Mechanisms underlying receptor-effector coupling of guanylyl cyclase C
Guanylyl cyclase C (GCC), an intestinal receptor for E. coli heat-stable enterotoxin (ST) and guanylin, possesses a domain structure, including extracellular ligand binding (ECD), a single transmembrane (TMD), juxtamembrane (JD), regulatory kinase homology domain (KHD), a hinge region (HD) and catalytic guanylyl cyclase (CD) domains. Intestinal cells exhibit binding sites with different affinities for ST, suggesting the existence of different receptors 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. The studies herein confirmed that GCC, a single gene product, exhibits multiple ligand binding affinities, including high and low affinity sites (R scH and R scL) with picomolar and nanomolar affinities respectively, and a ligand-induced conversion of low affinity sites from a higher to lower affinity state (R scL1 to R scL2). Occupancy of R scL2 is coupled to ligand-induced catalytic activation, and the ligand-induced affinity shift appears to be an important mediator in ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC, expressed in COS-7 cells, were examined to identify the structural domains underlying expression of R scH and R scL 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 R scH. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for the expression of the ligand-induced shift from R scL1 to R scL2. 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 JD and KHD are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC resulted 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 demonstrate that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.
0307: Molecular biology