CDNA Cloning Of Intracellular Signaling Molecules Coupled With Endothelin Receptors By Yeast Two-hybrid System

The three known members, ET-1, ET-2, and ET-3 in endothelin family, have potentvasoconstrictive activity. In addition, endothelins control many other cellularprocesses including gene expression, cytoskeletal reorganization, cell differentiation andgrowth. They can also stimulate secretion of neuropeptides, pituitary hormones, andatrial natriuretic peptide from neural and neuroendocrine cells Endothelins exert their biological effects through G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) are the largest group of cell surface moleculesinvolved in signal transduction and are receptors for a wide variety of stimuli rangingfrom light, calcium and odourants to biogenic amines and peptides. However, theprecise mechanism of downstream signaling and trafficking of the receptors is largelyunknown. The late study showed that the histone acetyl transferase Tip60 and thehistone deacetylase HDAC7 interact with one of the ET receptors, ETA. To clarify thenew protein molecule interacting with ET receptors (ETAR and ETBR) and study theirinteraction for downstream signal pathway of ET receptors, we employed yeast two-hybrid system to screen novel proteins interacting with ETAR and ETBR C-terminusfrom human fetal brain cDNA library. The interaction was determined by glutathioneS-transferase pull-down assays and co-immunoprecipitation from transfected HEK293Tcells. We found a novel zinc finger protein which interact with ETAR C-terminus. Mainmethods and results are as follow. 1.Yeast two-hybrid screening To find novel proteins that may bind to endothelin A and B type receptors (ETARand ETBR) and investigate their interactions with an expectation to provide new leadsfor the function study of the receptors, yeast two-hybrid assay was performed to screena human fetal brain cDNA library using the C-terminus of ETAR and ETBR as baits.X-Gal assay was subsequently conducted to further qualitatively confirm theinteractions between receptors and the identified proteins. The result showed thatselection medium screening identified approximately 6.4×105 and 2.75×106 coloniesamong all the 5×107 human fetal brain cDNA library colonies as binding partners ofETAR and ETBR C-terminus in yeast cells respectively. Besides, in X-Gal assay, 90colonies of ETAR and 279 colonies of ETBR turned blue while other positive coloniesdidn't develope blue color. The colonies developing blue color are the candidates in thefurther study. 2.Interaction between a novel zinc finger protein(FLJ) and ETAR To confirm the interaction of FLJ with ETAR, GST conjugated peptidecorresponding to the C-terminus of ETAR was used in GST pull-down experiments.The results demonstrated that FLJ bound to ETAR-C-GST but not to GST alone. Theseresults confirmed the direct interaction between the ETAR C-terminus and FLJ invitro. The interaction was further verified by co-immunoprecipitation and western blotexperiments using transfected HEK293T cells. ETAR with HA epitope at theN-terminus (HA-ETAR) was expressed in HEK293T cells alone or co-expressed withFLJ (in the pcDNAFLAG vector) and immunoprecipitated with anti-HA antibody.Western blot with anti-FLAG antibody showed that FLJ could interact with ETAR invivo. Our results suggested that a novel zinc finger protein FLJ interacts with ETARC-terminus as determined by yeast two-hybrid system, glutathione S-transferasepull-down assay and co-immunoprecipitation from transfected HEK293T cells. Theseworks provide a new insight for clarifying the mechanism of ETAR downstream signaltransduction. The function of FLJ and the effect of the interaction on ETARdown-stream signal pathway need further study.