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dc.contributor.advisorHagenbuch, Bruno A
dc.contributor.authorZhang, Yuchen
dc.date.accessioned2018-11-13T23:34:33Z
dc.date.available2018-11-13T23:34:33Z
dc.date.issued2017-12-31
dc.date.submitted2017
dc.identifier.otherhttp://dissertations.umi.com/ku:15566
dc.identifier.urihttp://hdl.handle.net/1808/27333
dc.description.abstractMany transporters are expressed at the basolateral membrane of human hepatocytes, including Organic Anion Transporting Polypeptide 1B1 (OATP1B1), OATP1B3, Organic Cation Transporter 1 (OCT1), Na+/Taurocholate Cotransporting Polypeptide (NTCP) and more. These transporters are part of the absorption system of the liver, which is responsible for the uptake of chemicals from the portal vein into hepatocytes for further metabolism and elimination. Extensive studies have characterized the function of these transporters, and results suggest that liver uptake transporters, like OATP1B3 and OATP1B1, or OATP1B3 and NTCP, have many overlapping substrates. Because these transporters seem to play an essential role in the protection of the body from xenobiotics, it is important to better understand their function and how they interact with each other. In this dissertation, I focused on one of these transporter, OATP1B3, as a model transporter to improve the understanding of liver uptake transporters. OATP1B3 is responsible for the uptake of many endogenous compounds like bile acids and hormones as well as xenobiotics, including numerous drugs. Recent studies demonstrated that some of the liver uptake transporters like OATP1B1, OCT1 and NTCP can form homo-oligomers. In the first specific aim, I evaluated the hypothesis that OATP1B3 also can form homo-oligomers. To address this aim, co-immunoprecipitation and proximity ligation assays of differently tagged OATP1B3 were performed in transiently transfected HEK293 cells. The results demonstrated that OATP1B3 indeed can form homo-oligomers. In addition, uptake assays with wild-type and non-functional OATP1B3 suggested that the OATP1B3 unit in the homo-oligomers works as individual functional unit. Besides that, by using proximity ligation assays, the interaction between OATP1B3 and OATP1B1, and between OATP1B3 and NTCP was demonstrated in HEK293 cells. Interactions between OATP1B3 and NTCP were also confirmed in frozen liver sections. In the second specific aim, I evaluated the hypothesis that OATP1B3 can form hetero-oligomers with other transporters and that these interactions can influence their expression and function. I was able to extend the findings of hetero-oligomerization to include OCT1 using both immunoprecipitation and proximity ligation assays. Uptake assays and surface biotinylation experiments were performed with HEK293 cells co-expressing OATP1B3 and OCT1, OATP1B3 and OATP1B1, or OATP1B3 and NTCP. The results demonstrated that these interactions between OATP1B3 and the other transporters lead to changes in both, function and expression of OATP1B3 in a transporter-dependent manner. In the third specific aim, I evaluated the hypothesis that photoaffinity labeling can be used to study the binding sites and translocation pathways of OATP1B3. The known OATP1B3 substrate 8-fluorescein-cAMP (8-FcA) was used to perform photoaffinity labeling experiments with CHO Flp-In cells stably expressing His-tagged OATP1B3. The results suggested that 8-FcA can label proteins but background labeling was very high even without UV activation. Purification of OATP1B3 was also attempted to improve chances for a successful follow-up mass spectrometry analysis. Although relatively pure protein was obtained, the low yield prevented further analysis. In summary, this dissertation reveals that 1) OATP1B3 can from homo- and hetero-oligomers, and that the homo-oligomers of OATP1B3 work as separate functional units; 2) hetero-oligomerization of OATP1B3 can result in changes of function and surface expression of OATP1B3 in a transporter-dependent manner; 3) photoaffinity labeling may be a good method to study the binding sites of OATP1B3, after the purification methods have been improved.
dc.format.extent129 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsCopyright held by the author.
dc.subjectPharmacology
dc.titleIDENTIFICATION AND CHARACTERIZATION OF THE OLIGOMERIZATION AND STRUCTURAL FUNCTIONAL RELATIONSHIP OF ORGANIC ANION TRANSPORTING POLYPEPTIDE 1B3
dc.typeDissertation
dc.contributor.cmtememberBlanco, V. Gustavo
dc.contributor.cmtememberKasturi, Partha
dc.contributor.cmtememberLampe, Jed N
dc.contributor.cmtememberReed, Gregory A
dc.thesis.degreeDisciplinePharmacology, Toxicology & Therapeutics
dc.thesis.degreeLevelPh.D.
dc.identifier.orcid
dc.rights.accessrightsopenAccess


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