Human Guanylyl Cyclase Research Articles

Guanylyl cyclase activity in plants?

Cyclic nucleotides have defined signal transduction roles in animals, fungi, and other diverse eukaryote kingdoms. However, the situation in plants is still not clear after more than 3 decades of investigations reporting evidence for and against a regulatory role for cyclic nucleotides in higher plants. No sequenced higher plant genome contains homologs of the nucleotidyl cyclase genes that are recognizable in diverse eukaryotic kingdoms, including, curiously, the Chlorophyta phylum of the Viridiplantae kingdom. The report of Qi et al. (1) is a recent example of claims that recombinant plant proteins (or their fragments) from diverse protein families have guanylyl cyclase (GC) activity in vitro—claims that have led to the hope that the cyclic nucleotide regulatory system in plants is not a molecular house of cards. However, the GC activity reported was ≈150 fmol cGMP per μg of 14-kDa protein fragment (AtPepR1-GC) per 30 min. This activity is extraordinarily low. Each molecule of this putative recombinant GC synthesizes one cGMP molecule every 10 d. By contrast, recombinant and native human GC molecules synthesize 103–104 cGMP molecules per second, 109- to 1010-fold faster (2). This very low activity is typical of the other putative noncanonical GCs reported in plants, including fragments of other plant receptor kinases cited by Qi et al. (1) and Ludidi and Gehring (AtGC1, ref. 3). GC comprises 0.005–0.1% of bovine lung protein (2), but even if AtPepR1 was 99% of total Arabidopsis protein the GC activity of the plant would be <10−6 of the activity in some animal tissues.

The Membrane Proximal Extracellular Domain of Human hGC-B Folds Independently

Human Guanylyl Cyclase B (hGC-B) is a single-transmembrane receptor protein which upon binding C-type natriuretic peptide (CNP) to its extracellular domain catalyzes the intracellular conversion of GTP to the second messenger cGMP. cGMP in turn affects various physiological processes such as smooth muscle contraction, cell proliferation, phototransduction, and salt as well as fluid homeostasis. The 3-dimensional binding site of the peptide hormone is unknown, and the binding mechanism is not yet understood. Therefore, a model of the C-terminal moiety of the extracellular domain of human GC-B containing the potential binding site was derived from the crystal structure of (GC-A). The selected protein sequence was provided with an N-terminal TEV-cleavage site and fused with a 109 aa thioredoxin-tag and a hexahistidine-tag. The identity of the purified 25 kDa protein was confirmed by protein mass fingerprint and its secondary structure was determined by CD- and NMR-spectroscopy. The protein proved to be properly folded with the observed secondary structure matching the predicted secondary structure and the homologous structure in the extracellular domain of GC-A. Size exclusion chromatography confirmed the monomeric state of P-hGC-B.

Functional inactivation of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain.

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.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Intestine-specific activity of the human guanylyl cyclase C promoter is regulated by Cdx2.

The heat-stable enterotoxin receptor, guanylyl cyclase C, exhibits an intestine-specific pattern of expression. The aim of this study was to identify the transcriptional activator that mediates intestine-specific expression of guanylyl cyclase C. Fragments of the promoter were assayed to isolate regions directing intestine-specific gene activation. Deoxyribonuclease I footprinting was used to identify a site of intestine-specific protection. Electrophoretic mobility shift assays (EMSAs) and supershift analyses were used to characterize the protein that bound to the protected site. The protected site was mutated to analyze its role in promoter activity. Reporter gene assays revealed that intestine-specific expression of guanylyl cyclase C is directed by the proximal promoter. Deoxyribonuclease I footprinting identified a specific site in the proximal promoter that exhibited intestine-specific protection. EMSAs and supershift analyses revealed that the transcription factor Cdx2 bound to an intestine-specific site of protection. Mutation of the Cdx2-protected site of the promoter eliminated binding of Cdx2 and reduced reporter gene activity to the level of extraintestinal cells. These data show that Cdx2 and its consensus-binding site in the promoter are required for intestine-specific expression of the guanylyl cyclase C gene.

Evaluation of heterodimeric guanylyl cyclase genes as candidates for human hypertension

Both physiologic and pharmacological data have implicated the nitric oxide (NO) signaling cascade in the regulation of blood pressure in humans and its impairment in the pathogenesis of hypertension. In biological systems, the principal receptor for NO is NO-stimulated guanylyl cyclase. NO-stimulated guanylyl cyclases are obligate heterodimers (alpha/beta). The genes for guanylyl cyclase subunits alpha1, beta, and beta2 are likely candidates for causing hypertension in the Dahl rat as their expression is altered and their gene loci are closely linked to known quantitative trait loci for blood pressure in Dahl rat crosses. The objective of the current study was to test whether markers near guanylyl cyclase subunit genes were linked to hypertension in Caucasians. To test for linkage of genetic markers in or near the guanylyl cyclase genes to hypertension in Caucasians, a sample of 124 Utah hypertensive sib pairs was genotyped. Four highly polymorphic markers in or near the human guanylyl cyclase subunits homologous to the rat alpha1 (human chromosome 8), rat beta1 (human chromosome 4), and rat beta2 (human chromosome 13) genes showed no evidence of excess allele sharing in the set of hypertensive sibships. We conclude that the heterodimeric guanylyl cyclase subunit loci do not appear to be linked to hypertension in Caucasians.

Thermodynamic Analyses Reveal Role of Water Release in Epitope Recognition by a Monoclonal Antibody against the Human Guanylyl Cyclase C Receptor

The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.

Cell line-specific transcriptional activation of the promoter of the human guanylyl cyclase C/heat-stable enterotoxin receptor gene

The guanylyl cyclase C protein, expressed primarily in the intestine, is the receptor for the heat-stable enterotoxin of Escherichia coli. We have isolated and sequenced the promoter region and the first exon of human guanylyl cyclase C and determined the major site of transcription initiation. Transfection of a − 1973 + 124 promoter/luciferase gene fusion construct in the Caco-2 intestinal cell line resulted in a high level of expression; results with deletion constructs indicate the presence of multiple positive-acting sequence elements. These promoter elements were not active upon transfection into NIH/3T3 and LLC-PK 1 cell lines which do not express GC-C.

Human guanylyl cyclase C promoter directs intestinal expression of luciferase reporter gene in transgenic mice

Human guanylyl cyclase C promoter directs intestinal expression of luciferase reporter gene in transgenic mice