We previously expression cloned a unique rat liver mRNA encoding a high affinity Na+independent organic anion transport protein (oatpl). Oatp1 is the first member of a new family of transport proteins with differing tissue distributions and substrate specificities and is responsible for a substantial fraction of organic anion transport by the hepatocyte. We showed that Oatpl distribution in hepatocytes is limited to the basolateral (sinusoidal) plasma membrane and, using transient and stable Oatp 1-transfected cells, we demonstrated that it is an organic anion/HCO3 exchanger and that transport activity of Oatp 1 is down regulated quickly following serine phosphorylation in response to stimulation of a purinergic receptor. Determinants of oatp 1 function and regulation are not known. The proposed studies will utilize state of the art technologies, including mass spectrometry, that we have recently successfully adapted for characterization of oatp 1 and other members of the oatp family to examine this issue.
Three specific aims are proposed: (1) To elucidate specific regulatory and binding domains of oatpl. We have applied mass spectrometry based analytical procedures to examine structural features of oatp 1. This technology will now permit us to define regulatory phosphorylation sites as well as ligand-binding sites. Functional importance of specific domains will be further tested by mutagenesis and deletion techniques. (2) To test the hypothesis that oligomerization of oatpl is essential for its proper expression and function. Although previous studies have assumed that oatpl functions as a monomer, our recent studies suggest that this protein exists as an oligomer. We will determine the identity and functional significance of oatp 1 binding partners, especially PDZ domain proteins. The proposed studies will utilize cotransfection, immunoprecipitation, and cross-linking techniques that we have developed. (3) To test the hypothesis that oatpl is a 10 transmembrane domain protein. The working model, based upon computer but not experimental analysis, is that the oatp's have 12 transmembrane domains. Our preliminary data suggest that oatpl actually has 10 transmembrane domains. This results in a substantially different model in which the extracellular domain is markedly expanded. Establishing the correct model has high potential importance for understanding the mechanism by which this protein functions. These studies should provide fundamental mechanistic information that is applicable to the oatp family of transporters. Ultimately, this work should permit insight into the mechanism and regulation of organic anion transport in normal and diseased liver.
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