Protein acetylation and deacetylation as a novel regulatory mechanism for endothelin receptor A-mediated downstream effects

Endothelin (ET), a potent vasoconstrictive and mitogenic agent, exerts its biological effects via the seven transmembrane receptors, endothelin receptor A (ETA) and endothelin receptor B (ETB). In order to better understand its signaling mechanism, we initiated a search for ETA-interacting protein(s) using the yeast two-hybrid system. A human cDNA library was screened using the C-terminal cytosolic tail of ETA as a bait. Four strong positive clones were identified and two of these were chosen for further analysis. The first clone was Tip60 (Tat-interactive protein of 60 kDa), an enzyme with histone acetyltransferase activity. The second clone corresponded to a novel member of the histone deacetylase family and was named ERIDA (E&barbelow;TA R&barbelow;eceptor I&barbelow;nteracting D&barbelow;eA&barbelow;cetylase).; The interaction of ETA with Tip60 and ERIDA was confirmed in vitro by the Glutathione-S-Transferase pull-down assay and in vivo by co-immunoprecipitation in transfected COS-7 cells. In addition, we showed that Tip60 and ERIDA mRNAs are expressed in tissues that also express ETA. Confocal laser scanning fluorescence microscopy showed that in the absence of ET-1, both Tip60 and ERIDA localize in the nucleus, while ETA is predominantly confined to the plasma membrane. The addition of ET-1 caused internalization of ETA and its localization in the perinuclear compartment. At the same time, ET-1 administration induced nuclear export of Tip60 and ERIDA into the same perinuclear compartment, where each protein co-localized with ETA.; In order to test the effect of Tip60 and ERIDA on MAP kinase signaling, these proteins were co-transfected with ETA into COS-7 cells, and the phosphorylation state of MAPK was analyzed. We found that Tip60 increased ET-1 induced MAPK phosphorylation whereas ERIDA had no significant effect.; Thus, we propose that protein acetylation and deacetylation may represent a novel regulatory event, which may affect downstream signaling and sorting and/or trafficking of the receptor.