Endocrinology Abstracts

Reversed pharmacology in insects: finding novel orphan G protein-coupled receptors and their ligands

C. J. P. Grimmelikhuijzen 1 (Home Page), F. Hauser 1 , K. Eriksen 1 , M. Schiøtt 2 , L. Søndergaard 2 , F. Staubli 1 & P. Svane 1

1 Dept. of Cell Biology, Zoological Institute, and 2 Dept. of Genetics, Institute of Molecular Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark

In mammals, reproduction is controlled by the hypothalamic neuropeptide GnRH (gonadotropin-releasing hormone), the pituitary glycoprotein hormones LH (luteinizing hormone) and FSH (follicle-stimulating hormone), and a variety of sex steroid hormones. In insects, the molecular mechanisms behind reproduction are, so far, only poorly understood. Using several approaches, we have now found that Drosophila melanogaster produces at least two receptors that are structurally and evolutionarily related to the LH and FSH receptors from mammals: DLGR-1 (Drosophila Leu-rich repeats containing G protein-coupled receptor-1) and DLGR-2. As with the mammalian glycoprotein hormone receptors, DLGR-1 and -2 contain a large extracellular N terminus with Leu-rich repeats that probably form a horseshoe-like structure to which the ligands bind. DLGR-1 has nine of these repeats (the same number af the LH and FSH receptors), whereas DLGR-2 has 18-19, which is an unusual large number. DLGR-1 is expressed in Drosophila embryos, all three larval stages, pupae and adult flies, and could be involved in reproduction. DLGR-2 is only expressed in embryos and pupae, which points to a role in development. This role is confirmed by the isolation of a DLGR-2 knock-out (P-element insertion) mutant of Drosophila, which dies in the late phases of embryogenesis (just around hatching). A receptor similar to DLGR-1 has also been cloned from Bombyx mori. Furthermore, we have also cloned a receptor from Drosophila that is structurally and evolutionarily related to the mammalian GnRH receptor. We have permanently transfected mammalian CHO cells with the DNA coding for this receptor together with DNA coding for the G protein G-16, and transiently with DNA coding for aequorin. When a homogenate of Drosophila larvae was added to these transfected CHO cells, we could measure a very strong rise in intracellular Ca 2+ concentration (luminescence), indicating ligand binding to the Drosophila GnRH receptor. Using this system as a bioassay, we have now purified the ligand (with HPLC) and we are presently elucidating its structure.

Index terms: Drosophila melanogaster, Bombyx mori, neurohormone, reproduction, development.

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