Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.
Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state. Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites. These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites. Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA). Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity. These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.
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