Exchanging the Extracellular Domain of Amyloid Precursor Protein for Horseradish Peroxidase Does Not Interfere with α-Secretase Cleavage of the β-Amyloid Region, but Randomizes Secretion in Madin-Darby Canine Kidney Cells

doi:10.1074/jbc.270.51.30310

Exchanging the Extracellular Domain of Amyloid Precursor Protein for Horseradish Peroxidase Does Not Interfere with α-Secretase Cleavage of the β-Amyloid Region, but Randomizes Secretion in Madin-Darby Canine Kidney Cells(*)

From the Experimental Genetics Group, Center for Human Genetics, Campus Gasthuisberg O & N, KULeuven, B-3000 Leuven, Belgium

^¶To whom correspondence should be addressed. Tel.: 32-16-345862; Fax: 32-16-345871; fredvl{at}cc3.kuleuven.ac.be.

Abstract

Secretory processing and polarized sorting of horseradish peroxidase fused to the amyloid precursor protein transmembrane domain were compared with those of wild-type amyloid precursor protein in COS and polarized Madin-Darby canine kidney (MDCK) cells. The cellular and secreted forms of the chimeric protein were enzymatically active in colorimetric and cytochemical assays after reconstitution with hemin and Ca²⁺. The peroxidase enzyme was secreted by a proteolytic process, similar to the parent amyloid precursor protein. In polarized MDCK cells, amyloid precursor protein was secreted exclusively in the basolateral compartment, while the peroxidase chimeric protein was secreted in both compartments. The basolateral sorting determinant for secretion must therefore be located in the extracellular domain of amyloid precursor protein. On the other hand, cell surface-associated peroxidase chimeric protein was similar to cell surface-associated wild-type amyloid precursor protein, mainly expressed at the basolateral side. The basolateral cell-surface expression, in contrast to the basolateral secretion, is therefore controlled by determinants in the cytoplasmic domain. Methylamine inhibited and bafilomycin slightly increased the basolateral secretion of both proteins, but both drugs strongly increased apical secretion. The default secretory pathway of COS cells and the basolateral (but not the apical) secretory pathway of MDCK cells are therefore comparably sensitive to methylamine and not to bafilomycin.

Footnotes

↵^§ Postdoctoral Fellow of the “Nationaal Fonds voor Wetenschappelijk Onderzoek.”
↵* This work was supported by Grants 3.0069.89 and 3.0073.93 from the “Fonds voor Geneeskundig Wetenschappelijk Onderzoek,” by European Community Contracts BIOT-CT94-2065 and COF 94/12, and by a grant from the Inter-university Network for Fundamental Research (1991-1996). Part of this work was supported by Contract ETC-008 from the Action Program for Biotechnology of the Flemish Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

APP

amyloid precursor protein

APP

soluble APP

MDCK

Madin-Darby canine kidney

HRP

horseradish peroxidase
- Received July 6, 1995.
- Revision received September 8, 1995.