HCOM Biomedical Sciences
Life Sciences Building 235
Education: Ph.D. University of California, San Diego 1982
Neuropeptides are an important class of regulatory molecules in insects that regulate a variety of processes, including development, homeostasis, metamorphosis, ion transport, and muscle activity. The insect that we study is the tobacco hornworm, Manduca sexta, which has been an important model system for many years in investigations of endocrinology, physiology and neurobiology. We are focusing on the action of a peptide, allatotropin (Manse-AT), that was first shown to increase the rate of juvenile hormone (JH) synthesis by the adult corpora allata (CA), the source of JH.JH is an important hormone that has a wide spectrum of activities, the best known of which are its actions to prevent metamorphosis in larval insects and to stimulate egg maturation in the adult.
We localized the cells in the central and enteric nervous systems that contain Manse-AT mRNA by in situ hybridization. In larvae, the highest levels of Manse-AT mRNA were found in two cells in the frontal ganglion, that project their axons to the muscle surrounding the foregut, and also in two cells in the terminal abdominal ganglion. In the adult insect, additional cells contain Manse-AT mRNA, including neurosecretory cells in the abdominal ganglia and a cluster of cells in the posterior of the terminal ganglion. This widespread distribution suggests that Manse-AT may have diverse roles during different developmental stages.
Since Manse-AT was present in cells project toward the gut, we tested its effects on gut physiology in collaboration with Mary Chamberlin. Manse-AT rapidly inhibits active ion transport across the midgut epithelium. This active transport normally generates a large potential difference across the epithelium that provides energy for the absorption of nutrients.
We have used a molecular biological approach to characterize the gene for Manse-AT and its expression. Manse-AT is derived from a larger precursor that is cleaved within the secretory pathway prior to it release. We found that the Manse-AT gene is expressed as at least three mRNAs that differ from one another by alternative splicing. The location at which the alternative exons are included in the mature mRNAs occur within the open reading frame, so that three different propeptides are predicted as translation products. Each mRNA can encode a Manse-AT as well as other peptides, including some that are unique to each precursor. Some of these peptides share limited sequence identity with Manse-AT and possess overlapping bioactivity, both on JH synthesis in the adult and on ion transport in the larval midgut. The alternative splicing of Manse-AT transcripts is dynamically controlled in a tissue- and stage-specific manner, so the regulation of alternative splicing may have an important biological role.
An important question concerning the action of Manse-AT are the routes by which it mediates its action on the known target tissues. To begin to address this point, we are currently using an ELISA assay to quantitate the levels of Manse-AT in different tissues throughout development. We are also testing whether the peptide is secreted into the hemolymph where it can act as a hormone.
Our present and future interests include the characterization of the receptor protein that mediates the action of Manse-AT and other neuropeptides. We have already isolated clones for putative biogenic amine receptors and peptide receptors using a PCR approach. We will also use expression cloning as a method to identify membrane-bound receptors that bind Manse-AT. Our goals include the characterization of the tissue distribution of the receptor and the elucidation of the biochemical changes that occur in the target cells. Once neuropeptide receptors have been identified, agonists and antagonists of these receptors can be identified for use in disrupting important physiological events in insects.