RAT GUANYLYL CYCLASE-C EXPRESSED IN COS-7 CELLS EXHIBITS MULTIPLE AFFINITIES FOR ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN

Intestinal cells exhibit binding sites with different affinities for E scherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cycl ase C from intestinal cells has been identified as one receptor for th ese peptides. Equilibrium and kinetic binding characteristics of rat g uanylyl cyclase C expressed in COS-7 cells were examined, employing ST , to determine if this receptor exhibited multiple affinities. Scatcha rd analysis of equilibrium binding yielded curvilinear isotherms consi stent with the presence of high (pM) and low (nM) affinity sites. Kine tic analysis of binding demonstrated that these sites exhibited simila r dissociation but different association kinetics. In addition, two di stinct affinity states of low affinity sites were identified with diss ociation constants of 0.15 and 5.85 nM. Association of ST and low affi nity sites was biphasic, while dissociation from these sites was unimo dal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state a t equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC(50) = 110 nM) with receptor occupancy revealed that bind ing of ST to the lowest affinity state of low affinity sites (EC(50) = 80 nM) is directly coupled to catalytic activation. These studies sug gest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guan ylyl cyclase C, rather than different receptors for the ligand. The sh ift in affinity state of low affinity sites and its correlation with c atalytic activation suggest a central role for this phenomenon in mech anisms mediating receptor-effector coupling of membrane guanylyl cycla ses.