A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.
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.
Scott
Robert O
RO
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Thelin
William R
WR
Milgram
Sharon L
SL
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015815:Cell Adhesion Molecules; D002460:Cell Line; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D004768:Enterotoxins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; C423943:PDZD2 protein, human; D011506:Proteins; D018000:Receptors, Peptide; D005982:Glutathione Transferase; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP