A Drosophila Juvenile Hormone Receptor

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The proposed work will investigate the hormonal control of development and reproduction in insects by probing the molecular action of juvenile hormone (JH) in the model insect Drosophila melanogaster. Important roles for JH in insects have been identified, but the action of this hormone remains an enigma because a JH receptor has not been identified in any insect. A genetic approach identified the Methoprene-tolerant (Met) gene, and found that the protein that it encodes (MET) is involved in the action of JH in this insect, probably as a component of a JH receptor. In the proposed work Met will serve as a focus for understanding this receptor. The proposed work consists of two objectives: (1) Identification of the partner protein of MET. Met+ in D. melanogaster is a member of the bHLH-PAS family of transcriptional regulators. PAS proteins function with a partner protein in a heterodimeric complex, and there is no reason to believe that MET is an exception. An understanding of JH binding and reception must include an identification of the partner protein. One promising candidate is a homologous gene, Met-like (Met-l), recently cloned fromD. melanogaster. Yeast two-hybrid technology using Met+ as bait will be employed to directly test MET-L as the partner of MET as well as search for other proteins that interact with MET. In this proven technology candidate genes are expressed together in yeast cells. Physical interaction between their gene products will drive transcription of one or more reporter genes engineered into the yeast strain, allowing ready visualization of this interaction. Interaction of each protein will be verified in vitro using antibody to MET. Any novel gene(s) will be identified, possibly by function, from the D. melanogaster sequence database, for further characterization. (2) Identification and characterization of the JH binding protein. Previous binding studies demonstrated that JH III binding affinity was much poorer in tissue extracts from Met mutants, accounting for the resistance to methoprene, and implicating MET as a receptor component. To identify the JH-binding protein and establish its binding characteristics, JH III binding to the individual proteins and to the MET: PARTNER heterodimeric complex will be measured. Additionally, a photoaffinity analog of JH III, epoxyfarnesyl diazoacetate, will be used to photoaffinity label and establish/confirm which protein is the high-affinity binding protein in the heterodimeric complex. Results from these studies will define the components of the putative JH receptor and any associated proteins that physically interact with MET. The JH-binding protein will also be identified, thus clarifying the poorly understood molecular basis for JH action. These results will provide the necessary knowledge and molecular tools for further analysis of transcriptional control by JH.