RESUMO
Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.
Assuntos
Clatrina/metabolismo , Detergentes/farmacologia , Endocitose , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Receptores de Interleucina-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Resistência a Medicamentos , Dinaminas , Endocitose/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Microscopia Eletrônica , Mutação/genética , Octoxinol/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores da Transferrina/metabolismo , Transfecção , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The Ras-related GTP-binding protein Cdc42 has been implicated in a diversity of biological functions including the regulation of intracellular trafficking and endocytosis. While screening for Cdc42 targets that influence these activities, we identified the protein-tyrosine kinase ACK2 (for activated Cdc42-associated kinase 2) as a new binding partner for clathrin. ACK2 binds clathrin via a domain that is conserved among a number of other clathrin-binding proteins including the arrestins and AP-2. Overexpression of ACK2 in NIH3T3 cells results in an inhibition of transferrin receptor endocytosis because of a competition between ACK2 and AP-2 for clathrin. Activated Cdc42 weakens the interaction between ACK2 and clathrin and thus reverses the ACK2-mediated inhibition of endocytosis. Overexpression of ACK2 increases the amount of clathrin present in fractions enriched in clathrin-coated vesicles. Taken together, our data suggest that ACK2 may represent a novel clathrin-assembly protein and participate in the regulation of receptor-mediated endocytosis.