Functional Inactivation of the Human Guanylyl Cyclase C Receptor:  Modeling and Mutation of the Protein Kinase-like Domain

Abstract

Receptor guanylyl cyclases possess an extracellular ligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and stable toxin family of peptides, presumably as a result of binding to the kinase homology domain (KHD). Modeling of the KHD of GC-C indicated that it could adopt a structure similar to that of tyrosine kinases, and sequence comparison with other protein kinases suggested that lysine 516 was positioned in the KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did not recognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491-568 of GC-C. Mutation of lysine 516 to an alanine in full-length GC-C (GC-C K516A ) dramatically reduced the ligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-type GC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-C converted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C K516A did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domain and the catalytic domain in regulation of activity in this family of receptors, and the presence of K 516 is critical for the possible proper orientation of ATP in this domain.

Guanylin, Uroguanylin, and Heat-stable Euterotoxin Activate Guanylate Cyclase C and/or a Pertussis Toxin-sensitive G Protein in Human Proximal Tubule Cells

“…The role of this phosphorylation in the GC-C function is not known yet (35,36). In our study, an inhibitor of tyrosine kinases, genistein (10 M), was able to block all depolarizations of V m of IHKE-1 cells induced by GN (n ϭ 7) or UGN (n ϭ 11, each 10 nM).…”

“…This pH dependence of the effects especially for UGN in the IHKE-1 cells may be physiologically relevant within the kidney as the luminal pH becomes more acidic along the nephron and preliminary data indicate that UGN and GN also act in the collecting duct. 2 The role of tyrosine phosphorylation of the kinase homology domain in GC-C may play a role in regulation of GC-C signaling (35,36). For the guanylate cyclase activation of guanylate cyclase A and B (GC-A, GC-B), phosphorylation of the kinase homology domain appears to be essential for ligand-induced activation (for review, see Ref.…”

“…GC-C is phosphorylated in the basal state and undergoes a STa-induced time-dependent switching in the activity in the intestine (20). Phosphorylation of GC-C could play a similar role in its activity (35,36). However, a possible dephosphorylation of GC-C leading to inactivation of the guanylate cyclase and consecutive activation of a coupled G protein appears unlikely as this should influence the effects of all three peptides.…”

Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

“…However, a subsequent report indicated that this region is not involved in the regulation of NPR-A (22). More recently, the mutation of Lys 516 in GC-C, which is conserved in all human receptor guanylyl cyclases (29), was shown to dramatically inhibit the activation of this receptor (5).…”

A novel membrane guanylyl cyclase expressed in medaka (Oryzias latipes) intestine

“…14 (5), 820-828. (Yu et al, 1997) and Lys residues (Bhandari et al, 2001). N-linked glycosylation sites are underlined, and the glycosylation sites at Asn 195 and Asn 402 in porcine GC-C (Hasegawa et al, 1999a) are indicated by open circles.…”