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Carboxypeptidase M, a Glycosylphosphatidylinositol-anchored Protein, Is Localized on Both the Apical and Basolateral Domains of Polarized Madin-Darby Canine Kidney Cells

1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês

10.1074/jbc.274.44.31632

1083-351X

Gerd McGwire, Robert P. Becker, Randal A. Skidgel,

Receptor Mechanisms and Signaling

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor andN-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane γ-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4–8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells. Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor andN-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane γ-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4–8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells. carboxypeptidase M glycosylphosphatidylinositol Madin-Darby canine kidney epidermal growth factor fetal bovine serum Dulbecco's modified Eagle's medium Hanks' balanced salt solution phosphate-buffered saline sulfo-N-hydroxysuccinimidobiotin 5-dimethylaminonaphthalene-1-sulfonyl-l-alanyl-l-arginine phosphatidylinositol-specific phospholipase C bovine serum albumin polyacrylamide gel electrophoresis Regulatory B-type carboxypeptidases play important roles by specifically cleaving C-terminal Arg or Lys residues from peptides and proteins (1Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor & Francis Ltd., London1996: 241-283Google Scholar). Carboxypeptidase M (CPM),1 a member of this family of enzymes, is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane enzyme, widely distributed in human tissues (1Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor & Francis Ltd., London1996: 241-283Google Scholar, 2Skidgel R.A. Johnson A.R. Erdös E.G. Biochem. Pharmacol. 1984; 33: 3471-3478Crossref PubMed Scopus (86) Google Scholar, 3Skidgel R.A. Trends Pharmacol. Sci. 1988; 9: 299-304Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 4Nagae A. Deddish P.A. Becker R.P. Anderson C.H. Abe M. Tan F. Skidgel R.A. Erdös E.G. J. Neurochem. 1992; 59: 2201-2212Crossref PubMed Scopus (44) Google Scholar, 5Nagae A. Abe M. Becker R.P. Deddish P.A. Skidgel R.A. Erdös E.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 221-229Crossref PubMed Scopus (67) Google Scholar) and often highly expressed in epithelial cells (1Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor & Francis Ltd., London1996: 241-283Google Scholar, 5Nagae A. Abe M. Becker R.P. Deddish P.A. Skidgel R.A. Erdös E.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 221-229Crossref PubMed Scopus (67) Google Scholar), including Madin-Darby canine kidney (MDCK) cells (6Deddish P.A. Skidgel R.A. Kriho V.B. Li X.Y. Becker R.P. Erdös E.G. J. Biol. Chem. 1990; 265: 15083-15089Abstract Full Text PDF PubMed Google Scholar). The MDCK cell line is speculated to have originated from distal renal tubular epithelial cells (7Rodriguez-Boulan E. Salas P.J.I. Annu. Rev. Physiol. 1989; 51: 741-754Crossref PubMed Scopus (24) Google Scholar, 8Cereijido M. Contreras R.G. Gonzalez-Mariscal L. Annu. Rev. Physiol. 1989; 51: 785-795Crossref PubMed Scopus (45) Google Scholar) and has been used extensively as a model of polarized renal tubular epithelium. These cells were also used to show that all GPI-anchored surface proteins are specifically localized to the apical surface (9Lisanti M.P. Sargiacomo M. Graeve L. Saltiel A.R. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9557-9561Crossref PubMed Scopus (290) Google Scholar). Additional studies on the sorting of GPI-anchored proteins revealed their apical localization to be a conserved feature of polarized epithelial cells from other tissues and species (10Lisanti M.P. Le Bivic A. Saltiel A.R. Rodriguez-Boulan E. J. Membr. Biol. 1990; 113: 155-167Crossref PubMed Scopus (110) Google Scholar). These data, together with studies using genetically engineered GPI fusion proteins (11Brown D.A. Crise B. Rose J.K. Science. 1989; 245: 1499-1501Crossref PubMed Scopus (305) Google Scholar, 12Lisanti M.P. Caras I.W. Davitz M.A. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar), resulted in the classification of the GPI anchor as a dominant apical sorting signal (13Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar, 14Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar). More recently, the MDCK cell line was used as a model system to show that N-linked carbohydrate is an additional apical targeting signal (13Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar, 15Gut A. Kappeler F. Hyka N. Balda M.S. Hauri H.-P. Matter K. EMBO J. 1998; 17: 1919-1929Crossref PubMed Scopus (176) Google Scholar). CPM activity and mRNA are found in human kidney (5Nagae A. Abe M. Becker R.P. Deddish P.A. Skidgel R.A. Erdös E.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 221-229Crossref PubMed Scopus (67) Google Scholar, 16Tan F. Chan S.J. Steiner D.F. Schilling J.W. Skidgel R.A. J. Biol. Chem. 1989; 264: 13165-13170Abstract Full Text PDF PubMed Google Scholar), which also secretes CPM into urine (17McGwire G.B. Skidgel R.A. J. Biol. Chem. 1995; 270: 17154-17158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Skidgel R.A. Davis R.M. Erdös E.G. Anal. Biochem. 1984; 140: 520-531Crossref PubMed Scopus (46) Google Scholar). Although the roles of CPM in kidney function have not been clearly defined, erythropoietin, bradykinin, and epidermal growth factor (EGF) are potential CPM substrates that are generated in the kidney and excreted into urine. Kinetic studies with bradykinin (19Skidgel R.A. Davis R.M. Tan F. J. Biol. Chem. 1989; 264: 2236-2241Abstract Full Text PDF PubMed Google Scholar) and EGF (17McGwire G.B. Skidgel R.A. J. Biol. Chem. 1995; 270: 17154-17158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) showed that these peptides are good substrates of CPM in vitro. Bradykinin induces natriuresis, diuresis, and prostaglandin synthesis in the kidney; thus, inactivation by CPM could play a role in the regulation of salt and water balance (1Skidgel R.A. Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor & Francis Ltd., London1996: 241-283Google Scholar, 20Carretero O.A. Scicli A.G. Laragh J.H. Brenner B.M. Kaplan N.M. Endocrine Mechanisms in Hypertension. Raven Press, Ltd., New York1989: 219-239Google Scholar). The biological role of CPM in renal tubular epithelium will depend on its apical or basolateral localization because its endogenous peptide substrates and corresponding receptors can also have polarized distributions. For example, CPM is responsible for the initial metabolism of EGF to des-Arg53-EGF at the surface of MDCK cells (17McGwire G.B. Skidgel R.A. J. Biol. Chem. 1995; 270: 17154-17158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Because the EGF receptor is predominantly expressed on the basolateral domain of these cells (21Maratos-Flier E. Kao C.-Y.Y. Verdin E.M. King G.L. J. Cell Biol. 1987; 105: 1595-1601Crossref PubMed Scopus (95) Google Scholar), the functioning of CPM in this pathway would be possible only if it was expressed on the same domain. CPM was positively identified on the apical surface of MDCK cells (6Deddish P.A. Skidgel R.A. Kriho V.B. Li X.Y. Becker R.P. Erdös E.G. J. Biol. Chem. 1990; 265: 15083-15089Abstract Full Text PDF PubMed Google Scholar), but a possible basolateral localization could not be determined because of the inaccessibility of the antibodies to this surface in the techniques that were employed. However, the fact that CPM is both GPI-anchored and N-glycosylated would argue against a substantial basolateral distribution. In this study, the cell-surface distribution and sorting of CPM were investigated. We show that CPM is present on both the apical and basolateral domains of MDCK cells, in contrast to the apical localization reported for other GPI-anchored proteins. Fetal bovine serum (FBS) was from Atlanta Biologicals, Inc. Dulbecco's modified Eagle's medium (DMEM), Ham's nutrient mixture F-12, Hanks' balanced salt solution (HBSS), phosphate-buffered saline (PBS), reduced glutathione, iodoacetamide, Triton X-100, and Triton X-114 were from Sigma. Sulfo-N-hydroxysuccinimido-LC-biotin (sulfo-NHS-LC-biotin) and sulfo-NHS-SS-biotin were from Pierce. Immobilized streptavidin was from Roche Molecular Biochemicals. 5-Dimethylaminonaphthalene-1-sulfonyl-l-alanyl-l-arginine (dansyl-Ala-Arg) was synthesized and purified as described (22Tan F. Deddish P.A. Skidgel R.A. Methods Enzymol. 1995; 248: 663-675Crossref PubMed Scopus (29) Google Scholar). The ProLong anti-fade kit and fluorescein isothiocyanate-conjugated goat anti-rabbit Alexa 488 were from Molecular Probes, Inc. (Eugene, OR). Redivue Pro-mix l-[35S] in vitrolabeling mixture was from Amersham Pharmacia Biotech. Phosphatidylinositol-specific phospholipase C (PI-PLC) fromBacillus thuringiensis was from ICN or Oxford Glycosystems. Most other chemicals were from Fisher. MDCK cells (CCL-34) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM containing 4.7 g/liter sodium bicarbonate, 25 mm Hepes, 100 units/liter penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated FBS. CPM activity was determined in a fluorometric assay with dansyl-Ala-Arg as the substrate as previously published (22Tan F. Deddish P.A. Skidgel R.A. Methods Enzymol. 1995; 248: 663-675Crossref PubMed Scopus (29) Google Scholar, 23Skidgel R.A. Conn P.M. Methods in Neurosciences: Peptide Technology. Academic Press, Inc., Orlando, FL1991: 373-385Google Scholar). For measurement of CPM activity in intact monolayers, cells were rinsed with HBSS containing 4.7 g/liter sodium bicarbonate, 25 mmHepes, 100 units/liter penicillin, and 0.1 mg/ml streptomycin (HBSS-BH). Buffer containing 0.2 mm dansyl-Ala-Arg was added apically or basolaterally to 12-mm (0.5 ml) or 24.5-mm (1 ml) inserts. An equal amount of buffer without dansyl-Ala-Arg was added to the opposite, control side. Cells were incubated at 37 °C in 5% CO2 for 0.5–1 h, and then 250 μl of buffer was collected from each side and added to 150 μl of 1 m citric acid. The product was extracted, and the fluorescence was measured as described (22Tan F. Deddish P.A. Skidgel R.A. Methods Enzymol. 1995; 248: 663-675Crossref PubMed Scopus (29) Google Scholar, 23Skidgel R.A. Conn P.M. Methods in Neurosciences: Peptide Technology. Academic Press, Inc., Orlando, FL1991: 373-385Google Scholar). Alkaline phosphatase was measured in a colorimetric end-point assay using a kit (Sigma) as described by the manufacturer, except that the reaction was scaled down to use 25 μl of sample in a total reaction volume of 275 μl. γ-Glutamyl transpeptidase was measured in a colorimetric end-point assay with γ-glutamyl-p-nitroanilide as the substrate essentially as described (24Orlowski M. Arch. Immunol. Ther. Exp. 1965; 13: 538-541Google Scholar, 25Naftalin L. Sexton M. Whitaker J.F. Tracey D. Clin. Chim. Acta. 1969; 26: 293-296Crossref PubMed Scopus (197) Google Scholar). Protein concentrations were measured as described (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using BSA as the standard. MDCK cells (5 × 104 or 2.5 × 105 cells) were seeded into 12- or 24.5-mm Transwell cell culture inserts, respectively, and grown for 5–7 days, after which experiments were performed. The integrity and tightness of the MDCK monolayers were routinely determined by transepithelial electrical resistance and occasionally by [3H]methoxyinulin (5000 Da) diffusion. Cells used for experiments had a transepithelial electrical resistance of >400 ohms·cm2 and an apical-to-basolateral [3H]methoxyinulin (1 μCi/ml) diffusion of <2%. Cell monolayers in 24.5-mm inserts were biotinylated apically or basolaterally as described (9Lisanti M.P. Sargiacomo M. Graeve L. Saltiel A.R. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9557-9561Crossref PubMed Scopus (290) Google Scholar) with some modifications. All treatments were performed on ice. Cells were washed five times with ice-cold PBS containing 0.1 mmCaCl2 and 1 mm MgCl2 (PBS-CM), and 1 ml of sulfo-NHS-LC-biotin (0.5 mg/ml) in PBS-CM was added to one or both sides of the inserts. Buffer alone was added to the opposite side when biotinylation was performed on only one side. Cells were incubated for 20 min, after which the solution was removed, fresh biotin solution was added, and the incubation was repeated. Free biotin was quenched by washing the cells three times for 2 min each with serum-free DMEM containing 50 mm NH4Cl. Membranes containing biotinylated cell monolayers were excised, and the cells were solubilized in 20 mm potassium phosphate buffer, pH 7.5, containing 0.15 m NaCl, 1% Triton X-100, and 60 mm n-octyl glucoside for 2–12 h at 4 °C with rotation. The filters were removed, and insoluble material was removed by a 1-h centrifugation at 100,000 × g. An either equal or double volume of immobilized streptavidin slurry (50%) was added to the lysates, and the mixture was incubated at 4 °C overnight. The streptavidin was removed by a 10-min centrifugation in a microcentrifuge, and CPM, γ-glutamyl transpeptidase, and alkaline phosphatase activities were measured in the supernatant. Activities in samples were compared with controls treated identically except for omission of the biotinylation reagent. The amount of each enzyme on the apical and basolateral domains was taken as the amount of activity precipitated by streptavidin following apical and basolateral biotinylation, respectively. Membranes containing biotinylated cell monolayers were excised, and the cells were solubilized in 1 ml of cell lysis buffer (25 mm Tris-HCl, pH 7.5, containing 0.1m NaCl, 2.5% Triton X-100, 60 mm n-octyl glucoside, 5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 0.1 mm leupeptin, 1 μm pepstatin A, and 10 μm E-64 (trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane)) for 1 h at 4 °C with rotation. The filters were removed, and insoluble matter was removed by a 5-min centrifugation in a microcentrifuge. The lysates were preadsorbed with 10 μl of normal rabbit serum (1:2 diluted) followed by 50 μl of a 1:3 protein A-Sepharose slurry. The precipitates were pelleted, and the supernatants were transferred to new tubes. CPM was immunoprecipitated by incubation overnight at 4 °C with 20 μl of polyclonal rabbit anti-human CPM antibody, purified as described (5Nagae A. Abe M. Becker R.P. Deddish P.A. Skidgel R.A. Erdös E.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 221-229Crossref PubMed Scopus (67) Google Scholar), followed by protein A-Sepharose precipitation. The precipitates were washed four times with mixed micelle buffer (25 mm Tris-HCl, pH 8.0, containing 0.15 mm NaCl, 5 mm EDTA, 8% sucrose, 1% Triton X-100, 0.2% SDS, and 0.2 mm phenylmethylsulfonyl fluoride) and then once with the same buffer without detergents. Bound protein was eluted in Laemmli buffer containing 0.1% dithiothreitol and resolved by SDS-PAGE (8% gel). Proteins were electroblotted onto Immobilon-P and detected with streptavidin-horseradish peroxidase followed by chemiluminescence using an ECL kit (Amersham Pharmacia Biotech). Cells were grown for 5 days on 6.25-mm diameter Falcon P.E.T. membrane cell culture inserts. The cells were rinsed twice with HBSS-BH and then fixed in 1% formaldehyde (depolymerized from paraformaldehyde) in the same buffer. The cells were rinsed 3 × 5 min with 0.1 m glycine in HBSS-BH and then 3 × 10 min with HBSS-BH, followed by preincubation in HBSS-BH containing 0.2% BSA and 5% normal goat serum for 30 min. Purified rabbit anti-human CPM antiserum (5Nagae A. Abe M. Becker R.P. Deddish P.A. Skidgel R.A. Erdös E.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 221-229Crossref PubMed Scopus (67) Google Scholar) was diluted 1:20 (final protein concentration = 5 μg/ml) in HBSS-BH containing 0.2% BSA, 5% normal goat serum, and 0.01% sodium azide and was then added to both sides of the insert. The cells were incubated for 36 h at 4 °C, when the antibody was removed, and the cells were washed 3 × 10 min with HBSS-BH. The cells were then again preincubated in HBSS-BH containing 0.2% BSA, 5% normal goat serum, and 0.01% sodium azide for 30 min, followed by a 2-h incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody diluted 1:300 in the same buffer (6.67 μg/ml final concentration) and added to the apical and basolateral sides. The cells were finally washed 3 × 10 min with HBSS-BH and mounted using the ProLong anti-fade mounting medium for Alexa dyes. The fluorescent staining was evaluated on a Zeiss 510 laser scanning confocal microscope. Confluent monolayers in 12-mm inserts were rinsed three times with HBSS-BH. Then, 0.5 ml of the same buffer, containing various concentrations of PI-PLC (0.002–0.5 units/ml) and 0.1% heat-inactivated BSA (56 °C for 30 min), was added to the apical or basolateral side of the inserts; and buffer without PI-PLC was added to the opposite side. After incubation for 4 h at 37 °C, the buffer was collected; any cells were removed by a 10-min centrifugation in a microcentrifuge; and the released CPM activity was determined in the supernatant. The basal spontaneous release of CPM was measured in control cells treated identically except that buffer without PI-PLC was added instead. To measure the release of CPM by PI-PLC from membrane fractions, cells were rinsed with PBS; scraped off; pelleted; resuspended in fractionation buffer (50 mm Hepes, pH 7.5, and 0.25m sucrose) containing 1 mm phenylmethylsulfonyl fluoride, 0.1 mm leupeptin, 1 μm pepstatin A, and 10 μm E-64; lysed by sonication for 3 × 10 s; and fractionated by sequential centrifugation as described (4Nagae A. Deddish P.A. Becker R.P. Anderson C.H. Abe M. Tan F. Skidgel R.A. Erdös E.G. J. Neurochem. 1992; 59: 2201-2212Crossref PubMed Scopus (44) Google Scholar). The final P3 membrane fraction was washed once, resuspended in fractionation buffer, and incubated with or without PI-PLC (0.5 units/ml final concentration) at 37 °C for 1 h. One-half of the volume was removed from each reaction, and the soluble and cell membrane-bound enzymes were separated by a 1-h centrifugation at 100,000 × g. New PI-PLC (0.5 units/ml; 1.0 unit/ml final concentration) or buffer was added to the remaining reaction mixtures; and the incubation was continued for an additional hour, followed by centrifugation as described above. CPM and alkaline phosphatase were measured in both the high speed sediments and supernatants. The surface distribution of GPI-anchored proteins in MDCK cells was determined by domain-specific biotinylation, Triton X-114 extraction, and PI-PLC release essentially as described (9Lisanti M.P. Sargiacomo M. Graeve L. Saltiel A.R. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9557-9561Crossref PubMed Scopus (290) Google Scholar). Samples were separated by SDS-PAGE (7.5% gel) and electroblotted onto Immobilon-P. Biotinylated proteins were detected by alkaline phosphatase-coupled streptavidin. Protein bands were quantitated with a Protein Design Institute scanning densitometer. Confluent monolayers of MDCK cells in 24.5-mm inserts were washed twice with HBSS-BH and then incubated for 30 min in Cys- and Met-deficient DMEM containing 5% dialyzed FBS (1000-Da cutoff). Cells were metabolically labeled for 30 min in 1 mCi/ml [35S]Met/[35S]Cys in the deficient medium. The labeling medium was removed, and the cells were washed twice with complete DMEM containing 10% FBS and then incubated in the same medium containing a 5× normal concentration of unlabeled Met and Cys. At the indicated times, the chase medium was removed; the cells were washed three times with ice-cold PBS-CM; and the apical or basolateral domains were selectively biotinylated as described above. The filters were excised, and the cells were solubilized in cell lysis buffer (1 ml/insert) for 1 h at 4 °C with rotation. The filters were removed, and insoluble matter was removed by a 5-min centrifugation in a microcentrifuge. CPM was immunoprecipitated with specific antiserum and protein A-Sepharose as described above. Precipitated proteins were eluted by boiling the samples for 5 min in 30 μl of 10% SDS. The supernatants were transferred to new tubes and diluted to 1.25 ml with 10 mm Tris-HCl, pH 7.4, containing 0.15 mm NaCl, 1% Triton X-100, and 1 mm EDTA. Biotinylated proteins were precipitated with 50 μl of streptavidin-Sepharose (1:3 slurry) for 1 h at 4 °C. The precipitates were washed twice with mixed micelle buffer and then once with the same buffer without detergents. Precipitated protein was eluted in Laemmli buffer containing 1% dithiothreitol, resolved by SDS-PAGE (8%), and analyzed by autoradiography using a BioMax TranScreen-LE intensifying screen (Eastman Kodak Co.). The endocytosis/transcytosis of basolateral CPM was investigated by biotinylation with sulfo-NHS-SS-biotin, which can be released by reduction with glutathione (27Le Bivic A. Real F.X. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9313-9317Crossref PubMed Scopus (150) Google Scholar). The basolateral side of confluent MDCK cell monolayers in 24.5-mm inserts was biotinylated at 4 °C with sulfo-NHS-SS-biotin using the procedure described above. The cells were incubated at 37 °C in DMEM containing 10% FBS for various amounts of time, after which they were put on ice and rinsed once with ice-cold PBS-CM. Any apical or basolateral surface biotin was then removed by a 30-min incubation with a glutathione-containing solution added to either the basolateral or apical side (1 ml/insert). The solution consisted of 50 mm glutathione, 90 mm NaCl, 1 mm MgCl2, and 0.1 mmCaCl2, to which NaOH (60 mm final concentration) and FBS (10% final concentration) were added just prior to use as described (28Bretscher M.S. Lutter R. EMBO J. 1988; 7: 4087-4092Crossref PubMed Scopus (47) Google Scholar). Control cells were incubated in buffer without glutathione. The cells were then rinsed once with PBS-CM, and free sulfhydryl groups were quenched by rinsing twice with 1 ml of iodoacetamide (5 mg/ml). Following a final rinse with PBS-CM, the membranes were excised, and the cells were solubilized in 1 ml of cell lysis buffer as described above. CPM was immunoprecipitated with polyclonal antiserum to recombinant human CPM, resolved by SDS-PAGE (8%) under nonreducing conditions, and electroblotted onto Immobilon-P. Biotinylated proteins were detected with streptavidin-horseradish peroxidase (1:1500) and the ECL chemiluminescence kit. When the CPM activity on the surface of intact MDCK cell monolayers was measured by the hydrolysis of dansyl-Ala-Arg added to the buffer on either the apical or basolateral side, the apical and basolateral domains contained approximately two-thirds and one-third of the extracellular CPM activity, respectively (TableI). A negligible amount (<2%) of hydrolyzed substrate was detected on the apical side when dansyl-Ala-Arg was added to the basolateral side and vice versa (TableI, Footnote c), indicating that the monolayer was impermeable to the substrate. The carboxypeptidase activity in MDCK cell membranes had previously been identified as CPM by means of enzymatic properties and reactivity with specific antiserum to purified CPM on Western blots (6Deddish P.A. Skidgel R.A. Kriho V.B. Li X.Y. Becker R.P. Erdös E.G. J. Biol. Chem. 1990; 265: 15083-15089Abstract Full Text PDF PubMed Google Scholar). However, it could not be ruled out that another, minor carboxypeptidase was also present on these cells and if, expressed solely on one side of the cells, it could affect the distribution determined for CPM. To exclude this possibility, we specifically immunoprecipitated CPM activity from either the apical or basolateral side by first removing either the apical or basolateral activity with domain-specific biotinylation and streptavidin precipitation, followed by immunoprecipitation of the activity remaining in the supernatant with antiserum specific for CPM. More than 95% of the remaining activity on either the apical or basolateral side was immunoprecipitated by specific anti-CPM antiserum (data not shown), eliminating the possibility of a significant contribution by another peptidase to the activity being measured.Table IExtracellular distribution of CPM in MDCK cellsExtracellular distribution of CPMaResults are the means ± S.E. (n= 6).Domain-selective biotinylationbPercent of total extracellular CPM activity precipitated by streptavidin after either apical or basolateral biotinylation. Total extracellular CPM activity is defined as the combined activity precipitated by streptavidin after biotinylation on both sides. Of the total cellular CPM activity, 45 ± 9 and 35 ± 4% were precipitated after apical and basolateral biotinylation, respectively. The nonprecipitable activity is presumed to be intracellular.Dansyl-Ala-Arg hydrolysiscPercent of total extracellular CPM activity measured by addition of dansyl-Ala-Arg substrate to either the apical or basolateral side. Total extracellular CPM activity is defined as the combined apical and basolateral hydrolysis of dansyl-Ala-Arg. The amount of product (dansyl-Ala) detected on the opposite side was 0.72 ± 0.47% and 1.93 ± 1.20% of that on the test side when dansyl-Ala-Arg was added to the basolateral and apical sides, respectively.%Apical56 ± 767 ± 3Basolateral44 ± 733 ± 3a Results are the means ± S.E. (n= 6).b Percent of total extracellular CPM activity precipitated by streptavidin after either apical or basolateral biotinylation. Total extracellular CPM activity is defined as the combined activity precipitated by streptavidin after biotinylation on both sides. Of the total cellular CPM activity, 45 ± 9 and 35 ± 4% were precipitated after apical and basolateral biotinylation, respectively. The nonprecipitable activity is presumed to be intracellular.c Percent of total extracellular CPM activity measured by addition of dansyl-Ala-Arg substrate to either the apical or basolateral side. Total extracellular CPM activity is defined as the combined apical and basolateral hydrolysis of dansyl-Ala-Arg. The amount of product (dansyl-Ala) detected on the opposite side was 0.72 ± 0.47% and 1.93 ± 1.20% of that on the test side when dansyl-Ala-Arg was added to the basolateral and apical sides, respectively. Open table in a new tab The relatively non-polarized distribution of CPM was also assessed by measuring CPM activity precipitable by streptavidin after domain-selective biotinylation of the cells. The distribution of CPM was even less polarized (56% apical and 44% basolateral) in these experiments (Table I), which may more accurately reflect its true distribution. This is because cells growing on the plastic wall of the inserts are excluded when the filters are excised (see "Experimental Procedures" for details), whereas when substrate is added to each domain, the cells on the wall contribute to the apical (but not the basolateral) activity. Similar results were obtained when apically or basolaterally biotinylated CPM was subjected to SDS-PAGE and visualized with streptavidin-horseradish peroxidase and chemiluminescence (Fig. 1). Quantification of the bands by densitometry showed the apical and basolateral distribution to be 66 and 34%, respectively (average of two experiments). The 52–53-kDa protein detected on both domains is consistent with the previously published molecular