Eager, Nicholas Stanley Charles; (1993) Characterisation of chicken RXR-γ. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The clone pR2 was isolated from a chick cDNA library and its insert was sequenced. The predicted amino acid sequence, encoded by the insert, was analysed and the protein was found to be a new member of the steroid/thyroid receptor superfamily. Further analysis revealed that it was a member of a newly discovered class of retinoid receptors, the retinoid X receptors (RXRs). By comparing the amino acid identities with other RXRs isolated from the mouse it was confirmed that our clone encoded chicken RXR-γ (cRXR-γ). At this time the ligand for the RXRs was unknown. All-trans retinoic acid had been shown to activate members of the RXR family but this was considered to represent a spurious cross-reaction. In order to identify the nature of the ligand for RXRs, co-transfections were carried out in Baby Hamster Kidney cells with a construct encoding cRXR-γ and a reporter construct consisting of the chloramphenicol acetyl transferase (CAT) gene under the control of a promoter containing a response element which will bind to the RXRs. Using this system a number of retinoids were tested for their ability to activate the reporter. Of all the retinoids tested, none were found to bring about reporter activation at physiological levels. It was later reported that an isomer of retinoic acid, 9-cis retinoic acid, was the ligand for the RXRs. Structural similarities between 9-cis retinoic acid and the eicosanoids raised the possibility that eicosanoids may also activate RXRs in vivo. Using a similar transfection system it was demonstrated that lipoxygenase metabolites of arachidonic acid (the precursor of eicosanoids) are capable of activating cRXR-γ at submicromolar concentrations. Consistent with this, molecular modelling predicts conformational similarities between some lipoxygenase products and 9-cis retinoic acid. This may explain why certain eicosanoids (the hydroxyeicosatetraenoic acids) mimic some of the actions of retinoids in cell-based assays. As a preliminary step in the analysis of the function of cRXR-γ, a system was set up to block the effect of the receptor by co-expression of a dominant negative mutant. QT6 cells were co-transfected with an expression construct for cRXR-γ and for the ligand binding domain of cRXR-γ. It was shown that by co-expressing this dominant negative mutant, cRXR-γ mediated transactivation was inhibited. A genomic clone was isolated for cRXR-γ and partially sequenced. This allowed the identification of an exon/intron boundary within the cDNA. From this genomic clone a region of 5'-sequence was subcloned into a CAT construct and transfected into QT6 cells. This showed that the sequence contained promoter activity. In summary, these results have lead to the identification of a novel clone as encoding a member of the RXRs. The activation of the receptor by a number of retinoid and non-retinoid compounds has been determined and a genomic clone isolated leading to the discovery of a region containing promoter activity.

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