Role of Amphiphysin II in Somatostatin Receptor Trafficking in Neuroendocrine Cells
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m310792200
ISSN1083-351X
AutoresPhilippe Sarret, M. James Esdaile, Peter S. McPherson, Agnes Schönbrunn, Hans‐Jürgen Kreienkamp, Alain Beaudet,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoAmphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of 125I-[d-Trp8]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (Bmax) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst2A and sst5 receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway. Amphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of 125I-[d-Trp8]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (Bmax) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst2A and sst5 receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway. The formation of carrier vesicles is an important aspect of membrane transport along the secretory and endocytic pathways (for review, see Ref. 1Kirchhausen T. Nat. Rev. Mol. Cell. Biol. 2000; 1: 187-198Crossref PubMed Scopus (422) Google Scholar). In the past few years, studies on clathrin-mediated receptor endocytosis have led to the identification of novel protein components of the endocytic regulatory machinery. The budding and fission of clathrin-coated vesicles from the plasma membrane, initiated by the recruitment of the adaptor protein-2 (AP-2) 1The abbreviations used are: AP, adaptor protein; TGN, trans-Golgi network, Bin, bridging integrator; DMEM, Dulbecco's modified Eagle's medium; WT, wild type; BSA, bovine serum albumin; SRIF, somatostatin; TBS, Tris-buffered saline; ACTH, adrenocorticotropic hormone; CRF, corticotrophin-releasing factor. 1The abbreviations used are: AP, adaptor protein; TGN, trans-Golgi network, Bin, bridging integrator; DMEM, Dulbecco's modified Eagle's medium; WT, wild type; BSA, bovine serum albumin; SRIF, somatostatin; TBS, Tris-buffered saline; ACTH, adrenocorticotropic hormone; CRF, corticotrophin-releasing factor. and clathrin, were shown to be regulated by several accessory proteins including dynamin, synaptojanin, amphiphysin, Eps15, intersectin, and endophilin (for review, see Refs. 2Marsh M. McMahon H.T. Science. 1999; 285: 215-220Crossref PubMed Scopus (481) Google Scholar, 3McPherson P.S. Kay B.K. Hussain N.K. Traffic. 2001; 2: 375-384Crossref PubMed Scopus (186) Google Scholar, 4Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (424) Google Scholar, 5Takei K. Haucke V. Trends Cell Biol. 2001; 11: 385-391Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The trans-Golgi network (TGN) is a dynamic organelle through which nascent secretory and transmembrane proteins are sorted and packaged into distinct carrier vesicles for transport either to the plasma membrane or to endosomal compartments. Three major pathways out of the TGN have been identified thus far: the regulated secretory pathway, which delivers surface-destined cargo via clathrin-coated vesicles; the constitutive secretory pathway, which mediates sorting from the TGN via non-clathrin-coated vesicles; and the clathrin-mediated trafficking route to the endosomal/lysosomal compartments (for review, see Refs. 6Traub L.M. Kornfeld S. Curr. Opin. Cell Biol. 1997; 9: 527-533Crossref PubMed Scopus (194) Google Scholar, 7Corda D. Hidalgo Carcedo C. Bonazzi M. Luini A. Spano S. Cell. Mol. Life Sci. 2002; 59: 1819-1832Crossref PubMed Scopus (52) Google Scholar, 8Allan V.J. Thompson H.M. McNiven M.A. Nat. Cell. Biol. 2002; 4: E236-E242Crossref PubMed Scopus (173) Google Scholar, 9Gu F. Crump C.M. Thomas G. Cell. Mol. Life Sci. 2001; 58: 1067-1084Crossref PubMed Scopus (140) Google Scholar). Whereas a growing number of proteins have been identified to function in the sorting of proteins toward the endosome/lysosome, such as the adaptor protein-1 (AP-1) and the GGA family (10Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (442) Google Scholar, 11Wasiak S. Quinn C.C. Ritter B. de Heuvel E. Baranes D. Plomann M. McPherson P.S. J. Biol. Chem. 2001; 276: 26622-26628Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12Le Borgne R. Hoflack B. Biochim. Biophys. Acta. 1998; 1404: 195-209Crossref PubMed Scopus (116) Google Scholar, 13Collins B.M. Praefcke G.J. Robinson M.S. Owen D.J. Nat. Struct. Biol. 2003; 10: 607-613Crossref PubMed Scopus (49) Google Scholar, 14Lui W.W. Collins B.M. Hirst J. Motley A. Millar C. Schu P. Owen D.J. Robinson M.S. Mol. Biol. Cell. 2003; 14: 2385-2398Crossref PubMed Scopus (67) Google Scholar, 15Mills I.G. Praefcke G.J. Vallis Y. Peter B.J. Olesen L.E. Gallop J.L. Butler P.J. Evans P.R. McMahon H.T. J. Cell Biol. 2003; 160: 213-222Crossref PubMed Scopus (202) Google Scholar, 16Puertollano R. Randazzo P.A. Presley J.F. Hartnell L.M. Bonifacino J.S. Cell. 2001; 105: 93-102Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17Puertollano R. van der Wel N.N. Greene L.E. Eisenberg E. Peters P.J. Bonifacino J.S. Mol. Biol. Cell. 2003; 14: 1545-1557Crossref PubMed Scopus (104) Google Scholar), relatively little is known regarding proteins that function in the regulated or constitutive secretory pathways. Recent studies have suggested that proteins that regulate membrane budding at the cell surface may also be involved in the production of transport vesicles at the TGN. For example, dynamin II has been localized to the TGN of mammalian cells and shown to be required for the formation of transport vesicles (18Maier O. Knoblich M. Westermann P. Biochem. Biophys. Res. Commun. 1996; 223: 229-233Crossref PubMed Scopus (49) Google Scholar, 19Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (108) Google Scholar, 20Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (274) Google Scholar, 21Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (341) Google Scholar, 22McNiven M.A. Cao H. Pitts K.R. Yoon Y. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 23Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (355) Google Scholar). Accordingly, introduction of the dynamin II mutant (K44A) was found to interfere with the formation of both clathrin-coated and non-clathrin-coated vesicles from the TGN (24Cao H. Thompson H.M. Krueger E.W. McNiven M.A. J. Cell Sci. 2000; 113: 1993-2002Crossref PubMed Google Scholar, 25Kreitzer G. Marmorstein A. Okamoto P. Vallee R. Rodriguez-Boulan E. Nat. Cell. Biol. 2000; 2: 125-127Crossref PubMed Scopus (206) Google Scholar). Different isoforms of the same accessory proteins could also be involved in clathrin-mediated endocytosis versus production of secretory vesicles. Thus, whereas AP-2, endophilin A1, and synaptojanin are essential for synaptic vesicle endocytosis, AP-1, AP-3, and AP-4, endophilin B1, and synaptojanin-like Inp53p have been implicated in vesicle budding from the TGN (10Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (442) Google Scholar, 26Bensen E.S. Costaguta G. Payne G.S. Genetics. 2000; 154: 83-97Crossref PubMed Google Scholar, 27Farsad K. Ringstad N. Takei K. Floyd S.R. Rose K. De Camilli P. J. Cell Biol. 2001; 155: 193-200Crossref PubMed Scopus (487) Google Scholar, 28Modregger J. Schmidt A.A. Ritter B. Huttner W.B. Plomann M. J. Biol. Chem. 2003; 278: 4160-4167Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Amphiphysins belong to the BAR (Bin/Amphiphysin/Rvs) family of proteins, which includes the mammalian bridging integrators (Bin1, Bin2, and Bin3), amphiphysin I and II, and the yeast Rvs161p and Rvs167p (for reviews, see Refs. 29Ramjaun A.R. McPherson P.S. J. Neurochem. 1998; 70: 2369-2376Crossref PubMed Scopus (134) Google Scholar and 30Zhang B. Zelhof A.C. Traffic. 2002; 3: 452-460Crossref PubMed Scopus (72) Google Scholar)). Amphiphysin I and II act as multifunctional adaptor proteins that cooperate in the recruitment and targeting of other key endocytic proteins. Through direct protein-protein interactions, both isoforms bind their C-terminal SH3 domains to proline-rich sequences of dynamin and synaptojanin (Fig. 1) (31David C. McPherson P.S. Mundigl O. de Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (350) Google Scholar, 32de Heuvel E. Bell A.W. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (491) Google Scholar, 34Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Amphiphysins I and II have also been reported to interact with AP-2 and endophilin through an SH3 domain-independent sequence (31David C. McPherson P.S. Mundigl O. de Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (350) Google Scholar, 35Wang L.H. Sudhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 36Micheva K.D. Ramjaun A.R. Kay B.K. McPherson P.S. FEBS Lett. 1997; 414: 308-312Crossref PubMed Scopus (69) Google Scholar, 37Leprince C. Romero F. Cussac D. Vayssiere B. Berger R. Tavitian A. Camonis J.H. J. Biol. Chem. 1997; 272: 15101-15105Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The insert region of amphiphysin I and II was reported to interact with clathrin-binding sites, but at sites distinct from the AP-2 and endophilin-binding domains (Fig. 1) (4Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (424) Google Scholar, 29Ramjaun A.R. McPherson P.S. J. Neurochem. 1998; 70: 2369-2376Crossref PubMed Scopus (134) Google Scholar). Furthermore, the N terminus of amphiphysin II mediates the dimerization and plasma membrane targeting of this protein (Fig. 1) (38Ramjaun A.R. Philie J. de Heuvel E. McPherson P.S. J. Biol. Chem. 1999; 274: 19785-19791Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Seven different splice isoforms of amphiphysin II have so far been isolated (Fig. 1) (34Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 37Leprince C. Romero F. Cussac D. Vayssiere B. Berger R. Tavitian A. Camonis J.H. J. Biol. Chem. 1997; 272: 15101-15105Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 39Sparks A.B. Hoffman N.G. McConnell S.J. Fowlkes D.M. Kay B.K. Nat. Biotech. 1996; 14: 741-744Crossref PubMed Scopus (215) Google Scholar, 40Tsutsui K. Maeda Y. Seki S. Tokunaga A. Biochem. Biophys. Res. Commun. 1997; 236: 178-183Crossref PubMed Scopus (63) Google Scholar, 41Sakamuro D. Elliott K.J. Wechsler-Reya R. Prendergast G.C. Nat. Genet. 1996; 14: 69-77Crossref PubMed Scopus (312) Google Scholar, 42Butler M.H. David C. Ochoa G.C. Freyberg Z. Daniell L. Grabs D. Cremona O. De Camilli P. J. Cell Biol. 1997; 137: 1355-1367Crossref PubMed Scopus (213) Google Scholar, 43Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (209) Google Scholar). These splice variants have pleiotropic roles, including clathrin-mediated endocytosis, apoptosis, stress signaling processes, and regulation of membrane dynamics, perhaps through interaction with the actin cytoskeleton (for review, see Refs. 4Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (424) Google Scholar, 5Takei K. Haucke V. Trends Cell Biol. 2001; 11: 385-391Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 30Zhang B. Zelhof A.C. Traffic. 2002; 3: 452-460Crossref PubMed Scopus (72) Google Scholar, and 44Wigge P. McMahon H.T. Trends Neurosci. 1998; 21: 339-344Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 45McPherson P.S. Cell Signalling. 1999; 11: 229-238Crossref PubMed Scopus (89) Google Scholar, 46Wakeham D.E. Ybe J.A. Brodsky F.M. Hwang P.K. Traffic. 2000; 1: 393-398Crossref PubMed Scopus (15) Google Scholar). Much of the variability in the splice forms comes from a central region, encoded by exons 12a-12d (Fig. 1) (47Wechsler-Reya R. Sakamuro D. Zhang J. Duhadaway J. Prendergast G.C. J. Biol. Chem. 1997; 272: 31453-31458Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The latter may convey different binding specificities and functions to the different splice forms. In particular, amphiphysin IIb, which is lacking an important determinant for plasma membrane targeting and does not bind to clathrin, may have functions unrelated to endocytosis. To test this hypothesis, we investigated the effect of amphiphysin IIb overexpression on the trafficking of somatostatin receptors sst2A and sst5 in the pituitary corticotrope cells AtT-20 that express both receptor subtypes endogenously (48Patel Y.C. Panetta R. Escher E. Greenwood M. Srikant C.B. J. Biol. Chem. 1994; 269: 1506-1509Abstract Full Text PDF PubMed Google Scholar, 49Sarret P. Nouel D. Dal Farra C. Vincent J.P. Beaudet A. Mazella J. J. Biol. Chem. 1999; 274: 19294-19300Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Our results demonstrate a role for amphiphysin IIb in the constitutive trafficking of these receptors from the TGN. Culture and Transfection of AtT-20 Cells—AtT-20 cells (a mouse ACTH-secreting tumor cell line) were grown and subcultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose (4.5 g/liter) supplemented with 10% fetal bovine serum and 10% horse serum in the presence of 100 units/ml penicillin/streptomycin (Invitrogen) as previously described (49Sarret P. Nouel D. Dal Farra C. Vincent J.P. Beaudet A. Mazella J. J. Biol. Chem. 1999; 274: 19294-19300Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Cell monolayers were grown in T75 cm2 flasks, maintained in a humidified atmosphere of 95% O2 and 5% CO2 at 37 °C and passaged when the monolayer achieved 90% confluence. An amphiphysin IIb-overexpressing AtT-20 cell line was established according to procedures described previously (50Botto J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; 243: 585-590Crossref PubMed Scopus (56) Google Scholar). Briefly, 24 h after plating, semi-confluent AtT-20 cells were transfected with the recombinant pcDNA3 plasmid (5 μg/35-mm dish; Invitrogen) containing a BamHI-EcoRI insert of amphiphysin IIb cDNA using the DAC-30 reagent according to the manufacturer's recommendation (Eurogentec, Seraing, Belgium). After 2 days, the medium was changed for growth medium containing 0.75 mg/ml geniticin (G418). Surviving colonies were isolated 2 weeks later and separately cultivated in 24-well plates. Clones were then checked for expression of amphiphysin IIb by immunoblotting and immunofluorescence microscopy using an antibody raised against amphiphysin II (34Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The clone expressing the highest level of amphiphysin IIb was selected for this study. Cells were maintained in standard growth medium supplemented with 0.5 mg/ml G418. They were plated in 16-mm multiwell dishes for binding and ACTH release experiments at an initial plating density of 105 cells per well. Immunoblotting Analysis—Polyclonal antibodies raised against amphiphysin II have been extensively characterized elsewhere (34Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 38Ramjaun A.R. Philie J. de Heuvel E. McPherson P.S. J. Biol. Chem. 1999; 274: 19785-19791Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). For blots, AtT-20 cells were homogenized in 20 mm HEPES-OH, pH 7.4, containing 0.83 mm benzamidine, 0.23 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml aprotinin, and 0.5 μg/ml leupeptin. Insoluble material was removed from the extract by centrifugation for 15 min at 13,000 × g. Proteins (100 μg/lane) were then separated on a 3–12% gradient SDS-PAGE, transferred to nitrocellulose, and processed for Western blotting analysis using antibodies raised against amphiphysin II. The immunoreactivity was visualized using an enhanced chemiluminescent detection system (PerkinElmer Life Sciences). Morphological Parameters—To compare the morphological characteristics of amphiphysin IIb overexpressing cells with those of wild-type, non-transfected cells (WT), perimeter, area, and form factor (defined as 4π × area ÷ perimeter2) were determined in 100 cells (50 WT, 50 amphiphysin IIb), using a computer-assisted image analysis system (Biocom, Les Ulis, France). Both WT and amphiphysin IIb-transfected cells were first fixed for 20 min with 4% paraformaldehyde (Poly-sciences, Warington, PA) in 0.1 m phosphate buffer (phosphate buffer), pH 7.4, and rinsed twice with 0.1 m phosphate buffer. They were then stained with 0.05% toluidine blue, mounted, and analyzed under a 50 oil immersion objective on a Leitz Diaplan microscope. All calculations and statistical analyses were performed using Excel 5.0 (Microsoft Corp., San Francisco, CA). Statistical significance was verified using Student's t test. Preparation of Cell Homogenates—Confluent AtT-20 cells were washed and scraped off the culture dishes with ice-cold Tris-buffered saline, pH 7.5. Subsequently, the cells were centrifuged at 15,000 × g for 5 min at 4 °C in microcentrifuge tubes and resuspended in hypotonic TE buffer (5 mm EDTA and 10 mm Tris-HCl, pH 7.5). Membrane homogenates were then sonicated, recentrifuged at 15,000 × g for 30 min at 4 °C, and resuspended in the same buffer. Binding and internalization of 125I-Tyr0[d-Trp8]-Somatostatin Association Kinetics—To investigate somatostatin (SRIF) binding and internalization, WT and amphiphysin IIb-overexpressing AtT-20 cells were equilibrated for 10 min at 37 °C in Earle's buffer (140 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 0.9 mm MgCl2, and 25 mm HEPES, pH 7.4) supplemented with 0.1% glucose and 1% bovine serum albumin (BSA)). The equilibration medium was then replaced by 250 μl of Earle's buffer containing 0.1 nm125I-Tyr0-[d-Trp8]SRIF (2000 Ci/mmol) in the presence of 0.8 mm 1,10-phenanthroline for 5–45 min at 37 °C. After various incubation periods, the cells were washed twice with either 0.5 ml of Earle's buffer, or with 0.5 ml of a hypertonic acid buffer (Earle's buffer containing 0.2 m acetic acid and 0.5 m NaCl, pH 4) for 3 min to strip off surface-bound radioactivity (but retain intracellularly sequestered SRIF). Cells were then harvested with 1 ml of 0.1 m NaOH and cell associated radioactivity was counted in a γ-counter. Nonspecific binding, as measured in the presence of 1 μm unlabeled [d-Trp8]SRIF (Neosystem, Lyon, France), represented less than 5% of the total binding. Equilibrium Binding Experiments on Whole Cells—After equilibration for 10 min at 37 °C in Earle's buffer, saturation experiments were performed by incubating cells for 30 min at 37 °C with increasing concentrations (0.5 to 16 nm) of 125I-SRIF isotopically diluted with unlabeled [d-Trp8]SRIF in the binding buffer. At the end of the incubation, cells were washed twice with 0.5 ml of equilibration buffer and harvested with 1 ml of 0.1 m NaOH. Nonspecific binding was measured in the presence of 1 μm nonlabeled [d-Trp8]SRIF. Dissociation constant (Kd) and maximal binding capacity (Bmax) were derived from Scatchard analysis of the data. Equilibrium Binding Experiments on Crude Cell Homogenates— Crude cell homogenates (50 μg) were incubated with increasing concentrations (0.5 to 1.6 nm) of 125I-SRIF for 30 min at 25 °C in 250 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 2 mm MgCl2 containing 1% BSA and 0.8 mm 1,10-phenanthroline). Binding experiments were terminated by addition of 3 ml of ice-cold buffer followed by filtration through glass microfiber filters (GF/C, Whatman, Clifton, NJ) preincubated with 0.5% polyethylenimine. After washing twice with 3 ml of ice-cold buffer, the radioactivity retained on the filter was counted in a γ-counter. Nonspecific binding was measured in the presence of 1 μm unlabeled [d-Trp8]SRIF. All binding/internalization data were calculated and plotted using Prism 3.02 (Graph Pad Software) and represent the mean ± S.D. of n determinations (as indicated in “Results”). Internalization of α-Bodipy Red [d-Trp8]SRIF (fluo-SRIF) in AtT-20 Cells—For confocal microscopic tracking of the internalized ligand, the pH-insensitive dye Bodipy 576/589 (Molecular Probes, Inc., Eugene, OR) emitting red fluorescence was covalently conjugated to the degradation-resistant SRIF analog [d-Trp8]SRIF in the α-position (for more detail, see Ref. 51Nouel D. Gaudriault G. Houle M. Reisine T. Vincent J.P. Mazella J. Beaudet A. Endocrinology. 1997; 138: 296-306Crossref PubMed Scopus (103) Google Scholar). AtT-20 cells, grown on 12-mm polylysine-coated glass coverslips in 18-mm Petri dishes were equilibrated for 10 min at 37 °C in Earle's buffer containing 1% BSA and 0.1% glucose. They were then incubated for 10 or 40 min in the same buffer with 20 nm fluo-SRIF (kindly provided by Prof. J. P. Vincent), in the presence or absence of 10-5m non-fluorescent [d-Trp8]SRIF. For selective visualization of internalized fluo-SRIF, cells were washed with hypertonic acid buffer, pH 4, for 3 min. Labeled cells were then mounted on glass slides with Aquamount, air-dried, and examined under a Zeiss laser scanning confocal microscope equipped with an Axiovert 100 inverted microscope and an argon-krypton laser. Samples were scanned at 568 nm wave-length excitation. Images were acquired as single transcellular optical sections and averaged over 16 scans/frame and processed using the Carl Zeiss CLSM software 3.1 version. The final composites were adjusted for contrast and brightness using Adobe Photoshop 6.0 software (Adobe, San Jose, CA) and processed using Deneba's Canvas 7.0 imaging software (Deneba Software, Miami, FL) on an Apple Powerbook G3. Immunodetection of sst2A and sst5 Receptors in AtT-20 Cells—AtT-20 cells, plated on poly-l-lysine-coated glass coverslips, were fixed for 20 min with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, rinsed twice with 0.1 m Trizma (Tris base)-buffered saline (TBS), pH 7.4, and preincubated for 30 min at room temperature with a blocking solution consisting of 5% normal goat serum, 2% BSA, and 0.1% Triton X-100 (BDH Inc., Toronto, Ontario, Canada) in 0.1 m TBS. Immunostaining was performed by incubating cells overnight at 4 °C in TBS containing 0.05% Triton X-100 with one of the following rabbit antibodies: 1) 1:1000 dilution of sst5 serum; 2) 1:2000 dilution of sst2A antibody. The specificity of each of these antibodies has been fully established elsewhere (52Gu Y.Z. Brown P.J. Loose-Mitchell D.S. Stork P.J. Schonbrunn A. Mol. Pharmacol. 1995; 48: 1004-1014PubMed Google Scholar, 53Dournaud P. Gu Y.Z. Schonbrunn A. Mazella J. Tannenbaum G.S. Beaudet A. J. Neurosci. 1996; 16: 4468-4478Crossref PubMed Google Scholar, 54Hipkin R.W. Friedman J. Clark R.B. Eppler C.M. Schonbrunn A. J. Biol. Chem. 1997; 272: 13869-13876Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 55Roth A. Kreienkamp H.J. Nehring R.B. Roosterman D. Meyerhof W. Richter D. DNA Cell Biol. 1997; 16: 111-119Crossref PubMed Scopus (64) Google Scholar, 56Stroh T. Kreienkamp H.J. Beaudet A. J. Comp. Neurol. 1999; 412: 69-82Crossref PubMed Scopus (69) Google Scholar). After three rinses (5 min each) in TBS, cells were incubated for 1 h at room temperature with an Alexa 594-conjugated goat anti-rabbit IgG (1:750; Molecular Probes). They were then washed twice in TBS and mounted on glass slides with Aquamount for confocal microscopic examination. Images were acquired, stored, and archived as described above. Double Immunofluorescence Labeling—To identify the intracellular compartments of somatostatin receptor sequestration, WT and amphiphysin IIb-transfected AtT-20 cells were fixed with 4% paraformaldehyde for 20 min at room temperature, washed twice with 0.1 m TBS, and preincubated for 30 min in the same buffer containing 5% normal goat serum (normal goat serum), 2% BSA, and 0.1% Triton X-100. They were then rinsed twice with TBS and incubated in a mixture of primary antibodies in TBS containing 0.5% normal goat serum and 0.05% Triton X-100 overnight at 4 °C. The mixture contained the mouse anti-syntaxin 6 antibody (3 μg/ml; Transduction Laboratories, Mississauga, Ontario, Canada) and either sst5 (1:1000 of serum) or sst2A (1:2000) antibodies raised in rabbit. After rinsing three times (5 min each) with TBS, bound primary antibodies were revealed by simultaneous incubation with goat anti-mouse Alexa 488- (1:500; Molecular Probes) and goat anti-rabbit Alexa 594-conjugated secondary antibodies (1:750; Molecular Probes) for 60 min at room temperature. After washing, the coverslips were mounted on glass slides using Aquamount and viewed with a confocal microscope. ACTH Release Studies—WT and amphiphysin IIb-overexpressing AtT-20 cells were plated in 16-mm multiwell culture dishes and allowed to form monolayers for 48 h prior to experiments. ACTH release was measured on intact and attached cells as previously described (57Guild S.B. Murray A.T. Wilson M.L. Wiegand U.K. Apps D.K. Jin Y. Rindler M. Roder J. Jeromin A. Mol. Cell. Endocrinol. 2001; 184: 51-63Crossref PubMed Scopus (7) Google Scholar, 58Gully D. Geslin M. Serva L. Fontaine E. Roger P. Lair C. Darre V. Marcy C. Rouby P.E. Simiand J. Guitard J. Gout G. Steinberg R. Rodier D. Griebel G. Soubrie P. Pascal M. Pruss R. Scatton B. Maffrand J.P. Le Fur G. J. Pharmacol. Exp. Therap. 2002; 301: 322-332Crossref PubMed Scopus (72) Google Scholar). Each well was washed twice with 1 ml of DMEM supplemented with 0.1% BSA (DMEM/BSA) and then incubated for 1 h in 1 ml of fresh DMEM/BSA at 37 °C in a humidified atmosphere of 10% CO2 in air. The DMEM/BSA was then decanted and replaced with 500 μl of fresh DMEM/BSA in the absence or presence of corticotrophin-releasing factor 41 (CRF-41; Neosystem, Lyon, France) and [d-Trp8]SRIF, alone or in combination. Zero time samples were taken at this point and the remaining cells were incubated for 2 h at 37 °C in a humidified atmosphere of 10% CO2 in air. Incubations were terminated by collecting the DMEM/BSA medium, centrifugating this medium for 30 s at 10,000 × g, and removing of the supernatant. The ACTH content of the supernatant was measured using a radioimmunoassay kit (Diasorin, Still-water, MN). Values were expressed as the mean ± S.E. of three determinations performed in duplicate. Calculations and statistical analyses were performed using Prism 3.02 (Graph Pad Software, San Diego, CA). Statistical significance was verified using a repeated Measures analysis of variance with a Bonferroni's Multiple Comparison Test. Detection of Amphiphysin IIb in AtT-20 Cells—To investigate amphiphysin IIb function, we generated an AtT-20 cell line overexpressing amphiphysin IIb. The expression of amphiphysin IIb was examined by Western blotting on cell homogenates prepared from both WT and amphiphysin IIb-transfected AtT-20 cells using a polyclonal antib
Referência(s)