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J. Biol. Chem., Vol. 279, Issue 22, 23542-23549, May 28, 2004

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The Acidic Cluster of the CK2 Site of the Cation-dependent Mannose 6-Phosphate Receptor (CD-MPR) but Not Its Phosphorylation Is Required for GGA1 and AP-1 Binding^*

Jacqueline Stöckli, Stefan Höning, and Jack Rohrer¶

From the Institute of Physiology, University of Zurich, Zurich 8057, Switzerland and the Institute for Biochemistry II, University of Göttingen, 37073 Göttingen, Germany

Lysosomal biogenesis depends on proper transport of lysosomal enzymes by the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the trans-Golgi network (TGN) to endosomes. Trafficking of the CDMPR is mediated by sorting signals in its cytoplasmic tail. GGA1 (Golgi-localizing, -ear-containing, ARF-binding protein-1) binds to CD-MPR in the TGN and targets the receptor to clathrin-coated pits for transport from the TGN to endosomes. The motif of the CD-MPR that interacts with GGA1 was shown to be ⁶¹DXXLL⁶⁵. Reports on increased affinity of cargo, when phosphorylated by casein kinase 2 (CK2), to GGAs focused our interest on the effect of the CD-MPR CK2 site on binding to GGA1. Here we demonstrate that Glu⁵⁸ and Glu⁵⁹ of the CK2 site are essential for high affinity GGA1 binding in vitro, whereas the phosphorylation of Ser⁵⁷ of the CD-MPR has no influence on receptor binding to GGA1. Furthermore, the in vivo interaction between GGA1 and CD-MPR was abolished only when all residues involved in GGA1 binding were mutated, namely, Glu⁵⁸, Glu⁵⁹, Asp⁶¹, Leu⁶⁴, and Leu⁶⁵. In contrast, the binding of adaptor protein-1 (AP-1) to CD-MPR required all the glutamates surrounding the phosphorylation site, namely, Glu⁵⁵, Glu⁵⁶, Glu⁵⁸, and Glu⁵⁹, but like GGA1 binding, was independent of the phosphorylation of Ser⁵⁷. The binding affinity of GGA1 to the CD-MPR was found to be 2.4-fold higher than that of AP-1. This could regulate the binding of the two proteins to the partly overlapping sorting signals, allowing AP-1 binding to the CD-MPR only when GGA1 is released upon autoinhibition by phosphorylation.

Received for publication, December 10, 2003 , and in revised form, March 24, 2004.

^* This work was supported in part by a Prof. Dr. Max Cloetta Fellowship (to J. R.) and a Roche Research Fellowship (to J. S.) as well as a Swiss National Foundation Grant 31-67274. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Tel.: 41-1-635-51-34; Fax: 41-1-635-68-14; E-mail: rohrer.jack{at}access.unizh.ch.

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