Exquisite ligand stereoselectivity of a Drosophila juvenile hormone receptor contrasts with its broad agonist repertoire

Abstract

The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in Drosophila melanogaster known as germ cell–expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand–binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.

Transcription‐inducing activity of natural and synthetic juvenile hormone agonists through the Drosophila Methoprene‐tolerant protein

“…In insect cells, the intrinsic activity of natural JHAs can be affected by JH‐inactivating enzymes, such as JH esterase and JH epoxide hydrolase . In fact, the pEC 50 values of natural JHAs (JH I, JH III and MF) in the S2 cell‐based system were substantially lower than those obtained in our reporter system, despite the similar potencies of fenoxycarb in these two assay systems (Table ). Thus, insect cell‐based systems have a potential risk of underestimating the potency of natural JHAs.…”

“…Table compares the pEC 50 values of JHAs measured in three different reporter systems: (i) a reporter assay in HEK293T cells transfected with DmMet and DmTai (this study), (ii) a two‐hybrid reporter assay in Chinese hamster ovary (CHO) cells transfected with VP16AD–DmMet and GAL4DBD–DmTai, and (iii) a reporter assay in Drosophila Schneider 2 (S2) cells that express endogenous JH receptors . The pEC 50 values obtained in this study were two orders of magnitude higher than those obtained in the two‐hybrid system.…”

“…The identification of JH receptor proteins, however, has enabled the development of efficient in vitro bioassays for JHAs. These in vitro assays include (i) a ligand–receptor binding assay based on a cell‐free protein synthesis system, (ii) a reporter gene assay employing JH receptors expressed constitutively in cultured insect cell lines, and (iii) a two‐hybrid reporter gene assay using engineered Met and Tai proteins fused to the VP16 activation domain (VP16AD) and GAL4 DNA binding domain (GAL4DBD), respectively . These systems are quantitative and easy to handle but still have several drawbacks.…”

Intraluminal Farnesol and Farnesal in the Mealworm's Alimentary Canal: An Unusual Storage Site Uncovering Hidden Eukaryote Ca2+-Homeostasis-Dependent “Golgicrine” Activities

“…In addition to the cited emerging questions, this paper also tries to answer the still only partially answered question on how endogenous sesquiterpenoids act at the molecular level. Is it only at the level of transcription (29,31) and/or on the whole integrated system of Ca 2+ -homeostasis (1,28,69)?…”

“…To date, the most cited mode of action of JH (and farnesol as well) is that JH directs the whole physiology of its target cells through its binding to the transcription factor Germ cell-expressed (Gce) and its duplicate paralog Methoprene-tolerant (Met) in Drosophila (29). Although the advocates of this thesis do not deny the existence of a functional link between a possible membrane receptor and the activation of Met [e.g., (30,31)], the role of a membrane receptor is often thought to be of minor importance, even to be non-existent. Considering the so far absence of convincing experimental evidence that JH can-and doesenter the nucleus, De Loof and Schoofs (1) recently proposed an alternative for the MET-mediated mode of action.…”