Abstract

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET–TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Highlights

  • Juvenile hormone (JH) and 20-hydroxyecdysone are the two major hormones controlling insect molting and metamorphosis [1, 2]

  • We present high-yield insect cell– based expression and purification of recombinant JH receptor (JHR) MET–TAI complexes from T. castaneum and A. aegypti that are active in both hormone and target juvenile hormone response element (JHRE) DNA binding

  • While T. castaneum methoprene-tolerant was expressed in its entirety, Tribolium castaneum taiman (TcTAI) sequence was limited to include the bHLH and the two tandem PAS domains, that is, regions that are required and sufficient for TcTAI to engage in the JH-stimulated interaction with T. castaneum methoprenetolerant [23]

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Summary

Results

The AaJHR protein purified as a complex of His6-AaMET and FLAG-AaTAI subunits of apparent molecular weights consistent with those calculated from their sequences in the expression construct (95.1 and 46.8 kDa, respectively) (Fig. S1B). The persisting protein–DNA complex (Fig. 3B; lane 12) suggests that once formed in the presence of methoprene during the expression and purification steps, the T. castaneum methoprene-tolerant–TcTAI dimer is sufficiently stable to display k-JHRE binding in the EMSA without addition of hormone. This result is consistent with previous findings that JH was not necessary for binding of a bacterially expressed A. aegypti MET–TAI complex to DNA [24].

24 ΔScoref
26 Probabilityd
Discussion
Experimental procedures
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