Novel Modulators of Rickets Receptor Mediated Signaling In Vitro and In Vivo.
Harwood, Bejamin.
2014
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Abstract: My initial efforts focused on extending the utility of membrane tethered ligands (MTLs) to enable exploration of the function of rickets (rk), a Drosophila melanogaster G protein-coupled receptor (GPCR). Rk mediates wing expansion, cuticle hardening, and melanization during the transition from the pupal to adult stages. The endogenous rk ligand is bursicon, a heterodimeric cystine-knot p... read morerotein comprised of an and a subunit. As a probe of rk function, I developed a bursicon MTL by genetically fusing the and subunits. Subsequent experiments showed that the bursicon MTL activates rk in vitro. Tissue specific expression of the bursicon MTL using the Gal4/UAS system demonstrates that this construct can modulate rk signal transduction in vivo. Notably, in vivo expression of a bursicon MTL results in decreased viability and defects in wing expansion, phenotypes expected when bursicon/rk signaling is disrupted. Subsequent in vitro studies have shown that chronic exposure to a bursicon MTL results in rk desensitization. Expression of a bursicon MTL with a selection of tissue specific Gal4 drivers has shown that rk is critical for Drosophila development, specifically in muscle. Since our previous studies had shown an important role for bursicon/rk signaling during eclosion in conjunction with the fact that the pathway is conserved among insects; it became clear that rk is a putative target for a novel insecticide. To investigate this hypothesis, we conducted a high throughput screen for chemical modulators of rk; more than 350,000 compounds were assessed. The highest affinity ligands are currently being evaluated and further optimized. Finally, as another extension of MTL technology, we have developed a second type of membrane anchored ligand. These constructs, soluble membrane anchored ligands (SMALs), are comprised of a peptide conjugated to a lipid. In vitro cell based studies have shown that SMALs recapitulate the desirable properties of MTLs (e.g. increased efficacy, prolonged activity). The combination of MTL and SMAL technologies creates a powerful system to develop and optimize membrane anchored ligands as molecular probes of GPCRs. In addition, SMALs hold promise as a new form of therapeutics.
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Genetics.
Advisor: Alan Kopin.
Committee: Pamela Yelick, Rob Jackson, Janis Lem, and Hans-Willi Honegger.
Keywords: Pharmacology, Molecular biology, and Genetics.read less - ID:
- 9w032f90q
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