Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts 02111, USA.
Mol Pharmacol. 2013 Apr;83(4):814-21. doi: 10.1124/mol.112.081570. Epub 2013 Jan 22.
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 (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled 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 composed 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. Coexpression 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. Coexpression 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.
对复杂异二聚体肽配体的研究受到缺乏药理学工具的阻碍。为了促进这些研究，我们探索了膜锚定配体（MTL）的应用。我们重点研究了双翅目昆虫保幼激素，这是一种异二聚半胱氨酸环蛋白，能激活 G 蛋白偶联受体 rickets（rk）。rk/保幼激素信号通路是昆虫中一种进化上保守的通路，对于翅膀扩张、表皮硬化和发育过程中的黑化是必需的。我们最初设计了两种不同的 MTL 构建体，每个构建体都由一个 II 型跨膜结构域、一个肽接头和一个 C 末端细胞外配体组成，分别对应于保幼激素的α或β亚基。两种互补的保幼激素 MTL 的共表达在体外引发了 rk 介导的信号转导。然后，我们能够生成具有功能活性的保幼激素 MTL，其中两个亚基融合成一个单一的异二聚体肽，定向为α-β或β-α。β-α MTL 构建体中的羧基末端 32 个氨基酸的缺失导致激动剂活性丧失。与 rk 共表达这种构建体抑制了可溶性保幼激素的受体介导的信号转导。因此，我们已经生成了可以激活或抑制 rk 信号转导的膜锚定保幼激素构建体。这些探针可用于未来的研究，以探索保幼激素在遗传上可操作的果蝇模型生物中的组织和/或发育阶段依赖性效应。此外，我们在生成功能多样的保幼激素 MTL 方面的成功为这项技术可以广泛应用于其他复杂配体，包括哺乳动物半胱氨酸环蛋白家族，提供了希望。
