Meeting Abstract
The physiological importance of sesquiterpenoid signaling has been recognized in both insects and crustaceans, particularly in the regulation of reproduction. Crustaceans use methyl farnesoate (MF) as the innate ligand whereas insects use an epoxide form of MF, JH III. In insects, the Methoprene-tolerant (Met) protein has been characterized as a JH receptor. The met gene belongs to the bHLH –PAS family of transcription factors, and Drosophila Met protein binds JH III with high affinity. Subsequent experiments have shown that Met heterodimerizes with steroid receptor coactivator (SRC). SRC in turn may function as a co-activator for ecdysone signaling. A met homolog was recently identified in cladocerans, and like insects, Met was found to interact with a crustacean homolog of SRC in the presence of JH III. The Met/SRC interaction was also found to be 10 times more sensitive to MF than to JH III. We are interested in characterizing the molecular basis of MF signalling in brachyuran crabs. We have identified a Met ortholog in Uca pugilator which is expressed during limb regeneration. Transcriptome analysis of developing limb bud RNA-seq libraries show hits throughout the molt cycle; a detailed analysis of met expression is in progress. BlastP results reveal conserved regional similarities to that of cladoceran met (34%), and similar correspondence to numerous insects. Homology modelling and structural validation of the PAS-B domain show a favorable ligand binding pocket with 8 specific amino acid (aa) residues that interact with JH III; there are 2 aa substitutions in Uca in comparison to insects. A SRC homolog, the heterodimer partner of Met-tolerant, is also observed in the limb bud transcriptome library. We are currently producing expression vector constructs to examine protein/protein and protein/ligand binding.
