MOLECULAR ASPECTS OF RETINOL UPTAKE AND ACTIVATION

Retinoids (vitamin A derivatives) are essential for a wide variety of physiological processes, including embryonic development, growth, vision, reproduction, and maintenance of various epithelia and the immune system. 11-Cis-retinal serves as the chromophore of the visual pigments in the retina, whereas the hormonal retinoids, alltrans and 9-cis retinoic acid (RA), regulate the non-visual functions via activation of nuclear retinoid receptors (RARs and RXRs). All vitamin A-dependent physiological functions require metabolic activation of the common precursor, all-trans retinol. Different forms of retinol are transported in plasma in complex with retinol-binding protein (RBP) and chylomicrons, and taken up into target cells in various tissues. Following uptake and possible isomerization, retinol is oxidized into retinal by retinol dehydrogenases (RDHs). The second oxidation step that irreversibly converts retinal into RA, is catalyzed by retinal dehydrogenases (RalDHs). To date, several key components involved in the generation of RA and 11-cis retinal have been identified. However, our knowledge of the cellular mechanisms that control the production of these highly active retinoids is limited. Our aim has been to functionally characterize events in retinoid activation, including cellular uptake, isomerization, and oxidation of retinol. In this work, we have reconstituted the whole biosynthesis pathway generating 9-cis RA using a coupled/enzyme reporter system. Mutations in the gene encoding RDH5 are associated with fundus albipunctatus, an autosomal recessive eye disease that causes stationary night blindness. In order to understand the molecular pathology of the disease we have characterized naturally occurring mutants of RDH5. Most RDH5 mutants showed loss of enzymatic activity and subcellular mislocalization. Interestingly, we found an enzymatically active mutant in one patient, in combination with an inactive mutant expressed from the other allele. Our functional studies suggest that a trans-dominant negative effect may occur in this patient as a result of formation of inactive heterodimers of RDH5. Previous work in our group has shown that the RDHs, RDH5 and cis-retinol/androgen dehydrogenase 1 (CRAD1), are ER resident proteins facing the lumen, and carrying short cytosolic tails in their extreme C-termini. In further investigations we have verified the membrane topology of CRAD1 and mapped the C-terminal determinants for ER retention and in vivo functional activity, respectively. Analyses using chimeric CD4 proteins as markers for subcellular localization showed that the C-terminal transmembrane segment of CRAD1 confers ER localization. Studies of CRAD1 tail mutants have shown that the cytosolic tail is necessary for the functional activity of the enzyme in vivo, but not in vitro. This indicates that additional component(s), interacting with, or modifying the cytosolic tail, are required for the activity of the enzyme in vivo. Our studies using the reporter assay have also shown that RBP-bound retinol is taken up into cells by a process that can be efficiently inhibited by blocking antibodies, suggesting that the uptake is indeed receptor-dependent. Moreover, we have characterized a trans/9-cis isomerase activity supporting 9-cis RA generation in vivo. Similar assays have also been employed in combination with immunohistochemistry, to identify sites of co-localized CRAD1 expression and retinoid receptor ligand generation in the mouse embryo. Notably, CRAD1 may potentially contribute to 9-cis RA synthesis in the developing kidney and heart. Taken together, the development and application of reporter assays has generated novel data regarding retinol uptake and activation, and provides a useful tool for addressing cell biological aspects of retinoid activation. LIST OF PUBLICATIONS This thesis is based on the following original papers, which are referred to in the text by their corresponding roman numerals: I Martin Liden, Anna Romert, Kristian Tryggvason, Bengt Persson and Ulf Eriksson. Biochemical defects in 11-cis retinol dehydrogenase mutants associated with fundus albipunctatus. J Biol Chem. 2001, 276, 49251-7 II Martin Liden, Kristian Tryggvason and Ulf Eriksson. The C-terminal region of cis-retinol/androgen dehydrogenase 1 (CRAD1) confers ER localization and in vivo enzymatic function. Exp Cell Res. 2005, In press (e-published ahead of print) III Martin Liden and Ulf Eriksson. Development of a versatile reporter assay for studies of retinol uptake and metabolism in vivo. Exp Cell Res. 2005, 310, 401-408 IV Martin Liden, Kristian Tryggvason and Ulf Eriksson. Analysis of CRAD1 expression and the generation of retinoid receptor ligands in developing mouse tissues.