Catalytic region mimetics in Na+/H+ exchanger regulatory factor 4 suppress guanylate cyclase 2C activity to regulate enterotoxin triggered diarrhea

Abstract

Guanylate cyclase 2C (GCC) upon binding to the bacterial heat-stable enterotoxin ST, generates excessive cGMP, driving intestinal chloride and fluid secretion that manifests as diarrhea. We investigated the regulatory mechanism of GCC through its interactions with scaffolding proteins sodium-hydrogen exchanger regulatory factor (NHERF)1 to 4. PSD95, Dlg1, ZO-1 (PDZ) domain in NHERF4 inhibited GCC catalytic activity, while NHERF1-3 binary binding had no impact. NHERF4-mediated inhibition was mimicked by two synergistically acting peptides, (N4-110 [NH2-LERPRFCLL-COOH] and N4-195 [NH2-RHAHDVARAQL-COOH]), localized in close proximity within the PDZ1 domain. These peptides, which showed high sequence homology to the GCC catalytic domain, were mapped via 3-D structural modeling to the GCC dimer interface. Fluorescence resonance energy transfer (FRET) analysis confirmed that NHERF4-PDZ1 domain binding interfered with GCC oligomerization. In mouse and human enteroid models, NHERF4 peptides dose-dependently reduced GCC-mediated fluid secretion. Additionally, NHERF4-GCC interaction was enhanced upon ST stimulation, suggesting that NHERF4 functions as a negative regulator of aberrant GCC activity during enterotoxin-induced diarrhea. Furthermore, we described a macromolecular complex of GCC with multi-drug resistance protein 4 (MRP4), a cAMP/cGMP efflux transporter, in the regulation of fluid secretion through NHERF3-mediated assembly. Overall, our findings reveal novel regulatory mechanisms for GCC, offering insights into targeted therapies for enterotoxin-triggered diarrheas.