 1992; 268: 763-766Abstract Full Text PDF Google Scholar, 11von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 12Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (174) Google Scholar, 13Kurose H. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10093-10099Abstract Full Text PDF PubMed Google Scholar, 14Eason M.G. Liggett S. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 15Jones S. Leone S. Bylund D.B. J. Pharmacol. Exp. Ther. 1990; 254: 294-300PubMed Google Scholar). In addition, selective itineraries for the α2AR subtypes are observed in polarized Madin-Darby canine kidney (MDCKII) renal epithelial cells. Thus, the α2AAR subtype is targeted directly to the basolateral surface (16Keefer 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 rapidly lost from the apical (t½ = 5–15 min) and selectively retained on the basolateral (t½ = 10–12 h) surface (17Wozniak 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 domain of MDCK cells that may be shared by all three α2AR subtypes, as they manifest comparable half-lives on that surface (17Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the α2AAR, since deletion of this loop, creating the mutant α2AΔ3iAR, results in accelerated basolateral turnover (t½≅ 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 at½ of 10–12 h) (18Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). The accelerated turnover of the α2AΔ3iAR when compared with the wild-type α2AAR structure suggests that the third intracellular loop interacts with proteins that either tether α2AAR to a particular surface domain or, alternatively, mask the α2AAR from interacting with endocytosis machinery. Other functional roles have been attributed to the third intracellular loop of α2AAR. The N- and C-terminal 10–15 residues of the 3i loop, predicted to form amphipathic helices, are involved in coupling to G proteins (19Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 20Eason M.G. Moreira S.P. Liggett S.B. J. Biol. Chem. 1995; 270: 4681-4688Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 21Eason M.G. Liggett S.B. J. Biol. Chem. 1996; 271: 12826-12832Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Wade S.M. Scribner M.K. Dalman H.M. Taylor J.M. Neubig R.R. Mol. Pharmacol. 1996; 50: 351-358PubMed Google Scholar). The C-terminal third of the 3i loop of the α2AAR subtype is implicated in the interaction with β-arrestin, a protein that preferentially associates with G protein-coupled receptor kinase-phosphorylated receptors sustaining agonist-elicited homologous desensitization (23Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). For the α2AR subtypes, G protein-coupled receptor kinase phosphorylation sites are in the N-terminal region of the α2AAR 3i loop (24Jewell-Motz E. Liggett S.B. Biochemistry. 1995; 34: 11946-11953Crossref PubMed Scopus (42) Google Scholar, 25Eason M.G. Liggett S.B. J. Biol. Chem. 1995; 270: 24753-24760Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), widely distributed throughout the α2BAR 3i loop, and presumed to be absent in the α2CAR 3i loop (13Kurose H. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10093-10099Abstract Full Text PDF PubMed Google Scholar, 26Liggett S.B. Ostrowski J. Chesnut L.C. Kurose H. Raymond J.R. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 4740-4746Abstract Full Text PDF PubMed Google Scholar). The present studies were undertaken to identify interacting proteins for the intracellular 3i loops of the α2AR subtypes.In vitro translation of Gen10-α2AR 3i loop fusion proteins (gen10-α23i) served as a means to create [35S]methionine-radiolabeled 3i loops as ligands for identifying interacting proteins via a gel overlay strategy. Our findings reveal that these loops, in a subtype-selective fashion, interact with the ζ isoform of 14-3-3 proteins (14-3-3ζ). Using a solid phase binding assay with [35S]Met-14-3-3ζ as a probe and solubilized α2AR as the target indicates that 14-3-3 proteins can bind to native α2AR subtypes in a way that relies on the 3i loop in the receptor structure. Further evidence for the functional relevance of these interactions is the ability of a Raf peptide, corresponding to a 14-3-3-interacting domain, to block Gen10-α23i loop interactions with 14-3-3ζ in its phosphorylated, but not in its non-phosphorylated, state. 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 NEN Life Science Products. PVDF nylon membranes were from Millipore (Bedford, MA). The FPLC and DEAE-Sephacel columns were from Amersham Pharmacia Biotech. Dodecyl-β-maltoside and cholesterol hemisuccinate were purchased from Calbiochem and Sigma, respectively. Staph A immunoprecipitin was obtained from Life Technologies, Inc. 12CA5 monoclonal antibody against the hemagglutinin epitope engineered into the α2AR structures was obtained from Babco; the M2 monoclonal antibody against the FLAG epitope engineered into the N terminus of 14-3-3ζ was from Eastman Kodak Co., and the rabbit anti-14-3-3β (or pan) and anti-ζ isoform antibodies were from Santa Cruz Laboratories (Santa Cruz, CA). Protein A beads were from Vector (Burlingame, CA). Centricon-10 concentrating filters were purchased from Amicon (Beverly, MA). The tube gel adapter kit was from Hoefer Scientific instruments (San Francisco, CA). 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 Jr., E.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 pGEMEX-2 vector. The residues utilized are shown schematically in Fig. 2B. These 3i loop sequences were inserted in frame within the polylinker located downstream of the sequence encoding the Gen10 protein, a methionine-rich phage structural protein. The sequence encoding an epitope of the c-Myc protein was inserted 3′ to the sequence of the α2AR 3i loop sequences. The Gen10–3i loop fusion proteins and 14-3-3ζ were produced and [35S]Met-labeled using an in vitro T7 RNA polymerase-coupled translation system in reticulocyte lysates as follows: 25 μl of TNT lysate were added to 1 μl of amino acid mix (1 mm, minus methionine, TNT kit), 2 μl of TNT reaction buffer, 1 μl of TNT T7 RNA polymerase, 4 μl of [35S]methionine (1000 Ci/mmol, at 10 mCi/ml), 1 μl of RNasin ribonuclease inhibitor (40 units/μl). Then, 1 μg of the appropriate DNA template (presented as the circular plasmid DNA) was added, and the volume was adjusted to 50 μl with nuclease-free water. The mixture was incubated for 90 min at 30 °C. Products were analyzed and quantitated following each synthesis by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, and the band representing each probe was cut out of the dried gel and counted in scintillation mixture. If smaller molecular weight species were generated during the translation reaction, they were eliminated by P30 size exclusion chromatography before use as probes. These 35S-labeled Gen10-α23i loop fusion proteins were used as radioactive ligands in subsequent gel overlay assays as described previously (30Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (542) Google Scholar) and detailed below. Frozen pig brain cortex (2 g/preparation) was suspended in 20 ml of ice-cold lysis buffer (20 mm HEPES, 50 mmKCl, 2 mm MgCl2, 1 mmCaCl2 with the following protease inhibitors: 0.1 mm phenylmethylsulfonyl fluoride, 20 μg/ml soybean trypsin inhibitor, 5 μg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml benzamidine) and homogenized using a Brinkmann Polytron (two 5-s bursts separated by 30 s on ice). The lysate was filtered through cheesecloth to remove debris and then centrifuged at 38,000 ×g in an SS34 rotor (Sorvall RC 5B centrifuge) for 20 min. The supernatant of this centrifugation was removed and designated as the cytosolic fraction. The pellet was resuspended in 4 ml of lysis buffer, homogenized again, this time using a Teflon/glass homogenizer, and centrifuged as before. In early experiments, this pellet was resuspended and an aliquot saved to permit analysis of 3i loop interacting proteins in membrane protein fractions. To resolve proteins in the particulate fraction that could be extracted into Triton X-100, the membrane pellet was re-homogenized into 4 ml of ice-cold detergent-containing buffer (20 mm HEPES, 150 mm KCl, 2 mm MgCl2, 0.5% Triton X-100, and the protease inhibitors indicated above). All fractions were stored at −70 °C. The protein concentration in each fraction was estimated using the Bradford assay. Cultured MDCK cells (two 100-mm dishes/preparation) were harvested at confluence by scraping into 1 ml of lysis buffer (see above) using a rubber policeman. MDCK cell lysates were disrupted further by 10 up and down passages through a 25-gauge needle mounted onto a 5-ml syringe. The supernatant of the 15 min, 4 °C centrifugation (estimated at 30,000 × g in an Eppendorf centrifuge) was saved and defined as the cytosolic fraction. A gel overlay procedure (31McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32Shieh B.H. Zhu M.Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar) was used to detect binding of [35S]Met-labeled Gen10-α23i loops to fractionated cellular proteins resolved via SDS-PAGE. Protein aliquots (2.5–3.0 mg/sample) were separated by SDS-PAGE using 7.5–20% polyacrylamide gradients on 16-cm long and 1.5-mm thick gels. Prestained molecular weight markers also were run to permit estimation of the approximate molecular weights of the proteins identified by gel overlay analysis. The resolved proteins were transferred at 4 °C to PVDF nylon membranes (Millipore) by electrophoresis overnight at 30 V in Tris/glycine buffer (25 and 192 mm, respectively). The membranes were then cut in 2–4-mm strips for gel overlay and Western blot analysis. For gel overlay analysis, PVDF membranes were blocked at least 1 h in blocking buffer: Tris-HCl/NaCl (50 and 200 mm, respectively, referred to hereafter as TBS), containing Tween 20 (3% v/v) and non-fat powdered milk (5% w/v). The PVDF membranes were then washed for 30 min in rinsing buffer: TBS containing Tween 20 (0.1% v/v) and non-fat powdered milk (5% w/v). The PVDF membrane strips were then incubated with 300,000 cpm of the appropriate [35S]Met-labeled Gen10-α23i loop structure in a 1-ml incubation for 4 h (to overnight) at 4 °C with constant rocking in rinsing buffer. Based on the concentration of methionine contributed to the [35S]Met-labeling reaction by the rabbit reticulocyte lysate (5 μm) and the specific activity of the [35S]Met radiolabel, we estimated that this 300,000 cpm of Gen10-α23i loop represents 5–10 pmol of probe. In experiments where the duration of the incubation or the amount of radioligand was varied, the times and concentrations evaluated are indicated in the figure legends. Following incubation with the various loop structures (or radiolabeled Gen10, as a control), membranes were washed 3 times with rinsing buffer, twice with cold TBS, and air-dried before autoradiography. Autoradiography was performed using a Molecular Dynamics PhosphorImager, and band intensities were calculated using the manufacturer's software, presented as arbitrary intensity units. Following quantitation, strips were exposed to x-ray film for 24–72 h. For Raf competition experiments, phosphorylated and non-phosphorylated peptides corresponding to a 14-3-3 binding region of Raf-1 (LSQRQRSTS(PO4)TPNVHMV and LSQRQRSTSTPNVHMV, respectively (33Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. J. Biol. Chem. 1998; 273: 16305-16310Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar)) were incubated with the membranes for 0.5 h prior to the addition of the [35S]Met-labeled Gen10-α23i loop probes, and the incubation was continued for 90 min followed by washing and detection, as described above. For each purification protocol, 15 ml of a cytosolic protein fraction prepared from 2 g of frozen pig brain cortex were loaded onto a 2-ml DEAE-Sephacel column equilibrated overnight with ice-cold column equilibration buffer (20 mmHEPES (pH 7.0), 50 mm KCl, 2 mmMgCl2, 1 mm CaCl2, and protease inhibitors as utilized above). The column pass-through was saved for evaluation of 3i loop binding activity. The DEAE column was washed with 100 ml of 150 mm KCl-containing lysis buffer. The proteins were eluted using a gradient of KCl (from 150 to 500 mmKCl) in lysis buffer, at a rate of 6 ml/h. One-ml fractions were collected and subsequently evaluated for 3i loop binding activity using gel overlay analysis. Preparative gel filtration using fast protein liquid chromatography (FPLC) was performed as follows: a 120-ml Superdex 200 column was equilibrated for 2 h with ice-cold buffer (20 m HEPES (pH 7.0), 150 mm KCl, 2 mm MgCl2, 1 mm CaCl2) at a rate of 2 ml/min. A 2-ml sample, corresponding to peak fractions from the DEAE-Sephacel column, was injected. Eluate fractions (2 ml/fraction) were collected at a rate of 2 ml/min for 2 h. The 3i loop binding activity in individual fractions was determined by assaying aliquots using sequential SDS-PAGE and gel overlay analysis; in some studies, the proteins were concentrated and desalted using Centricon-10 concentrators before assaying 3i loop binding activity. To be confident that the bands on SDS-PAGE manifesting 3i loop binding activity were not "contaminated" by underlying bands, two-dimensional gel electrophoresis was performed using protocols and the tube gel adapter kit provided by Hoefer Scientific instruments. Microsequencing was performed at the Harvard Microsequencing Laboratory facility (Dr. William Lane, Director) by Edman degradation of tryptic digests of the ∼30-kDa bands hydrolyzed in polyacrylamide gels, resolved by high pressure liquid chromatography, and assessed by mass spectrometry. MDCKII cells, parental or stably transfected with the α2A, α2B or α2CAR subtype, were grown to confluence on 150-mm plates, serum-starved overnight, harvested in lysis buffer (15 mm HEPES, 5 mmEGTA, and 5 mm EDTA (pH 7.6), containing 10 units/ml aprotinin and 100 μm phenylmethylsulfonyl fluoride), disrupted using a Teflon/glass homogenizer, split into 2 aliquots, and centrifuged at 30,000 × g. One aliquot was extracted with detergent, and the other was used to monitor receptor available for extraction. For detergent extraction, one pellet was resuspended in 2.25 ml/150-mm plate DβM/CHS extraction buffer (4 mg/ml dodecyl-β-d-maltoside (DβM), 0.8 mg/ml cholesterol hemisuccinate (CHS), 25 mm glycylglycine, 20 mmHEPES, 100 mm NaCl, 5 mm EGTA, 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, 10 units/ml aprotinin, and 100 μm phenylmethylsulfonyl fluoride), homogenized using a 27-gauge needle, and centrifuged at 100,000 ×g at 4 °C for 1 h. The resulting supernatant was defined as the detergent-solubilized receptor. To assess the α2AR binding capacity of these preparations, [3H]rauwolscine was used as a radioligand, and Sephacel G-50 chromatography was used to separate bound from free ligand, as described previously (34Nunnari J.M. Repaske M.G. Brandon S. Cragoe Jr., E.J. Limbird L.E. J. Biol. Chem. 1987; 262: 12387-12392Abstract Full Text PDF PubMed Google Scholar). To assess the relative efficiency of the detergent to extract receptor from membranes, the results of the G-50 chromatography binding assays were compared with radioligand binding assays performed on the membrane pellet, derived from a fraction of the original preparation, with [3H]rauwolscine. Based on these determinations we estimate that we extract >50% of the α2AR subtypes using this protocol. Equal concentrations of detergent-solubilized receptor were incubated with mouse anti-hemagglutinin antibodies for 1 h and then with 100 μl of protein A-agarose (1:1 slurry with DβM/CHS wash buffer) for a 2nd h. The protein A-agarose was rinsed twice with DβM/CHS wash buffer (1 mg/ml DβM, 0.2 mg/ml CHS, 25 mm glycylglycine, 20 mm HEPES, 100 mm Na, 5 mm EGTA, 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, 10 units/ml aprotinin and 100 μm phenylmethylsulfonyl fluoride) and incubated for 16 h with [35S]Met-14-3-3ζ rotating end over end at 4 °C. To terminate the incubation, the protein A resin was pelleted at 18,500 × g and rinsed twice with DβM/CHS wash buffer before resuspension in Laemmli buffer and fractionated by 12% SDS-PAGE. The gels were dried prior to autoradiography; the amount of [35S]Met-14-3-3ζ bound to receptor was quantitated by cutting the bands and counting in NEF 963 scintillation fluor. To assess the extent of non-receptor-dependent [35S]Met-14-3-3ζ binding, detergent extracts of parental MDCKII cells expressing no α2ARs were prepared, and volumes of this extract equal to the largest volume receptor-containing preparations was adsorbed to protein A resin and served as the control for these studies. Because the 3i loops of the α2AAR have been implicated in stabilization of these receptors on the basolateral surface of polarized renal epithelial cells, we sought to identify proteins that interact with these intracellular domains. We created fusion proteins of the 3i loops with the methionine-rich Gen10 protein.In vitro translation of these fusion proteins in the presence of [35S]methionine generated radiolabeled 3i loops that served as ligands for the identification of interacting proteins via gel overlay analysis. As can be seen in Fig. 1, the 3i loops of the α2BAR and α2CAR subtypes readily identified a doublet of apparent molecular mass of 30 kDa in cytosolic fractions of MDCKII cell lysate that was not detected by Gen10 protein or by the 3i loop of the α2AAR under these incubation conditions (see Fig. 2, later, for delayed binding by α2AAR 3i loop). The ∼30-kDa doublet identified by the α2B3i and α2C3i loops is enriched in the cytosolic fraction and is barely detected in the membrane fractions of MDCKII cells. Similar binding profiles were seen in fractions from porcine brain cortex, albeit with greater membrane-associated binding activity, and from lysates of MDCKII cells that had been grown in Transwell® culture to foster polarization (data not shown), consistent with the published experience that confluent MDCKII cells grown in regular culture dishes manifest many of the properties characteristic of the polarized cellular phenotype (16Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 35Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (810) Google Scholar). Fig. 2A demonstrates the time course for interaction of the α2AR 3i loops with the 30-kDa doublet in porcine brain cytosolic fractions. The 3i loops of the α2BAR and α2CAR interacted more readily and to a significantly greater extent than the 3i loop of the α2AAR subtype, whose binding to the 30-kDa doublet was detectable above background labeling only after longer (>4 h) incubations. When the ability of a 10× molar excess of unlabeled 3i loops to compete for binding of the35S-labeled 3i loops for each subtype was evaluated after a 2- or 4-h incubation, it was evident that competition for the binding of the α2AAR 3i loop was more facile than for the binding of the α2BAR or the α2CAR loop (data not shown), consistent with the apparent lower affinity of the α2AAR 3i loop for the 30-kDa interacting proteins in the gel overlay assay (Fig. 2). The Gen10 fusion protein (control probe) did not compete for any of the 3i loop-specific binding nor did a Gen10 fusion protein encoding 58 amino acids of the distal C-terminal tail of the β1 adrenergic receptor (data not shown), a region previously implicated in β1AR stabilization on the cell surface (36Hertel C. Nunnally M.H. Wong S.K. Murphy E.A. Ross E.M. Perkins J.P. J. Biol. Chem. 1990; 265: 17988-17994Abstract Full Text PDF PubMed Google Scholar, 37Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To reveal the molecular identity of the 30-kDa doublet, we undertook its purification from cytosolic fractions of porcine brain cortex. As shown in Fig. 3A, the interacting proteins were quantitatively adsorbed to DEAE-Sephacel and eluted, using a 50–500 mm KCl gradient, at approximately 250–300 mm KCl (fractions 82–95). Binding to these peak fractions showed a specificity of 3i loop binding characteristic of the unfractionated cytosol, as shown in Fig. 3B. The peak fractions were pooled and purified further using size exclusion FPLC, which removed most of the proteins migrating on SDS-PAGE at >50 kDa and <20 kDa (Fig. 4). The elution position of the 3i loop interacting proteins on FPLC corresponded to an M r of 50,000–80,000 (data not shown), suggesting that the ∼30-kDa proteins on SDS-PAGE may exist as a dimer, in a complex with other proteins, or both.Figure 4FPLC size exclusion chromatography of the 3i loop interacting proteins. A, silver-stained acrylamide gel showing the starting material (pooled DEAE eluate fractions), and FPLC eluate fractions. B, gel overlay analysis revealing the presence of the 3i loop interacting protein in the DEAE eluate starting material and in FPLC fraction 42. In this experiment, the 3i loop interacting proteins were identified using [35S]Met-labeled Gen10-α2CAR 3i loop as the ligand.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Material that had been purified by sequential chromatography on DEAE-Sepharose, FPLC, and concentrated by a second application to DEAE-Sepharose was subjected to two-dimensional isoelectric focusing and SDS-PAGE. As shown in Fig. 5A, the material migrating in the 30-kDa region on one-dimensional SDS-PAGE was resolved into three distinct spots upon two-dimensional gel analysis, as revealed by Zoion Coomassie staining. Gel overlay analysis indicated that two of the three spots, migrating at isoelectric points of 5 and 5.6, represented the α2AR 3i loop interacting proteins (Fig. 5B). In fact, the spot migrating at a pI of 5.0 has greater35S-Gen10–3i loop binding relative to its Coomassie labeling intensity than the protein migrating with a pI ≅ 5.6. Since the C-terminal tail of both the α2AAR and the β2AR interacts with a ∼30-kDa protein that corresponds to eIF2Bα (38Klein U. Ramirez M.T. Kobilka B.K. von Zastrow M. J. Biol. Chem. 1997; 272: 19099-19102Abstract Full Text Full Text PDF PubMed Scopu
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