The Ubiquitination Degradation Mechanism Regulates The Constitutive Activity Of GPR26 And Its Tumor Suppressor Effect In Liver Cancer

G-protein coupled receptors(GPCRs)are among the largest membrane protein families that play important roles in many physiological functions and numerous diseases.In addition to the classic ligand-stimulated receptor activity,an increasing number of studies have established that many GPCRs function constitutively in a receptor dose-dependent manner.Previous observations showed that following gene transfection,little or no protein was detectable for certain GPCRs(designated apparent state A),such as GPR26,GPR39,GPR78,GPR133,GPR139,BRS3 and LGR5,which showed strong constitutive activities.When we lysed cells in the immediate presence of Western blot loading buffer,a significant increase of protein levels was detected(actual state B),which was much closer to the true expression levels under physiological conditions.Indeed,treatment of the cell cultures with proteasome inhibitor MG132 increased protein levels further as well as the constitutive activity of the respective receptors.GPR26 was chosen for further functional experiments as the actual state B.We identified an important ubiquitination site,K286,as well as the ubiquitin ligase E3HECTD3 interacting with GPR26.The pronounced differences in the protein expression and constitutive activity of GPR26 were a consequence of the ubiquitin-mediated rapid degradation mechanism.Furthermore,we identified in vitro and in vivo anti-tumor activity associated with high expression levels and constitutive activity of GPR26 in liver cancer cells.The protein expression level of GPR26-K286 R mutant increased significantly,and so did the constitutive activity.Therefore,the GPR26-K286 R mutant had stronger anti-tumor functions than GPR26.By overexpressing ubiquitin ligase HECTD3,the expression level of GPR26 decreased,and the anti-tumor function was also weakened.Hence,GPR26 could act as an anti-tumor gene for hepatocellular carcinoma.This study also represents the actual state B of a batch of GPCRs that actually play potentially important roles in physiological functions by their constitutive activity,which is controlled by rapid ubiquitin-dependent degradation.