Abstract

Two distinct affinity states of low affinity Escherichia coli heat-stable enterotoxin (ST) receptors in rat intestinal membranes, with dissociation constants of 0.12 and 2.5 nM, were identified. Kinetic binding studies demonstrated biphasic association kinetics, whereas dissociation was unimodal. These studies also confirmed that ligand bound to each receptor state in an independent bimolecular reaction. In contrast, equilibrium binding studies yielded linear Scatchard plots, indicative of a single class of noninteractive binding sites, with a Kd = 2.3 nM. Close agreement of the dissociation constants determined by kinetic and equilibrium methods suggested that receptors were in the lower affinity state at equilibrium. Several models, including binding site heterogeneity, cooperativity, and ligand-induced alterations in receptor conformation were inconsistent with these observations. Indeed, these data were most consistent with a two-step binding process involving a third component. Comparison of the ligand dependence of enzyme activation (EC50 = 124 nM) and the calculated fractional receptor occupancy of the lower affinity component at 5 min (EC50 = 40 nM) demonstrated that occupation of the lower affinity state of low affinity ST receptors correlated with guanylate cyclase activation. The close correlation between receptor occupation and enzyme activation suggests that there are no spare receptors for ST in intestinal membranes. These data resolve the previously observed discrepancy between the affinity of receptors for ST and the potency of this ligand for activating guanylate cyclase. Receptor affinity state alterations may represent a common mechanism for receptor-effector coupling of particulate guanylate cyclases.