Mechanism Of ABCG2 Regulated By Its C Motif

Objective ABCG2,an important member of the ABC transporter superfamily,can pump its substrates out of the cells.The C motif is used to identify the members of ABC transporters.However,little is known about the C motif in ABCG2.Moreover,it has multiple post-translational modification sites and polymorphism sites.This study focused on the regulatory role of C motif on ABCG2.Methods 1)The mutants were generated using site-directed mutagenesis.The expression of m RNA and protein level were detected by RT-PCR and western blot.2)Protein trafficking were analyzed using deglycosylation,protein transport blocking,protein localization and amino acid charge analysis.3)Protein stability were analyzed by half-life measurement,degradation pathway development and dimer formation.4)ABCG2 ATPase activity were tested by ATP binding,ATPase activity assay,truncation and drug transport.Results 1)ABCG2 mutants T194 A,T194D,S195 A,S195D,AA,DD,R191 S,K192E,T194 I,KKK,AAA and EEE(AA: double mutant of T194 and S195 to AA,DD: double mutant of T194 and S195 to DD,KKK,AAA and EEE is respectively mutant from RKR)were generated and expressed in cells.2)Due to the loss of positive charge in C motif,the protein expression level of T194 D,S195D,DD,R191 S,K192E,AAA and EEE mutants is extremely lower than wild type(WT),while the m RNA expression levels are similar.The expression of T194 A,S195A,AA,T194 I and KKK have no significant change on both m RNA and protein level.3)C motif mutants lack matured glycosylation in Golgi and retained in the endoplasmic reticulum(ER),their accumulation in the ER induces ER stress and activates ER associated degradation(ERAD)pathway.On the other hand,WT is degraded in lysosomes.C motif mutants have very short half-life(< 4 h)compared with the long halflife of WT.4)C motif mutants still bind ATP.However,they lost ATPase activity which is coupled with transport function.Conclusion The absence of positive charge in the C motif blocked protein trafficking,decreased stability,lost ATPase activity and drug transport function of ABCG2.