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Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors
1Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, USA.
Abstract:
We have used a yeast genetic system to isolate cDNAs encoding proteins that specifically interact with the ligand-binding domain of human retinoid X receptor-alpha (RXR alpha). A number encoded portions of two known RXR heterodimer partners, the retinoic acid receptor (RAR) and the peroxisome proliferator activated receptor. Of four additional RXR-interacting proteins (RIPs) selected for further study two, RIP14 and RIP15, are previously unidentified orphan members of the nuclear receptor superfamily. Two others, RIP110 and RIP13, do not show significant similarities to previously reported proteins. RIP110 interacts with LexA-RXR only in yeast cells grown in the presence of the RXR ligand 9-cis-RA, while the interaction of the four receptor superfamily members and RIP13 is unaffected by the presence or absence of 9-cis-RA. RIP110 and RIP13 also interact in yeast with several other members of the receptor superfamily, but RIP14 and RIP15 interact only with RXR. Analysis of larger cDNA clones demonstrates that there are at least two isoforms of RIP14 that differ in the N-terminal (A and B) and hinge (D) domains. Northern blot analysis indicates that RIP14 is expressed specifically in liver and kidney, while RIP15 is expressed in every tissue tested. Both RIP14 and 15 bind as heterodimers with RXR to the RA response element (RARE) from the promoter of the RAR beta 2 isoform (the beta RARE), and RIP14 and RXR heterodimers also bind the ecdysone response element from the Drosophila heat shock protein 27 promoter. Both heterodimers also bind to several synthetic RAREs and other elements. In cotransfections, neither RIP14 nor RIP15 trans-activates a reporter containing multiple copies of the beta RARE under any of a variety of conditions, suggesting that their activities are dependent on the binding of as yet unidentified specific ligands or on activation by other processes.
Insights
Researchers identified novel proteins, RIP14 and RIP15, that interact with retinoid X receptor-alpha (RXR alpha). These new nuclear receptors bind DNA with RXR alpha but do not activate transcription, suggesting ligand-dependent regulation.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Retinoid X receptor-alpha (RXR alpha) is a crucial nuclear receptor involved in various cellular processes.
- Identifying specific interacting partners of RXR alpha is essential for understanding its regulatory mechanisms.
Purpose of the Study:
- To identify novel proteins that specifically interact with the ligand-binding domain of human RXR alpha.
- To characterize the interaction properties and potential functions of these newly identified RXR alpha-interacting proteins (RIPs).
Main Methods:
- Yeast genetic system for isolating interacting cDNAs.
- Analysis of cDNA clones and protein similarities.
- Northern blot analysis for tissue expression.
- Electrophoretic mobility shift assays (EMSAs) for DNA binding.
- Cotransfection assays for transcriptional activity.
Main Results:
- Two novel orphan nuclear receptors, RIP14 and RIP15, were identified as RXR alpha interacting proteins.
- RIP14 and RIP15 exhibit specific interactions with RXR alpha, unlike other identified RIPs.
- RIP14 and RIP15, as heterodimers with RXR alpha, bind to various response elements, including the beta RARE.
- RIP14 and RIP15 do not exhibit trans-activation activity in reporter assays, indicating potential ligand-dependent regulation.
Conclusions:
- RIP14 and RIP15 represent novel nuclear receptor superfamily members that specifically interact with RXR alpha.
- Their DNA-binding activity with RXR alpha suggests a role in retinoid signaling pathways.
- The lack of transcriptional activity implies that their function is regulated by as-yet-unidentified ligands or other signaling pathways.