Abstract
The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations we have explored the utility of membrane tethered ligands (MTL). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-couple receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs each comprised of a type II transmembrane domain, a peptide linker, and a C-terminal extracellular ligand that corresponded to either the α or β bursicon subunit. Co-expression of the two complementary bursicon MTLs triggered rk mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-β or β-α. Carboxy-terminal deletion of 32 amino acids in the β-α MTL construct resulted in loss of agonist activity. Co-expression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands including the family of mammalian cystine-knot proteins
- Neuropeptides
- Gs family
- cAMP
- Structure-activity relationships and modeling
- Fluorescence techniques
- Structure/function/mechanism
- Peptide hormones
- Received July 27, 2012.
- Revision received January 17, 2013.
- Accepted January 22, 2013.
- The American Society for Pharmacology and Experimental Therapeutics