Guanylyl cyclase C (GC-C) is a receptor-enzyme that is the major source of cyclic GMP at the brush border surface of the intestinal epithelial cell. GC-C has an extracellular ligand binding domain. Ligand binding activates an intracellular enzyme domain, ultimately leading to the opening of the cystic fibrosis transmembrane regulator (CFTR) chloride channels. GC-C can bind two different ligands. It can bind the STa enterotoxins produced by Escherichia coli and other diarrhetic bacteria. These toxins are a major cause of infant and travelers diarrhea world-wide. It can also bind the newly discovered intestinal peptide, guanylin, which is produced by enterochromaffin (EC) cells. Guanylin resembles the circulating natriuretic peptides (i.e., atrial natriuretic peptide -- ANP) in its precursor-product relationship, receptor structure, and activation of guanylate cyclase. Because guanylin and ST have the same receptor, the profuse diarrhea caused by the STa toxin is probably due to the unregulated activation of GC-C. Production of cyclic GMP at the brush border surface is associated with increased chloride secretion and decreased absorption, consistent with the secretory diarrhea associated with the STa toxin. However, the role of GC- C in normal intestinal physiology is unclear. Very little is known about the biology of GC-C and its natural ligand, guanylin. Yet the guanylin-STa receptor effector system may regulate enteric electrolyte balance or water loss, seeing as an intestinal counterpart to the brain and atrial natriuretic peptide systems. Further, the synthesis or activation of guanylin and GC-C may be controlled by the intestinal response to dietary factors, such as stretch, osmotic, or hormonal stimuli. The long term goal of the proposed research is to understand the guanylin- receptor interaction and its mechanisms of cell signaling using both biochemical and molecular approaches. Understanding the structure and function of this receptor system may contribute to the design of new anti- diarrheal drugs, vaccines or intestinal modulators. Because the rat intestinal response to STa is heightened by as much as ten-fold during starvation and because STa-associated secretory diarrhea is exacerbated in famished individuals, we will focus on this receptor system in the fasted rat.
The specific aims of this proposal are as follows: (l) To determine the dietary and circadian regulation of GC-C and GN at different intestinal sites. (2) To determine the molecular basis for the abrupt GC-C down- regulation from the enterocyte brush border surface that occurs when fasted rats are refed. (3) To define the localization and movement of GC-C during its synthesis, assembly, and transport to the brush border and after its binding to the STa toxin.
