Grants and Contracts Details
A major, unresolved question in insect endocrinology is whether the nuclear receptor ultraspiracle (USP) has an active, ligand binding function or, altematively, is a nonliganded, even passive, heterodimer partner. Our previous NIH-funded protein biochemical studies emphasized screening of many point mutants to the ligand-binding domain of the Drosophila USP. The specific aims were to identify optimal mutant USPs for future testing of an in vivo USP ligand binding function in the context of two transgenic fly assays. Our studies, supported by recent studies of others, were successful to demonstrate in cunured cell assays that the USP of Drosophila (flys) and Aedes (mosquitoes) can transduce transcriptional signaling by methyl epoxyfamesoate, a natural juvenile honnone (JH) in flys and mosquitoes. Our studies also show that this intracellular JH signaling requires a surface region on USP corresponding to a ligand-activated coactivator binding surface of the vertebrate counterpart, RXR. Using the cunured cell functional assay, we were successful in preparing point mutants to the USP ligand binding pocket and coactivator binding surface that act as dominant negatives to block USP-dependent JH signaling in the cunured cells. On this basis, the recent NIH study section panel summary directed that our next proposal move past the protein biochemical studies by using "the mutants already in hand rather than looking for more mutants" and to now orient the studies "into a direct biological context." Therefore, in this new proposal we take these Drosophila USP mutants now in hand (and corresponding Aedes USP mutants) into the following two transgenic fly assays: 1) Is the USP ligand binding pocket needed for USP function in JH/ecdysone-driven larval-larval moning? 2) Does the USP ligand binding pocket transduce JH effects on pupal-adun metamorphosis? These in vivo studies are the next logical step, building upon protein biochemical and cultured cell assays, and are insect endocrinology's first direct in vivo tests of a ligand-binding function for any USP. The outcome will have broad implications for insect endocrinology, enabling then specific tests on the Aedes USP, the resuns of which will yield to new insight on hormonal signaling in the more technically more difficun mosqu~o.
|Effective start/end date||8/1/05 → 7/31/11|
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