Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney 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 and N-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 gamma-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.
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Laboratory of Peptide Research, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Becker
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Skidgel
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D000469:Alkaline Phosphatase / Q000302:isolation & purification; D000818:Animals; D001692:Biological Transport; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity; D002478:Cells, Cultured; D004285:Dogs; D004705:Endocytosis; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000302:isolation & purification*; D008666:Metalloendopeptidases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010738:Type C Phospholipases / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification
D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D002268:Carboxypeptidases; C058479:carboxypeptidase M; D008666:Metalloendopeptidases; D043265:Phosphatidylinositol Diacylglycerol-Lyase