Mutational landscape of receptor guanylyl cyclase C: Functional analysis and disease-related mutations

Abstract

Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin, which causes diarrhea, and the endogenous ligands, guanylin and uroguanylin. GC-C is predominantly expressed in the intestinal epithelium and regulates fluid and ion secretion in the gut. The receptor has a complex domain organization, and in the absence of structural information, mutational analysis provides clues to mechanisms of regulation of this protein. Here, we review the mutational landscape of this receptor that reveals regulatory features critical for its activity. We also summarize the available information on mutations in GC-C that have been reported in humans and contribute to severe gastrointestinal abnormalities. Since GC-C is also expressed in extra-intestinal tissues, it is likely that mutations thus far reported in humans may also affect other organ systems, warranting a close observation of these patients in future.

Keywords: cGMP; guanylyl cyclase C; human mutation; meconium ileus; secretory diarrhea.

Figure 1

Schematic representation of domain organization of receptor guanylyl cyclase C (GC-C). GC-C, expected to be a homodimeric transmembrane receptor, has seven conserved functional domains including the extracellular domain that binds peptide ligands (heat-stable enterotoxins [ST], endogenous peptides like guanylin and uroguanylin, and the FDA approved ST analog, Linaclotide); a single transmembrane domain spanning the plasma membrane; a short juxtamembrane domain; the kinase-homology domain, which binds ATP; a linker region that may facilitate catalytic subunit dimerization and regulates catalytic activity; the catalytic guanylyl cyclase domain, which mediates the conversion of GTP to cGMP; and a carboxyl terminal tail (C-terminal domain) that modulates the guanylyl cyclase activity helps in cytoskeletal anchoring, and receptor internalization. The linear representation of the domain organization exhibits the domain boundaries with the single letter amino acid code at the particular position. The length of each domain has been mentioned within brackets. The figure has been created with Biorender. ATP, adenosine triphosphate; cGMP, guanosine 3’,5’-cyclic monophosphate; FDA, Food and Drug Administration; GTP, guanosine triphosphate; ST, heat-stable enterotoxin

Figure 2

GC-C signaling pathway in intestinal epithelial cells. GC-C is expressed on the surface of the enterocytes and acts as the receptor for uroguanylin and guanylin that are synthesized in the intestine and secreted into the lumen. GC-C is also activated by ST peptides produced by enterotoxigenic E. coli. Ligand-mediated activation of GC-C converts GTP to cGMP. Elevated levels of intracellular cGMP activates cGMP-dependent protein kinase II (PKGII) which in turn phosphorylates NHE3 thereby inhibiting its activity. Cyclic GMP inhibits the activity of the cAMP-phosphodiesterase PDE3, thereby cross-activating cAMP-dependent protein kinase A (PKA). PKGII and PKA phosphorylate the CFTR leading to chloride ion (Cl⁻) efflux. Cyclic GMP also activates the secretion of bicarbonate ion (HCO₃ ⁻) by an unidentified channel (marked “?” in the figure). These processes balance fluid-ion homeostasis in the intestine. Cyclic GMP also directly activates CNG channels leading to Ca²⁺ influx. Increased level of intracellular Ca²⁺ interacts with calcium-sensing receptors (CaRs), leading to cell differentiation and migration. Phosphorylation by protein kinase C (PKC) activates and phosphorylation by c-Src inhibits guanylyl cyclase activity of GC-C. PKGII on activation by cGMP activates p38 MAPK which phosphorylates the transcription factor Sp1 which in turn upregulates the transcription of p21 mRNA. Hydrolysis of cGMP to 5’GMP by the cGMP-specific phosphodiesterase, PDE5, can terminate GC-C signaling. The figure has been created with Biorender. cAMP, adenosine 3’,5’-cyclic monophosphate; cGMP, guanosine 3’,5’-cyclic monophosphate; CNG, cyclic-nucleotide-gated; GC-C, guanylyl cyclase C; GTP, guanosine triphosphate; MAPK, mitogen-activated protein kinase; ST, heat-stable enterotoxin

Figure 3. Alignment of receptor guanylyl cyclase C across species.

(a) Alignment of the extracellular domain (starting from residue 24 to 430 in human, ~406 amino acids). (b) Alignment of the KHD (starting from residue 490 to 735 in human, ~246 amino acids). (c) Alignment of the linker region (starting from residue 736 to 810 in human, ~75 amino acids). (d) Alignment of the GCD (starting from residue 811 to 1,010 in human, ~200 amino acids). Organisms selected are Homo sapiens (NP_004954.2), Mus musculus (NP_001120790.1), Rattus norvegicus (NP_037302.1), Danio rerio (XP_021329920.1), Xenopus tropicalis (XP_002934756.1), Gallus gallus (XP_416207.3), Chelonia mydas (XP_007064150.1), and Sus scrofa (NP_999270.1). Orthologs of GC-C were identified using BLAST and aligned using Kalign. Residues are colored depending on the level of conservation across the eight species using Jalview v2.11. Residues marked with red triangles identify reported mutations in the amino acid in patients causing duodenal atresia (DA), asplenia, congenital secretory diarrhea (CSD), meconium ileus (MI), and familial GUCY2C diarrhea syndrome (FGDS). Mutation N757K* is a result of an insertion of one base pair, resulting in conversion of Asn to Lys and generation of a stop codon immediately after the Lys residue. GC-C, guanylyl cyclase C; GCD, guanylyl cyclase domain; KHD, kinase-homology domain