Regulation of guanylyl cyclase C signalling : glycosylation, localisation and identification of downstream effectors

Abstract

Guanylyl cyclase C (GC-C) belongs to the receptor guanylyl cyclases that synthesize cGMP, a ubiquitous second messenger. GC-C is a glycoprotein receptor with an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cyclase domain. The intracellular domain can be further divided into a protein kinase–like domain that has homology to protein kinases, a guanylyl cyclase domain that catalyzes cGMP synthesis, and a short C-terminal tail.

GC-C is expressed predominantly in intestinal epithelial cells, but GC-C expression has also been detected in extraintestinal tissues such as the liver, kidney, and reproductive tissues. The ligands for GC-C include endogenous ligands such as the guanylin family of peptide hormones and bacterial heat-stable enterotoxins (ST). Binding of the ligands to the extracellular domain of GC-C leads to activation of the intracellular cyclase domain, which then synthesizes cGMP using MgGTP as a substrate. cGMP activates protein kinase A and protein kinase G, which in turn phosphorylate and activate the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). This leads to secretion of chloride ions followed by water in the intestinal lumen. GC-C has therefore been proposed to be involved in the regulation of ion homeostasis.

ST, which has a higher affinity for GC-C than the endogenous ligands, causes excessive salt and water secretion, leading to watery diarrhea, which is a major cause of infant mortality in developing countries. GC-C knockout mice, however, are apparently healthy and exhibit resistance to ST-induced diarrhea. This intriguing observation—that a gene responsible for infant mortality has been selected and maintained during evolution—indicates that GC-C perhaps provides protection against stressors not yet identified. This paradoxical phenotype of GC-C knockout mice, together with the observation that GC-C is expressed in extraintestinal tissues, including cells that do not express CFTR, suggests that GC-C could have additional downstream targets. Recent studies indeed suggest that GC-C exerts antiproliferative effects in colonic cell lines by activation of cGMP-gated calcium channels, and persistent exposure to ST during bacterial infection could provide protection against colonic cancer in vivo.

Cellular signaling pathways are in constant interaction with other signaling pathways in the cell. This interaction can lead to diverse biological responses following activation of a single receptor and allows fine-tuning of signaling pathways through regulation at multiple levels. The GC-C signaling pathway is also regulated under different physiological conditions at both the transcriptional and post-transcriptional levels. However, the molecular mechanisms of GC-C activation, subsequent regulation of GC-C–mediated signaling, and its cross-talk with other signaling pathways in vivo are not yet fully understood. This study was aimed at understanding the regulation of GC-C signal transduction and its role in cellular physiology.

Earlier studies carried out in the laboratory using the T84 colonic carcinoma cell line, which expresses GC-C, showed that prolonged ST treatment of T84 cells leads to cellular refractoriness to further ST stimulation. The cellular refractoriness to ST was due to desensitization of GC-C as well as activation of cGMP-dependent, cGMP-specific phosphodiesterase (PDE5A), leading to reduced accumulation of cGMP upon restimulation with ST.

In this work, studies were carried out to investigate the mechanism of GC-C desensitization using Caco-2 cells, which also express GC-C. Similar to T84 cells, after 9 hours of exposure to ST, Caco-2 cells became refractory to further ST treatment. In Caco-2 cells, the cellular refractoriness to ST was entirely due to GC-C desensitization, since inhibition of phosphodiesterases did not alleviate the refractoriness. GC-C desensitization was a cell-specific phenomenon and was not observed in HEK293 cells stably transfected with GC-C (HEK293-GCC), suggesting that GC-C is differentially regulated in different cell types.

In vitro guanylyl cyclase assays carried out on membranes prepared from control and desensitized Caco-2 cells showed a complete loss of ligand-mediated activation. However, there was only a partial decrease in GC-C content, as seen in ligand-independent activation of GC-C by MnGTP and non-ionic detergents, as well as in receptor-binding analysis. These observations indicated that only a fraction of the total GC-C receptor content was ligand-stimulatable, and desensitization could be due to selective downregulation of the ligand-stimulated form of GC-C.

Western blot analysis was then carried out on membranes prepared from control and desensitized cells. GC-C is expressed as two differentially glycosylated forms of 130 kDa and 145 kDa. Both forms were detected in membranes prepared from control cells. Interestingly, the 145 kDa form was not detected in desensitized Caco-2 cells. Upon incubation of desensitized cells without ST for 12 hours, cells regained ligand stimulatability, and concomitantly the 145 kDa form was again detected in Western blot analysis. These observations suggested that the 145 kDa form could be the ligand-stimulated form, and downregulation of the 145 kDa form was responsible for GC-C desensitization. The loss of the 145 kDa form upon desensitization was due to internalization and subsequent degradation, since desensitization was not observed in the presence of inhibitors of endocytosis.

Further studies were carried out in HEK293-GCC cells to investigate the functional significance of the two forms of GC-C. Treatment of HEK293-GCC cells with cycloheximide led to a decrease in the amount of the 130 kDa form with a corresponding increase in the 145 kDa form, indicating that the 130 kDa form was the precursor of the 145 kDa form, as suggested previously. Analysis of sugar residues using different glycosidases and lectins indicated that the 130 kDa form harbored high-mannose–type sugars, whereas the 145 kDa form contained complex sugars such as sialic acid and galactose, indicating processing in the Golgi complex.

The 145 kDa form localized to the plasma membrane, while the 130 kDa form was intracellular, as observed by surface biotinylation and trypsinization studies. Subcellular fractionation studies confirmed that the 130 kDa form was present in the endoplasmic reticulum (ER), while the 145 kDa form was present in plasma membrane fractions.

Interestingly, although both forms possessed guanylyl cyclase activity and could bind ST with similar affinity, only the 145 kDa form was activated by ST. The ER-localized 130 kDa form had high basal activity and did not show further ligand-mediated activation. The ligand stimulatability of the 145 kDa form was not due to the plasma membrane environment, since the fully mature form trapped intracellularly at 20°C was also ligand-stimulated. These results suggested that complex sugars present in the 145 kDa form are essential for ligand-mediated activation of GC-C, possibly by enabling the conformational change required for activation.

GC-C was expressed in HEK293 cells deficient in N-acetylglucosamine transferase I (GnTI), an enzyme essential for addition of complex sugars. As expected, GC-C expressed in HEK293 GnTI? cells did not undergo further glycosylation and migrated along with the 130 kDa form. Although catalytically active and localized to the plasma membrane, this form was not ligand-stimulatable, confirming that complex sugars are essential for ligand-mediated activation.

Removal of sialic acid and/or galactose from the mature 145 kDa form did not affect basal or ligand-stimulated activity, suggesting that sugars are required to establish a ligand-responsive conformation but are not directly involved in the activation process itself.

Immunofluorescence studies showed that intracellular localization of GC-C was not due to overexpression and was observed across different cell lines, suggesting physiological relevance. Fusion protein studies indicated that the intracellular domain plays a role in regulating GC-C localization.

To identify additional downstream effectors, differential display RT-PCR was performed on RNA from control and ST-treated Caco-2 cells. Several genes were differentially regulated, including pICln (a regulator of volume-activated chloride channels), a putative RNA methyltransferase similar to p120, and elmo2, a regulator of Rac GTPase activity. These findings suggest that GC-C may regulate cell cycle progression, cytoskeletal rearrangements, and cell motility.

In summary, this study provides insights into the activation and regulation of GC-C signaling. Glycosylation regulates both basal and ligand-mediated activity of GC-C. Complex sugars added in the Golgi are essential for ligand stimulatability. GC-C desensitization occurs via selective downregulation of the 145 kDa form. GC-C localization is regulated by its intracellular domain, and multiple downstream effectors suggest broader roles in cellular physiology than previously appreciated.