Interaction of the Cation-dependent Mannose 6-Phosphate Receptor with GGA Proteins

Abstract

The GGAs (Golgi-localizing, ␥-adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S-transferase pulldown assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.

Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

“…This latter finding is in agreement with observations from a previous study in which clathrin expression was reduced by antisense RNA (Iversen et al, 2003). The involvement of AP1 in the sorting of cathepsin D could depend on its role in the packaging of mannose 6-phosphate receptors into clathrin-coated vesicles at the TGN (Doray et al, 2002). However, AP1 has also been proposed to be localized to endosomes (Futter et al, 1998) and to play a role in the sorting of mannose 6-phosphate receptors from endosomes to the TGN (Meyer et al, 2000).…”

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

“…This requirement for serine phosphorylation to effect autoinhibition may relate to the internal location of the AC-LL motifs of GGA1 and GGA3. We and others have shown that the binding of the MPR AC-LL motifs to the VHS domains requires a precise spacing between the dileucines and the free C terminus of the receptors (12,18). The addition of alanines to the MPR C terminus greatly inhibited binding to the VHS domain.…”

Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors

“…2D). Furthermore, depletion of GAK had a marked effect on trafficking of MPR from the TGN to the lysosome, which normally occurs via vesicles containing clathrin, GGA and AP1 (Doray et al, 2002). Most of the MPR is distributed throughout the cytosol in the knockdown cells, whereas it is present at the TGN in mockdepleted cells (Fig.…”