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.
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, gamma-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.
Stöckli
Jacqueline
J
Institute of Physiology, University of Zurich, Zurich 8057, Switzerland.
Höning
Stefan
S
Rohrer
Jack
J
D020727:ADP-Ribosylation Factors / Q000502:physiology*; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000502:physiology; D016415:Sequence Alignment; D018808:Transcription Factor AP-1 / Q000502:physiology*
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C407306:GGA adaptor proteins; D017527:Receptor, IGF Type 2; D018808:Transcription Factor AP-1; D020727:ADP-Ribosylation Factors