Identification And Functional Research Of Eps15, A Novel Substrate Of G Protein-coupled Receptor Kinase 2

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form thelargest known receptor family of signal-transducing proteins. The range of signals actingthrough G protein-coupled receptors includes numerous neurotransmitters, chemoattractants,hormones, cytokines, and sensory stimuli such as photons or odorants. In the face ofcontinuing stimulation, signaling becomes attenuated by a process termed desensitization.G protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinasesthat specifically recognize activated GPCRs as substrates. Receptors phosphorylated by aGRK become targets for binding of β-arrestin. β-arrestin bound to a GRK-phosphorylatedreceptor prevents the further coupling of that receptor to its G protein and promotes theendocytosis of the receptor. Recent years studies about GRKs become a hotspot, and mostof them focused on their functions involved in GPCR desensitization. In the last few years,several non-GPCR substrates of GRKs such as tubulin, synucleins, PDGFR and EGFR havebeen reported, thus extending the physiological functions of GRKs beyond GPCRdesensitization. In this work, firstly using GRK2 as bait we screened mouse brain cDNA library forbinding proteins by the approach of yeast two-hybrid system. Totally 63 positive clonesincluding 6 kinds of cDNA were obtained. Among them, Eps15, Eps15R and β subunit ofCOPI were identified as candidates for GRK2 binding partners. We carried out a series ofin vitro experiment to study these three candidate proteins. Unexpectedly we found thatEps15 and Eps15R can be phosphorylated by GRK2, suggesting these two proteins are morelikely to be a true functional binding partner of GRK2. Because Eps15 and Eps15R aresimilar in both structure and function, we selected Eps15 as representative to study. Thekinetic analysis revealed that GRK2 had a K_m of 0.42±0.19 μM and a maximal velocity of4.8±1.4 nmol min^-1mg^-1 for Eps15. The analysis of the stoichiometry of GRK2-mediatedEps15 phosphorylation showed that there are more than ten phosphorylation sites. Throughmass spectrometry analysis four sites were found, but mutation of these four sites to alaninehas little effect on the phosphorylation of Eps15 by GRK2. Through an in vitro bindingexperiment, we delineated the EH domain of Eps15 and the pleckstrin homology (PH)domain of GRK2 were involved in the interaction.In the second part of the thesis, we tried to answer what is the upstream signal thatregulates the phosphorylation of Eps15 by GRK2, and what is the function of thisphosphorylation. Firstly,we detected whether the phosphorylation has any effects on twoimportant properties of Eps15, the ability to bind α subunit of AP-2 and oligomerization ofitself. But neither of these properties changed after GRK2-mediated phosphorylation. Weproved that GRK2 and Eps15 can colocalize in HEK293 cell observed by confocalmicroscope, and overexpression of GRK2 could result in Eps15 phosphorylation in HEK293 cell. These results strongly suggest that Eps15 could be a true substrate of GRK2 invivo. Because Eps15 is not constitutively phosphorylated in vivo, this phosphorylationmust be regulated by a certain signal. We stimulated several GPCRs, but did not detect anyphosphorylation of Eps15. Previous report demonstrated that Eps15 was phosphorylatedduring mitosis and the protein kinase responsible for this phosphorylation is still unknown.We tried to find out whether GRK2 is the kinase. Our results demonstrated that Eps15 hasa chromosomal localization during metaphase of mitosis, but the interaction of Eps15 withthe AP-2 is not inhibited by mitotic phosphorylation, suggesting that Eps15 may have aspecial function during mitosis but not to inhibit endocytosis by dissociation with AP-2. Inaddition, we proved that the phosphorylation sites of mitotic phosphorylation of Eps15 arequite different from GRK2-mediated phosphorylation. This result demonstrated that GRK2is not the kinase responsible for Eps15 phosphorylation during mitosis. Until now, wehavn't found the upstream signal that promotes GRK2 to phosphorylate Eps15 and what isthe function of this phosphorylation. We speculated this phosphorylation may be regulatedby a certain GPCR in a certain cell type or special condition that we didn't find out. It isalso possible that this phosphorylation is regulated by the other signaling pathways but notGPCR related signaling.