The guanylyl cyclase family at Y2K.

During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

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.

Functions of conserved cysteines of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considered to be important as they were thought to play a significant role in the regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen conserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes were able to synthesize cGMP. Mutation of two cysteines located in the N-terminal, putative heme-binding region of the beta1 subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with heme and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta1 subunit (Cys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of the corresponding cysteines on the alpha1 subunit did not alter NO responsiveness, indicating that heme-binding is mainly a feature of the N-terminal domain of the beta1 subunit.

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.

Functional domains of soluble guanylyl cyclase.

Soluble guanylyl cyclase is a heterodimer consisting of an alpha and beta subunit and stimulation occurs upon binding of NO to a prosthetic group. Little is known about the localization of catalytic and regulatory domains within the subunits of soluble guanylyl cyclase. We used deletion mutagenesis to identify the regions of alpha 1 and beta 1 subunits that are responsible for cGMP production or NO-heme-mediated activation. The amino terminus of the beta 1 subunit was necessary for NO stimulation since deletion of the 64 NH2-terminal amino acids resulted in a mutant with intact basal activity but complete loss of NO activation. The amino terminus of the alpha 1 subunit also appeared to be essential for NO sensitivity since deletion of 131 NH2-terminal amino acids of alpha 1 led to markedly reduced NO activation. These results suggest that NH2-terminal regions of alpha 1 and beta 1 are involved in NO-heme-mediated signal transduction. The NH2 terminally truncated beta 1 subunit exerted a dominant negative effect exclusively on the NO-stimulated activity of the wild type enzyme, further underlining that the regulatory domain is located within the NH2 terminus of the enzyme. Aside for the structural implications, the mutant represents a powerful tool to investigate nitric oxide-sensitive signaling pathways. Coexpression of the COOH-terminal halves of alpha 1 and beta 1 were sufficient for basal cGMP production while either of the halves expressed alone was inactive. Therefore the COOH-terminal regions appear to contain sufficient information for dimerization and basal enzymatic activity. Thus, we provide the first evidence that the regulatory and catalytic properties of soluble guanylyl cyclase can be attributed to different regions of the subunits and that the catalytic domain can be functionally expressed separately from the NH2-terminal regulatory domain. Taken together with findings on the membrane bound enzyme form, guanylyl cyclases, appear to resemble fusion proteins where different regulatory domains have been joined with a common cGMP-forming segment.

Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] is a hemoprotein that exists as a heterodimer; the heme moiety has been proposed to bind nitric oxide, resulting in a dramatic activation of the enzyme. Mutation of six conserved His residues reduced but did not abolish nitric oxide stimulation whereas a change of His-105 to Phe in the beta 1 subunit yielded a heterodimer that retained basal cyclase activity but failed to respond to nitric oxide. Heme was not detected as a component of the mutant heterodimer and protophorphyrin IX failed to stimulate enzyme activity. The activity of the His mutant was almost identical to that of the wild-type enzyme in the presence of KCN, suggesting that disruption of heme binding is the principal effect of the mutation. Thus, the mutation provides a means to inhibit the nitric oxide-sensitive guanylyl cyclase signaling pathway.

Molecular cloning and expression of a new alpha-subunit of soluble guanylyl cyclase. Interchangeability of the alpha-subunits of the enzyme.

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the alpha 1-subunit and was designated alpha 2 as the new subunit is very similar to the alpha 1-subunit in the middle and C-terminal part; it is quite diverse in the N-terminal part. Preceding experiments had shown that coexpression of the alpha 1- and beta 1-subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221-223]. The finding that the alpha 2-subunit was able to replace the alpha 1- but not the beta 1-subunit in expression experiments demonstrates the interchangeability of the alpha-subunit isoforms of soluble guanylyl cyclase.