Recycling of Furin from the Plasma Membrane

doi:10.1074/jbc.274.51.36781

Recycling of Furin from the Plasma Membrane

FUNCTIONAL IMPORTANCE OF THE CYTOPLASMIC TAIL SORTING SIGNALS AND INTERACTION WITH THE AP-2 ADAPTOR MEDIUM CHAIN SUBUNIT*

From the Institut für Virologie der Philipps-Universität Marburg, Robert-Koch Strasse 17, 35037 Marburg, Germany

Abstract

The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL⁷⁶⁵) motif, a leucine-isoleucine (LI⁷⁶⁰) motif, and a phenylalanine (Phe⁷⁹⁰) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (μ2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with μ2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.

Footnotes

↵* This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 286.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Both authors contributed equally to this work.
↵§ Performed essential parts of this work in partial fulfillment of the requirements for a Ph.D. degree from the Philipps-University, Marburg, Germany.
↵¶ To whom correspondence should be addressed: Institut für Virologie der Philipps-Universität Marburg, Robert-Koch Strasse 17, 35037 Marburg, Germany. Tel.: 49-6421-2865145; Fax: 49-6421-2868962; E-mail: garten@mailer.uni-marburg.de.
Abbreviations:

TGN

trans-Golgi network

AP(s)

assembly protein(s)

CKII

casein kinase II

GST

glutathione S-transferase

wt

wild type

GST-F

GST-furin fusion proteins

CDF

CD46-furin chimeras

NRK

normal rat kidney

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

PVDF

polyvinylidene difluoride
- Received April 20, 1999.
- Revision received September 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.