Receptor Structure and Function


The past two decades have seen the steady identification of mammalian receptors of well known ligands (steroids, thyroid hormone, all-trans retinoic acid (RA)), as well as the identification of endogenous ligands for initially orphaned receptors (e.g., RXR, PPAR, FXR, LXR).  Similarly, steroid nuclear receptors in invertebrate models of transcriptional regulation, such as the Drosophila melanogaster ecdysteroid receptor (EcR), have been isolated a decade ago and used to develop important concepts in hormone action.  Although the invertebrate sesquiterpenoid hormone epoxymethyl farnesoate (juvenile hormone) has been known for over 40 years, and despite reports of binding to hRXR of the acid metabolite of its analog methoprene, there has been considerable exasperation over the failure to identify a nuclear hormone receptor through which juvenile hormone (JH) can act to regulate promoter activity.  

Our research has now demonstrated that Drosophila USP (dUSP) specifically binds JH III, but not control  farnesol or JH III acid, and also specifically changes in conformation upon binding of JH III in a  fluorescence binding assay.  Juvenile hormone III binding causes intramolecular changes in receptor conformation, and stabilizes the receptorís dimeric/ oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competed with JH III for binding to dUSP, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor.  The recombinant dUSP bound with specificity to a DR12 hormone response element in a gel shift assay.  The same DR12 element conferred enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T 3.   The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to dUSP and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle.  The prospect is also offered that synthetic, terpenoid structures may be discovered that can selectively agonize or antagonize USP function in vivo without harmful interaction with the retinoic acid receptor of amphibians or other vertebrates.

We are currently performing mutagenic studies on the ligand binding pocket of USP, assessing the effect on JH III binding of point  mutations of various amino acids that line the ligand binding pocket.  We are also transforming these mutant USPs into Drosophila to assess the in vivo phenotype of mutants that biochemically show loss of binding to USP.












Drosophila USP

apo-human RXRa

Jones, G., Sharp, P. A. 1997. Ultraspiracle: an invertebrate nuclear receptro for juvenile hormones. Proc. Natl. Acad. Sci. U.  S.  A  94, 13499-503

Jones, G., Jones, D. 2000. Considerations on the structural evidence of a ligand -binding function of ultraspiracle, an insect homolog of vertebrate RXR. Insect  Biochem. Molec. Biol. 30, 671 -79

Jones, G., Wozniak, M., Chu, X. Y.,  Dhar, S., and Jones, D. (2001) Juvenile Hormone III - Dependent Confirmational Changes of the Nuclear Receptor Ultraspiracle. Insect Biochem. Molec. Biol. 32, 33-49

Xu Y, Fang F, Chu Y, Jones D, Jones G. (2002) Activation of transcription through the ligand-binding pocket of the orphan nuclear receptor ultraspiracle.  Eur J Biochem. 269, 6026-36

Wozniak M, Chu Y, Fang F, Xu Y, Riddiford L, Jones D, Jones G.(2004)  Alternative farnesoid structures induce different conformational outcomes upon the Drosophila ortholog of the retinoid X receptor, ultraspiracle.
Insect Biochem Mol Biol. 34(11):1147-62.

Fang F, Xu Y, Jones D, Jones G. (2005)  Interactions of ultraspiracle with ecdysone receptor in the transduction of ecdysone- and juvenile hormone-signaling.  FEBS J. 272(7):1577-89.