The cloning and expression of a new guanylyl cyclase orphan receptor.

A novel membrane form of guanylyl cyclase (GC-G) has been identified through the isolation of a full-length cDNA clone; it is predicted to contain an extracellular ligand binding domain, a single transmembrane segment, and intracellular protein kinase-like and cyclase catalytic domains. That GC-G represents a guanylyl cyclase was confirmed by both transient expression in COS-7 cells and stable expression in H293 cells. Endogenous cyclic GMP concentrations of transfected or stable cells, however, were much higher than control cells, suggesting an inability of the cells to effectively regulate GC-G cyclase activity. Of six Cys residues found within the extracellular domain of guanylyl cyclase-A (GC-A), the receptor for atrial natriuretic peptide, five are conserved within GC-G. Ligands for the other cyclase receptors, nevertheless, failed to stimulate GC-G expressed in transient or stable cells, suggesting that the unknown ligands possess a structure different from the natriuretic peptides or heat-stable enterotoxins. 125I-ANP also failed to bind to H293 cells overexpressing GC-G. Based on Northern hybridization, mRNA for GC-G was predominantly expressed in lung, intestine, and skeletal muscle. Using the candidate gene approach to potentially define function, the gene for GC-G was mapped to the distal region of mouse chromosome 19 (syntenic with human chromosome 10q), but no human genetic defect has been ascribed to the GC-G locus. The finding of a new membrane form of guanylyl cyclase in peripheral tissues suggests the existence of another family or subfamily of ligands that signal through elevations of cGMP.

Guanylyl cyclases: approaching year thirty.

Since its discovery in 1963, cyclic GMP (cGMP) has been shown to be a ubiquitous second messenger. The enzymes that catalyze the formation of cGMP from GTP, guanylyl cyclases, exist in soluble and particulate isoforms. An explosion in the number of known isoforms, gene disruption, identification of new inhibitors and activators and finally the resolution of the structure of adenylyl cyclases have all provided important clues about the structure and function of guanylyl cyclases. This article gives a brief review of the recent developments in the field of guanylyl cyclase research.

The cloning of a Caenorhabditis elegans guanylyl cyclase and the construction of a ligand-sensitive mammalian/nematode chimeric receptor.

Substantial guanylyl cyclase activity was detected in membrane fractions prepared from Caenorhabditis elegans (100 pmol cGMP/min/mg at 20 degrees C or 500 pmol cGMP/min/mg at 37 degrees C), suggesting the potential existence of orphan cyclase receptors in the nematode. Using degenerate primers, a cDNA clone encoding a putative membrane form of the enzyme (GCY-X1) was obtained. The apparent cyclase was most closely related to the mammalian natriuretic peptide receptor family, and retained cysteine residues conserved within the extracellular domain of the mammalian receptors. Expression of the cDNA in COS-7 cells resulted in low, but detectable guanylyl cyclase activity (about 2-fold above vector alone). The extracellular and protein kinase homology domain of the mammalian receptor (GC-B) for C-type natriuretic peptide (CNP) was fused to the catalytic domain of GCY-X1 and expressed in COS-7 cells to determine whether ligand-dependent regulation would now be obtained. The resulting chimeric protein (GC-BX1) was active, and CNP elevated cGMP in a concentration-dependent manner. Subsequently, a search of the genome data base demonstrated the existence of at least 29 different genes from C. elegans that align closely with the catalytic domain of GCY-X1, and thus an equally large number of different regulatory ligands may exist.

New insights on the functions of the guanylyl cyclase receptors.

The discovery of at least 29 genes encoding putative guanylyl cyclases in Caenorhabditis elegans has raised the question as to whether there are numerous receptors yet to be discovered in the mammal. The nematode, however, not only seems ideal to study guanylyl cyclase receptor localization and function, given the large variety of isoforms, but also leads to possible identification of ligands for orphan guanylyl cyclases by the use of genetic and behavioral assays. A recent powerful approach to describe the function of different guanylyl cyclase isoforms in mammals has been the disruption of the corresponding genes in the mouse. A salt resistant elevation of blood pressure, which corresponds to the phenotype of 50% of all human patients with essential hypertension, is observed in mice lacking the GC-A-receptor. Mice missing the GC-C receptor have been shown to be resistant to STa, an E. coli heat-stable enterotoxin, which is largely responsible for travellers diarrhea in adults and mortality due to diarrhea in infants.

A mutation of the atrial natriuretic peptide (guanylyl cyclase-A) receptor results in a constitutively hyperactive enzyme.

Mutation of an invariant glutamate residue found within the catalytic domain of guanylyl cyclases resulted in a dramatic 14-fold increase in the activity of the guanylyl cyclase-A receptor. Even in the presence of Mn2+/Triton X-100, a treatment previously thought to yield hormone-independent and maximum cyclase activity, the mutant enzyme remained 7-fold more active; to our knowledge, this is the first example of a protein modification or of an added agent that significantly increases cyclase activity in the presence of Mn2+/Triton X-100. Intracellular concentrations of cGMP in cells expressing the mutant (E974A) cyclase were only marginally elevated by the addition of atrial natriuretic peptide, and in broken-cell preparations, the mutant enzyme also was relatively insensitive to ligand/regulatory nucleotide. The marked increase in cyclase activity was not due to a relief of protein kinase domain inhibition, since the point mutation caused 7- to 13-fold elevations in guanylyl cyclase-A activity when the protein kinase homology domain was deleted. The E974A mutation also altered the kinetics from positive cooperative to linear with respect to MnGTP, suggesting disruption of subunit-subunit interactions. Thus, a single point mutation within the catalytic domain of a guanylyl cyclase results in a constitutively hyperactive enzyme that is independent of protein kinase domain regulation.