Mechanisms of ligand-induced internalization of receptor tyrosine kinases
Grant-in-Aid for Scientific Research (C)
OKABAYASHI Yoshinori Kobe University, University Hospital, assistant professor, 医学部・附属病院, 助手 (10233363)
|Project Period (FY)
1997 – 1998
Completed (Fiscal Year 1998)
|Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
|Shc / AP2 / dynamin II / EGF receptor / internalization / AP2 / PHドメイン / proline-richドメイン / 受容体チロシンキナーゼ
Several growth factors, such as epidermal growth factor (EGF), insulin, and platelet-derived growth factor, exert their pleiotropic effects by binding to and activating cell surface receptors with the intrinsic protein tyrosine kinase activity. Following ligand binding, the receptor is rapidly internalized and subsequently enters distinct cellular sorting pathways leading to either degradation or recycling to the cell surface. The internalization of cell surface receptor-ligand complexs occurs via a multi-step process termed receptor-mediated endocytosis. Receptor-mediated endocytosis involves clustering of the membrane receptors accompanied by coat assembly, invagination of the membrane into coated pits, and, finally, budding to form coated vesicles. In this study we investigated the role of Shc and dynamin II in this process.
Adaptor complex AP2, a component of plasma membrane clathrin-coated pits and vesicles, binds to the amino acid residues 346-355 of Shc. Peptide binding assay sho
wed that both phenylalanines at 349 and 354 of Shc are necessary for interaction with AP2. Mutagenesis analysis showed that EGF stimulates complex formation of Shc and AP2 and association of Shc-AP2 complex with EGF receptors. Internalization assay showed that ^<125>I-EGF internalization was reduced in cells overexpressing mutant Shc in which both phenylalanines at 349 and 354 were substituted with alanines. Immunocytochemical staining study showed that dense punctate labeling along the plasma membrane border as well as punctate pattern characteristic of cytoplasmic vesicles near the plasma membrane were enhanced in cells expressing wild-type Shc.
In order to investigate the functional role of pleckstrin homology (PH) and proline-rich domains of dynamin II, we did mutagenesis analysis. In cells expressing mutant dynamin II with deleted PH domain, ^<125> I-EGF internalization was markedly reduced. This mutant was able to bind to EGF receptors but failed to form dimers. Another mutant lacking C-terminal proline-rich domain was unable to bind to EGF receptors and was unable to form higher order oliomers although this mutant was present as a dimer. The GTPase activity of these mutant dynamin II was markedly reduced.
These results indicate that Shc and dynamin II which is expressed ubiquitously are implicated in ligand-induced receptor internalization in various cells. Shc seems to be implicated in the step of clustering of the membrane receptors accompanied by coat assembly and invagination of the membrane into coated pits. Both PH and proline-rich domains are necessary for dynamin II function in pinching off the nech of the coated pits to form vesicles. Less
Report (3 results)
Research Products (4 results)