Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain.
BACKGROUND
Receptor-mediated endocytosis appears to require the GTP-binding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several proline-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate protein-protein interactions. Amphiphysin, a protein that is highly expressed in the brain, interacts with dynamin in vitro, as do Grb2 and many 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 recruitment, we used a transferrin and epidermal growth factor (EGF) uptake assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence 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 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin could associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis.


CONCLUSIONS
Our results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. We propose that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis.
Wigge
P
P
Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK.
Vallis
Y
Y
McMahon
H T
HT
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D019556:COS Cells; D003034:Coated Pits, Cell-Membrane / Q000502:physiology; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D018909:src Homology Domains
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D014168:Transferrin; C075637:amphiphysin; D020558:GTP Phosphohydrolases; D034281:Dynamins