Basolateral Secretion of Amyloid Precursor Protein in Madin-Darby Canine Kidney Cells Is Disturbed by Alterations of Intracellular pH and by Introducing a Mutation Associated with Familial Alzheimers Disease

doi:10.1074/jbc.270.8.4058

Basolateral Secretion of Amyloid Precursor Protein in Madin-Darby Canine Kidney Cells Is Disturbed by Alterations of Intracellular pH and by Introducing a Mutation Associated with Familial Alzheimers Disease (*)

From the Experimental Genetics Group, Center for Human Genetics, Campus Gasthuisberg, KULeuven, B-3000 Leuven, Belgium and
Cephalon Inc., West Chester, Pennsylvania 19380

^¶ To whom correspondence should be addressed:
Experimental Genetics Group, Dept. of Human Genetics, Campus Gasthuisberg O & N 6, B-3000 Leuven, Belgium
. Tel.: 32-16-3458-62; Fax: 32-16-3458-71; fredvl{at}cc3.kuleuven.ac.be.

Abstract

The analysis of potential sorting signals in amyloid precursor protein (APP) by site-directed mutagenesis and the disturbance of metabolic pathways by drugs is used here to define the parameters that determine polarized secretion of APP in Madin-Darby canine kidney cells. Endogenously produced APP751/770 and APP695 produced from transfected constructs are secreted almost exclusively into the basolateral compartment. The sorting mechanism is highly dependent on intracellular pH as demonstrated by its sensitivity to primary amines and inhibitors of the acidifying vacuolar proton ATPase. The role of potential basolateral sorting signals in the cytoplasmic, transmembrane, and βA4 amyloid region of APP was investigated. Neither deletion of the endocytosis and putative basolateral sorting signal GY.NPTY nor complete deletion of the cytoplasmic domain causes apical secretion of soluble APP. Further deletion of the transmembrane domain and of the βA4 amyloid region confirmed that the major basolateral sorting determinant resides in the extracellular domain of APP. Increased β-secretase cleavage of APP after introduction of the “swedish” double mutation causes apical missorting of about 20% of β-secretase-cleaved APP. The data underline the complexity of processing and sorting APP in polarized cells and suggest a possible problem of protein sorting in Alzheimer's Disease.

Footnotes

↵^§ Postdoctoral fellow of the Nationaal Fonds voor Wetenschappelijk Onderzoek.
↵* This investigation was supported by Grants 3.0069.89 and 3.0073.93 from the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, by EC Contract BIOT-CT91-0302, by STW Contract NCH22.2726, by a grant Geconcerteerde Acties from the Ministerie voor Onderwijs of the Belgian Government, and by a grant from the interuniversity network for Fundamental Research (IUAP, 1991-1996). Part of this work was done under contract with the Action Program for Biotechnology of the Flemish government (VLAB-IWT, ETC-008). 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

AD

Alzheimer's disease

EOFAD

early onset familial Alzheimer's disease

TGN

trans-Golgi network

MDCK

Madin-Darby canine kidney

PMA

phorbol 12-myristate 13-acetate

Pdbu

phorbol 12,13-dibutyrate

TES

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid

RSV

Rous sarcoma virus.
- Received September 21, 1994.
- Revision received December 9, 1994.