Caveolin-1 interacts directly with dynamin-2.
Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.
Yao
Qing
Q
Center for Basic Research in Digestive Diseases, and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
Chen
Jing
J
Cao
Hong
H
Orth
James D
JD
McCaffery
J Michael
JM
Stan
Radu-Virgil
RV
McNiven
Mark A
MA
D000595:Amino Acid Sequence; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats
C495541:Cav1 protein, mouse; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D034285:Dynamin II