The ABC (ATP-Binding-Cassette)-type proteins SPGP and MDR3 are essential for bile formation. SPGP mediates ATP-dependent transport of conjugated bile acids across the canalicular membrane and MDR3 is a phospholipid flippase that mediates transfer of phosphatidylcholine to bile. The transporters, which reside primarily in the canalicular membrane, can be recruited or removed from the apical domain in response to signals such as bile acids, cAMP or changes in osmolarity. Mobilization and targeting of the transporters to and from the canalicular membrane is probably mediated by association with proteins that link SPGP and MDR3 to sorting and trafficking networks. However, outside of the interaction between CFTR and NHERF, which is essential for polarized sorting of the chloride channel, little is known about proteins that bind and regulate trafficking of ABC-transporters. We have identified two proteins that specifically bind MDR3 and SPGP. GST-pulldowns from liver homogenates, FRET analyses and co-immunoprecipitation of transporters with associated proteins, confirmed the validity of these interactions. The goal of the proposed research is to study the role of these binding proteins in regulation of SPGP trafficking in polarized cells. Experiments in Aim 1 will use immunofluorescence microscopy and FRET to establish the sites in hepatocytes where SPGP interacts with binding partners, and the effect of stimuli which induce SPGP recruitment to, or retrieval from, the canalicular membrane.
Aim 2 focuses on determining the function of the interacting proteins vis-a-vis SPGP trafficking. The specific amino acid motifs in SPGP which mediate its association with interacting proteins will be identified using yeast two hybrid assays. These moieties will be mutated with the objective of generating mutant transporters that do not bind interacting proteins. Trafficking of the SPGP mutants will be studied in polarized cell model systems to determine the function of the association with the interacting proteins. Mutations which cause abnormal trafficking of canalicular ABC-transporters have been associated with cholestasis of pregnancy and Dubin-Johnson syndrome. Therefore, elucidating the pathways that govern transfer and recruitment of ABC-transporters to the apical membrane, and identifying proteins which control these processes, will provide critical insight into mechanisms underlying cholestasis and suggest targets for therapeutic drug design.
|Chan, Wayne; Calderon, German; Swift, Amy L et al. (2005) Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells. J Biol Chem 280:23741-7|
|Ortiz, Daniel F; Moseley, James; Calderon, German et al. (2004) Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells. J Biol Chem 279:32761-70|