Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s.
Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.
Department of Pharmacology, Rush University Medical Center, Medical College, Vascular Biology Section, Chicago, IL 60612, USA.
Ivana
I
Knezevic
Dan
D
Predescu
Cristina
C
Bardita
Minhua
M
Wang
Tiffany
T
Sharma
Barbara
B
Keith
Radu
R
Neamu
Asrar B
AB
Malik
Sanda
S
Predescu
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000818:Animals; D021941:Caveolae / Q000502:physiology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002966:Clathrin / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism / Q000502:physiology*; D004705:Endocytosis; D042783:Endothelial Cells; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D008168:Lung; D051381:Rats; D018909:src Homology Domains
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; C485568:ITSN1 protein, human; D020558:GTP Phosphohydrolases; D034285:Dynamin II