Novel Modulators of Rickets Receptor Mediated Signaling In Vitro and In Vivo.
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-k... read morenot protein comprised of an α and a β subunit. As a
probe of rk function, I developed a bursicon MTL by genetically fusing the α and
β 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
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