A binding site for SH3 domains targets dynamin to coated pits.
Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, proline-rich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wild-type forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to nonclathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.
Shpetner
H S
HS
Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
Herskovits
J S
JS
Vallee
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015398:Signal Transduction; D018909:src Homology Domains
D020558:GTP Phosphohydrolases; D034281:Dynamins