AMSH regulates CXCR4 stability and trafficking through association with the ESCRT-0 complex

Reversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, as illustrated by the CXC chemokine receptor 4 (CXCR4). The deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM (AMSH ) has been implicated in the endocytic sorting of both r eceptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). We examined the role of AMSH in the regulation of CXCR4 stability and trafficking, and characterized protein-protein interactions critical for AMSH activity. Loss of AMSH catalytic activity or depletion of protein levels by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Truncation and point mutants of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the src homology 3 (SH3) domains of the signal-transducing adaptor molecule (STAM) and growth factor receptor-bound protein 2 (Grb2) families of adaptor proteins with high affinity. Interestingly, cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination of the endosomal sorting complexes required for transport (ESCRT) components STAM1 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) that is also dependant on the RXXK motif of AMSH. As ubiquitination of endocytic machinery modulates their activity, this implicates AMSH as a regulator of endocytic adaptor protein function. This, in turn, greatly affects the fate of endocytosed receptors and is reflected in CXCR4 trafficking. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery.