Abstract
We show here that alternative splicing influences the polarized secretion of amyloid precursor protein (APP) as well as the release of its proteolytic 3-4-kDa fragments betaA4 and p3. In Madin-Darby canine kidney II cells stably transfected with various APP isoforms and APP mutants, APPsec was consistently secreted basolaterally. In contrast, Madin-Darby canine kidney II cells transfected with L-APP677, which occurs naturally by alternative splicing of exon 15, secreted this isoform both apically and basolaterally, while maintaining the basolateral sorting of endogenous APPsec. This suggests that the alternative splicing of APP exon 15 modulates the polarized sorting of secretory APP. The same alternative splicing event also decreased the production of betaA4 relative to p3. This is the first example of alternative splicing regulating polarized trafficking of a secretory protein.
Highlights
We show here that alternative splicing influences the a 4-kDa (A4) and 3-kDa (p3) peptides (for a recent review, see polarized secretion of amyloid precursor protein (APP) Refs. 11 and 12). A4 is the principal component of the amyloid as well as the release of its proteolytic 3– 4-kDa fragments A4 and p3
Removal of this signal causes the transmembrane APP to become distributed on both sides and concomitantly increases the release of A4 to the apical rather than the basolateral side, to which it is usually restricted. This mutation does not affect the polarized secretion of APPsec to the basolateral domain, suggesting that an additional determinant of basolateral sorting is present in the luminal domain and that the majority of APPsec is generated intracellularly [14, 15, 18]
L-APP677 tended to be secreted apically (65% apical), the absolute values were variable between individual experiments (Fig. 3 and Fig. 4)
Summary
Madin-Darby canine kidney II cells transfected with L-APP677, which occurs naturally by alternative splicing of exon 15, secreted this isoform both apically and basolaterally, while maintaining the basolateral sorting of endogenous APPsec. In order to identify the luminal sorting signal, we expressed different APP constructs (Fig. 1) in stably transfected MDCK II cells and analyzed the amount of secreted APP in both the apical and basolateral chambers.
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