MYKLES, D.L.; Colorado State University: Proteomics and signal transduction in the crustacean molting gland

Molting in decapod crustaceans is controlled by the X-organ/sinus gland complex, a neurosecretory center in the eyestalks. The complex secretes a neuropeptide, molt-inhibiting hormone (MIH), that suppresses production of molting hormone (ecdysone), an ecdysteroid secreted by a pair of molting glands (Y-organs or YOs) located in the anterior of the body. Binding of MIH to a YO membrane receptor results in a cyclic nucleotide-dependent inhibition of ecdysteroidogenesis. Eyestalk ablation (ESA) induces a rapid increase of hemolymph ecdysteroid titers. Proteomics has become a powerful tool to identify proteins involved with specific physiological or disease states. We are using expression and cell-map proteomics to determine which proteins are involved in YO signaling, YOs from intact and ES-ablated land crabs (Gecarcinus lateralis) were analyzed by 2-D gel electrophoresis and mass spectrometry. ESA causes dramatic changes in the levels of proteins between 12 kDa and 27 kDa. These proteins were selected as putative candidates for molt-regulating factors from both silver and phosphoprotein-stained gels. Nitric oxide synthase (NOS) was identified by immunoblotting and peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry as one of the proteins transiently phosphorylated in response to ESA. In addition, MIH interacting proteins were detected using immunoprecipitation with an anti-MIH antibody. These putative MIHBPs (MIH binding proteins) were observed only in intact YO and not in YOs from ES-ablated animals. This suggests that these proteins could be involved in the binding of MIH to its receptor in the YO membrane. We are currently characterizing the MIHBPs by PMF and internal peptide sequencing using ion-trap tandem mass spectrometry. Supported by NSF (IBN-0342982).