| dc.description.abstract | Guanylyl cyclases are enzymes that catalyze the conversion of magnesium-bound GTP to cGMP. Cyclic GMP then acts as a second messenger to a variety of exogenous and endogenous ligands. Guanylyl cyclases are of two types: soluble and particulate guanylyl cyclases. The cytosolic guanylyl cyclases are activated by nitric oxide (NO) and the particulate forms of this enzyme serve as receptors for a diverse range of peptide ligands. Receptor guanylyl cyclases are characterized by the presence of an extracellular ligand binding domain, a single transmembrane domain, and the cytosolic domain, which is subdivided into a protein kinase-like domain and the catalytic guanylyl cyclase domain.
Among the particulate forms, guanylyl cyclase A (GCA) and guanylyl cyclase B (GCB) are the receptors for natriuretic peptides. Guanylyl cyclase C (GCC) was initially described as the receptor for stable toxin peptides secreted by pathogenic bacteria. The other members of the receptor guanylyl cyclase family include the sea urchin sperm guanylyl cyclase and the two isoforms of retinal guanylyl cyclase. However, in the recent past, new members of the particulate guanylyl cyclase family, such as GCD, GCE, and GCF in sensory neurons, have been identified. Another membrane guanylyl cyclase isoform, GCG, was cloned from the mammalian lung, jejunum, and skeletal muscles. The newly isolated receptor guanylyl cyclases are currently regarded as orphan receptors with no known extracellular ligands. Receptor guanylyl cyclases homologous to retinal guanylyl cyclase have been cloned from photoreceptor cells of the medaka fish and the nervous system of the insect, Manduca sexta.
Guanylyl cyclase C (GCC) is a membrane-bound guanylyl cyclase predominantly located on the apical surface of intestinal villus cells. GCC binds ligands such as the heat-stable enterotoxin peptide (ST) and endogenous ligands, guanylin, uroguanylin, and lymphoguanylin. Ligand binding to the extracellular domain of the receptor activates its cytoplasmic catalytic domain, leading to an elevation of intracellular cGMP. This activation opens the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). The opening of CFTR is followed by the secretion of chloride ions and water into the intestinal lumen, and excessive chloride secretion results in watery diarrhea. The ST peptide mimics the signaling cascade triggered by guanylin/uroguanylin binding to GCC.
Guanylin, the first endogenous ligand discovered for GCC, is a 15-amino acid peptide isolated initially from the rat jejunum and identified as a ligand for GCC based on its ability to enhance cGMP levels in the T84 human colonic carcinoma cell line. The affinity of GCC for guanylin is known to be 100-fold lower than for ST, and guanylin, at physiological concentrations, does not cause diarrhea. It is possible that guanylin and ST induce different conformational changes upon binding to the receptor, and the guanylin-induced conformational change does not activate the receptor maximally. However, the lower potency of guanylin bioactivity could be due to its lower affinity for GCC. It would then be possible to regulate guanylin-mediated GCC activation through cross-talk with other signaling pathways.
Studies were initiated to elucidate the mechanism and regulation of guanylin-GCC interaction and understand the physiological relevance of GCC-mediated signaling. To this end, a 15-amino acid sequence corresponding to the bioactive human guanylin was synthesized. Synthetic human guanylin was purified and air-oxidized to generate the bioactive peptide. Synthetic guanylin was able to bind and stimulate GCC in T84 cells in a dose-dependent manner with an affinity of 50 nM, which was 100-fold lower than the affinity of GCC for ST.
Receptor activity is also regulated by the ability of the ligand to induce receptor desensitization. In vitro, pre-incubation of GCC with ST, in the absence of the substrate Mg-GTP, resulted in the inactivation of GCC. Synthetic human guanylin, like ST, was able to induce the inactivation of GCC in T84 membranes. However, at equimolar concentrations, guanylin induced a lower extent of inactivation of GCC compared to ST-induced inactivation. This could be explained on the basis of receptor occupancy and the affinities of the ligands for GCC. Since receptor activation and its inactivation both require ligand binding, due to the lower affinity of guanylin, at the same concentration as ST, fewer ligand-binding sites on GCC were occupied, resulting in a lesser extent of GCC inactivation. Thus, it was evident that guanylin induced changes in receptor conformation, resulting in both activation and inactivation in a manner similar to ST, suggesting that guanylin is a full agonist for GCC.
It was clear that guanylin, because of its 100-fold lower affinity for GCC, would not cause a significant increase in intracellular cGMP at physiological concentrations in intestinal cells. Therefore, it was envisioned that guanylin bioactivity could be potentiated by cross-talk with other signaling pathways. Regulation of ST-stimulable activity by PKC-mediated phosphorylation of GCC at a critical serine residue (Ser-1029) has been reported. Therefore, the potentiation of guanylin-induced GCC activity by PKC activation was investigated.
T84 cells were treated with the potent PKC activator, phorbol myristate acetate (PMA), prior to the addition of guanylin. A 2.6-fold enhancement in guanylin-induced cGMP levels was observed upon PMA treatment of T84 cells. An important observation was that basal cGMP levels remained unchanged. PMA-mediated potentiation of guanylin-induced GCC activity was significantly inhibited by pre-incubation of T84 monolayers with staurosporine, a potent inhibitor of PKC, indicating that a phosphorylation-mediated mechanism was operative.
A similar level of potentiation of guanylin-induced cGMP synthesis in T84 cells was observed upon PKC activation in the presence or absence of a general phosphodiesterase inhibitor, isobutylmethylxanthine. Thus, potentiation of guanylin-induced cGMP levels was not due to reduced activity of phosphodiesterase. It was the synthesis of cGMP alone that was modulated. Interestingly, PMA-mediated potentiation of guanylin bioactivity was mimicked by carbachol, a cholinergic agonist that activated PKC via activation of PLC? and release of diacylglycerol. This suggested that cross-talk with signaling pathways that activate PKC could potentiate guanylin-induced GCC activity in vivo.
Potentiation of ligand-induced guanylyl cyclase activity of GCC was also observed in in vitro guanylyl cyclase assays performed with membranes from T84 cells treated with PMA, suggesting that no additional cytosolic factors were required. The enhancement of ligand-induced GCC activation was not due to increased ligand binding to the receptor, as no significant increase was observed in either the affinity or the binding sites of GCC for guanylin on PMA treatment. This indicated that the modulation of the catalytic domain of GCC was responsible for the increase in cGMP levels following ligand binding.
It was possible that the increased activation of GCC seen in PMA-treated cells was due to decreased ligand-induced inactivation. However, ligand-induced inactivation was not alleviated in PMA-treated cells, suggesting distinct mechanisms for ligand-induced activation and ligand-induced inactivation.
One of the characteristic features of PMA-mediated regulation of PKC is the downregulation of PKC activity upon prolonged exposure to PMA. However, in contrast with the potentiation observed in ligand-stimulated GCC activity upon exposure of T84 cells to PMA, both basal and ligand-induced GCC activity were inhibited by 50% upon prolonged exposure to PMA. Additionally, equilibrium binding data and Western blot analysis using a GCC-specific monoclonal antibody revealed a reduction in total receptor content upon prolonged PMA treatment. Therefore, the decrease in GCC content observed on prolonged PMA treatment was likely a reflection of reduced levels of GCC mRNA.
Northern analysis of GCC mRNA from T84 cells treated with PMA for various time periods showed a decline in the steady-state levels of GCC mRNA. The GCC mRNA levels were compared with the GCC protein content in T84 cells treated with PMA. There were several important inferences from this analysis. At the end of a 1-hour PMA treatment, although ligand-induced GCC activity was potentiated with no change in the receptor content, GCC mRNA levels were reduced by 35%. Therefore, there was dual and contrasting regulation of GCC by PKC upon short-term PMA treatment.
Staurosporine could completely reverse the PMA-mediated downregulation of GCC, proving conclusively that the reduction in GCC mRNA was due to a PKC-mediated phosphorylation event. Carbachol also mimicked the phorbol ester-mediated downregulation of GCC mRNA. The carbachol-mediated downregulation was reversed by staurosporine, suggesting that PKC-mediated downregulation of GCC mRNA could be initiated via signaling mechanisms that activate PKC under physiological conditions.
Reduction in steady-state levels of mRNA could be due either to increases in the rate of mRNA degradation or to decreases in the rate of transcription of the GCC gene. To exclusively monitor the rate of degradation of GCC mRNA, control and PMA-treated T84 monolayers were incubated with actinomycin D for various times. No change in the rate of degradation of GCC mRNA was observed, indicating that regulation occurred mainly at the level of transcription. Run-on analysis performed on nuclei prepared from control and PMA-treated T84 cells showed a 70% decrease in GCC transcription. Thus, activation of PKC by PMA treatment downregulated GCC by reducing the transcription of the GCC gene.
One of the mechanisms of transcriptional downregulation of GCC could be through differential binding of transcription factors to their target sites in the GCC promoter in control and PMA-treated cells. The sequence of the 5'-flanking region of the human GCC gene is available. In this 1.8 kb promoter of the GCC gene, binding sites are present for several transcription factors, including Cdx2, hepatic nuclear factor (HNF4), and GATA-4. However, known phorbol ester-responsive and PKC-regulatable elements, such as the AP-1 or AP-2 sites, were missing in the GCC promoter. It was conceivable that PKC could mediate changes in the phosphorylation status of Cdx2, HNF4, or GATA, thereby regulating their DNA binding activity and, consequently, GCC transcription. The multiple Cdx2 and GATA binding sites present in the GCC promoter are identical with known consensus binding sites for these factors. Two of the three HNF4 binding sites show 75% homology to the accepted consensus sequence for HNF4 binding. Modulation of binding of these transcription factors on PMA treatment of T84 cells was investigated by monitoring the binding of these transcription factors to their cognate sites in electrophoretic mobility shift assays (EMSA).
Oligonucleotides corresponding to consensus target sites for Cdx2, HNF4, and GATA were synthesized. EMSA was performed with nuclear extracts prepared from control and PMA-treated T84 cells to analyze changes in binding of Cdx2, HNF4, and GATA to their target sequences due to activation of PKC. Results of the EMSA using the consensus binding sites for Cdx2, GATA, and HNF4 demonstrated the presence of these transcription factors in T84 cells. Upon PMA treatment and activation of PKC, there was a dramatic decrease in DNA binding in all three transcription factors to their cognate sequences.
Interestingly, there was a varying extent of recovery in DNA binding of these transcription factors upon inhibition of PKC activity. Staurosporine significantly reversed the reduction in DNA binding ability of Cdx2 and GATA by 60% and 75%, respectively, but recovery was only to 30% of control in the case of HNF4 binding to its cognate site. Therefore, it was possible that PMA not only triggered the activation of PKC but perhaps also other kinases, such as PKA, which could modulate HNF4 binding. Moreover, PMA addition appears to negatively modulate the activity of Cdx2, HNF4, and GATA at the same time, which emphasizes the hypothesis that GCC gene regulation is mediated by multiple factors binding to its upstream promoter sequence.
The two HNF4 binding sites in the GCC promoter were only 75% identical to the consensus HNF4 binding site. Binding of HNF4 to one of the oligonucleotides that corresponded to the putative binding sites in the hGCC promoter with a critical substitution in the 5'-half site was studied by EMSA. Results showed that the interaction of this potential HNF4 binding site with HNF4 is low, suggesting that the regulation of GCC transcription could be mediated by HNF4 through the more conserved site.
Using antibodies specific to HNF4, the mechanisms of PMA-mediated regulation of its DNA binding activity were investigated. The complex formed in EMSA between the consensus HNF4 binding site and T84 nuclear extract was supershifted upon binding of HNF4-specific antibody, thus confirming the presence of HNF4 in the complex. It was possible that the reduction in DNA binding activity of HNF4 was either due to a reduction in total HNF4 protein content or a functional modulation of HNF4 activity, such as a conformational alteration that led to a loss in DNA binding activity. Western analysis of control and PMA-treated nuclear extract with HNF4-specific antibody showed no reduction in HNF4 content. Therefore, PMA-mediated activation of a kinase could regulate the DNA binding activity of HNF4 by modulating its phosphorylation status. Again, no difference in phosphorylation was observed upon 32P labeling of control and PMA-treated T84 cells and subsequent immunoprecipitation of HNF4 protein from the nuclear extracts. Therefore, it was not a direct regulation of HNF4 on PMA treatment that modulated its DNA binding activity. However, the possibility that the PMA-mediated regulation of DNA binding activity of HNF4 could occur through phosphorylation of an HNF4-associating factor could not be entirely ruled out.
In conclusion, the studies described in this thesis attempt to delineate the mechanisms of transcriptional and post-transcriptional regulation of GCC by PKC. PKC potentiated ligand-induced GCC activity but downregulated GCC transcription, allowing fine-tuning of the regulation of GCC-mediated signaling. The results reported herein suggest the possibility of in vivo regulation of GCC by cross-talk with numerous pathways that are reported to activate PKC. Studies on the regulation of GCC transcription could provide insight into the variety of mechanisms employed by organisms to maintain tissue-specific gene expression, a hallmark of development in metazoans. | |
