AMSH interacts with ESCRT-0 to regulate the stability and trafficking of CXCR4.
Reversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, such as the chemokine receptor CXCR4. The deubiquitinating enzyme AMSH has been implicated in the endocytic sorting of both G protein-coupled receptors and receptor-tyrosine kinases. Herein, we examine the role of AMSH in the regulation of CXCR4 stability and trafficking and characterize protein-protein interactions critical for this function. Loss of AMSH catalytic activity or depletion by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Analysis of truncation and point mutation of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the SH3 domains of the STAM and Grb2 families of adaptor proteins with high affinity. Cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination, but not increased degradation, of the ESCRT-0 components STAM1 and Hrs. This is dependent on the RXXK motif of AMSH. Ubiquitination of endocytic machinery modulates their activity, suggesting that AMSH may directly regulate endocytic adaptor protein function. This is reflected in CXCR4 trafficking and provides a mechanism by which AMSH specifies the fate of endocytosed receptors. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery.
Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637, USA.
Maria I
MI
Sierra
Michelle H
MH
Wright
Piers D
PD
Nash
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D004705:Endocytosis / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D055550:Protein Stability; D021381:Protein Transport / Q000502:physiology; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C527276:CXCR4 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010750:Phosphoproteins; D019718:Receptors, CXCR4; C109775:STAM protein, human; C486696:STAMBP protein, human; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D043222:Ubiquitin Thiolesterase