Vital cellular responses including cell metabolism, proliferation and differentiation are all regulated by specific interactions between extracellular ligands and their plasma membrane-anchored receptors. Accessibility of ligand to receptor, is therefore, an important facet in biological regulation. Receptor expression on the cell surface is dictated, at least in part, by vesicle trafficking via the regulation of receptor internalization. Internalization (also termed endocytosis) is an important regulatory mechanism, which is implicated in receptor resensitization, downregulation, and more recently in signal transduction. Endocytosis of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) is dependent on the invagination and fission of clathrin coated vesicles from the plasma membrane into the cytosol. Constriction of the pits and fission of vesicles from the plasma membrane requires the GTPase activity of dynamin. Recent evidence suggests that protein tyrosine phosphorylation is also required for agonist-regulated internalization of cell surface receptors, although the locus of this protein tyrosine phosphorylation remains undefined. Dynamin has recently been shown to directly interact with the non-receptor tyrosine kinase c-Src and to be a tyrosine phospho-protein. Therefore, we hypothesize that tyrosine kinase activity may regulate endocytosis of receptors by acting on molecules important for receptor internalization especially dynamin. The present proposal introduces a novel regulatory mechanism for dynamin's role in the process of receptor-mediated endocytosis. The central hypothesis is that agonist-regulated tyrosine phosphorylation of dynamin controls its function in receptor internalization and signaling. Experiments will focus on identifying the molecular determinants involved in receptor-regulated tyrosine phosphorylation of dynamin and elucidating the role of tyrosine phosporylation of dynamin on its enzymatic activity.
The specific aims are: (1) To demonstrate the beta2 adrenergic receptor-regulated tyrosine phosphorylation of endogenous dynamin I and to identify phosphorylated residues; (2) To determine the in vivo mechanism(s) by which beta2 adrenergic receptors regulate tyrosine phosphorylation of dynamin; and (3) To express and purify recombinant dynamin proteins for use in vitro assays aimed at determining the physiologic role for tyrosine phosphorylation of dynamin on its enzymatic activity and receptor-mediated endocytosis.
| Moniri, Nader H; Daaka, Yehia (2007) Agonist-stimulated reactive oxygen species formation regulates beta2-adrenergic receptor signal transduction. Biochem Pharmacol 74:64-73 |
| Wang, Gaofeng; Moniri, Nader H; Ozawa, Kentaro et al. (2006) Nitric oxide regulates endocytosis by S-nitrosylation of dynamin. Proc Natl Acad Sci U S A 103:1295-300 |
