Meeting Abstract

P1.105  Jan. 4  Characterization of MIH-binding proteins associated with activation of the crustacean molting gland LEE, SG; CHANG, ES; MYKLES, DL*; Colorado State University, Fort Collins, CO; Bodega Marine Lab, Bodega Bay, CA; Colorado State University, Fort Collins, CO holynine@lamar.colostate.edu

Regulation of the molting cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland (XO/SG) complex and the molting gland or Y-organ (YO). Molt-inhibiting hormone (MIH), is produced in XO/SG complex, and regulates ecdysteroidogenesis in the YO. In the conventional model, molting is induced by a reduction in the synthesis and secretion of MIH in the eyestalks. Eyestalk ablation (ESA) causes a rapid increase of ecdysteroid titers and induces molting. In an effort to better understand the signaling pathway, immunopreciptation and mass spectrometry were used to identify MIH-binding proteins in quiescent and activated YOs. YOs from intact and 1-day post-ESA land crabs (Gecarcinus lateralis) were homogenized in a lysis buffer that solubilized cytosolic and membrane proteins. Proteins were immunoprecipitated with an anti-blue crab MIH serum and separated by SDS-PAGE or 2D-PAGE; gels were either stained with silver or subjected to Western blotting. Surprisingly, ESA had no effect on the amount of MIH immunoprecipitated from YO extracts. Two proteins, at 32 kDa (pI 8) and 35 kDa (pI 7.6), co-precipitated with MIH in YO extracts from intact animals, but not in YO extracts from 1-day post-ESA animals. These data suggest that the MIH-binding proteins (MIHBPs) mediate MIH suppression of the YO. Tandem mass spectrometry was used to sequence tryptic peptides from the two MIHBPs. The sequences of two of the peptides from the 32- and 35-kDa proteins were identical, suggesting that these proteins are related. More complete sequence information will facilitate cloning cDNAs encoding these proteins. Supported by NSF (IBN-0342982).