INHIBITION OF RECEPTOR-MEDIATED ENDOCYTOSIS BY THE AMPHIPHYSIN SH3 DOMAIN

Background: Receptor-mediated endocytosis appears to require the GTP-b inding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several prol ine-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate pro tein-protein interactions. Amphiphysin, a protein that is highly expre ssed in the brain, interacts with dynamin in vitro, as do Grb2 and man y other SH3 domain-containing proteins. In this study, we examined the role of amphiphysin in receptor-mediated endocytosis in vivo. Results : To address the importance of the amphiphysin SH3 domain in dynamin r ecruitment, we used a transferrin and epidermal growth factor (EGF) up take assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immun ofluorescence revealed that cells transfected with the amphiphysin SH3 domain showed a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cell s were co transfected with both dynamin and the amphiphysin SH3 domain , here, transferrin uptake was efficiently rescued. importantly, the S H3 domains of Gr62, phospholipase Cy and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recr uiting dynamin was additionally tested in vitro: amphiphysin could ass ociate with both dynamin and alpha-adaptin simultaneously, further sup porting a role for amphiphysin in endocytosis. Conclusions: Our result s suggest that the. SH3 domain of amphiphysin recruits dynamin to coat ed pits in vivo, probably via plasma membrane adaptor complexes. We pr opose that amphiphysin is not only required for synaptic-vesicle endoc ytosis, but might also be a key player in dynamin recruitment in all c ells undergoing receptor-mediated endocytosis.