Abstract
Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.
Highlights
Guanylyl cyclase C (GCC) is the receptor for heat-stable enterotoxins (STa)1 secreted by Escherichia coli and other enteric bacteria
We report the cloning and initial characterization of IKEPP, a novel PDZ protein expressed at the apical membrane of human intestinal epithelial cells
IKEPP directly associates with the COOH terminus of guanylyl cyclase C (GCC), the heat-stable enterotoxin receptor found at the apical surface of intestinal epithelial cells
Summary
Guanylyl cyclase C (GCC) is the receptor for heat-stable enterotoxins (STa)1 secreted by Escherichia coli and other enteric bacteria. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. To determine whether the COOH terminus of GCC participates in protein-protein interactions that may regulate its targeting or function, we screened a human intestinal epithelial enriched yeast two-hybrid library using CTEP as “bait.” We found that GCC associates via its COOH terminus with a novel protein containing four PDZ domains.
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