Studies on a novel cytosolic/nuclear binding protein for the eicosanoid 12(S)-hydroxyeicosatetraenoic acid

• Hydroxyeicosatetraenoic acids (HETEs) are lipoxygenase metabolites of arachidonic acid. HETEs were long considered to have few or no important biological function - a view that proved to be incorrect. These compounds have now been identified in many different types of cells, and they have been attributed several important actions, the molecular mechanisms of which are largely unclear. This thesis describes the discovery of a putative receptor for 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE).Specific high-affinity binding sites for 12(S)-HETE were detected in the cytosol (52%) and the nuclei (18%) of cells of the Lewis lung carcinoma (LLC) line. Similar l2(S)-HETE binding sites were detected in the cytosol of several other kinds of cells and in human platelets. Gel permeation chromatography and density gradient centrifugation indicated an apparent molecular weight of about 650 kDa and a sedimentation coefficient of20.5 S for the binding sites in the cytosol ofLLC cells. Treatment with ATP caused the 650 kDa component to dissociate into subunits. The actuall2(S)-HETE binding subunit was subsequently shown to have a molecular weight of about 50 kDa.The subcellular distribution of l2(S)-HETE binding sites in LLC cells was found to resemble the distribution of some nuclear receptors of the steroid hormone receptor superfamily. The untransformed glucocorticoid receptor has been reported to be located in cytosol in association with heat shock proteins 70 and 90, and Western blot analyses and immunoprecipitation revealed the same two proteins in the cytosolic 650 kDa l2(S)-HETE binding complex.A possible relationship to the steroid hormone receptor superfamily was also suggested by the observation that the 50 kDa l2(S)-HETE binding protein interacted with steroid receptor coactivator-l (SRC-l), which is known to be recruited to the site of transcriptional activity by several nuclear receptors. The interaction with SRC-1 occurred only when l2(S)-HETE was bound to its 50 kDa binding protein.In summary, the research presented in this thesis led to the discovery of a novel type of eicosanoid receptor. This binding site resembles the nuclear receptors for steroids and related compounds by virtue of its subcellular distribution, the inclusion of heat shock proteins in its binding complex, and its strictly ligand-dependent interaction with SRC-l.

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