The concept of second messenger molecules in hormone signal transduction was developed in the 1950s by Earl W. Sutherland describing the cyclic nucleotide adenosine 3′, 5′-cyclic monophosphate (cAMP) as an intracellular messenger molecule. The main features of this concept are the binding of a hormone to the extracellular site of a transmembrane receptor protein which triggers an intracellular response mediated by a second messenger molecule (e.g., cAMP). Shortly after cAMP was discovered as second messenger in hormone signaling, another cyclic nucleotide abbreviated cGMP (guanosine 3′, 5′-cyclic monophosphate) was first detected in rat urine (Ashman et al. 1963) and soon after was found in a variety of other tissues and biological samples. Every second messenger system requires the presence of synthesizing and degrading enzymes. For cyclic purine monophosphates these are adenylate and guanylate cyclases and specific forms of phosphodiesterases, respectively. Guanylate cyclases (GCs) catalyze the conversion of GTP to cGMP according to the reaction scheme: GTP → cGMP + pyrophosphate (PP i). The development of suitable and effective enzymatic assays led in 1969 to several publications that report on the determination of GC activity. Consequent studies then revealed that GC activities cofractionated with soluble and particulate membrane fractions, but it was not before the mid to late 1980s that soluble and membrane-bound GCs were purified from mammalian sources to apparent homogeneity. Subsequent cloning studies in the late 1980s and early 1990s using tissue-specific cDNA libraries resulted in deduced primary structures of soluble and particulate GC isoforms. Studies on mammalian GCs were inspired by work on sea urchin sperm that is a rich source of a particulate GC and that is activated by certain peptides. In mammalians it was known already from reports in the 1970s that neuromediators like acetylcholine could regulate the cGMP level in perfused heart tissue, but the physiological pathways linking these steps remained unclear until the 1980s. Then several discoveries showed that cGMP is the second messenger of important physiological responses including smooth muscle relaxation, intestinal fluid and electrolyte homeostasis, and sensory physiology, in particular phototransduction. Several physiologically relevant molecules that regulate and/or trigger activation of GCs were identified and characterized in the last three decades. The most important so far are nitric oxide for the soluble isoforms and natriuretic peptides, paracrine intestinal hormones, and changes in cytoplasmic Ca2+ levels in combination with Ca2+-sensor proteins for the different particulate isoforms. The identification of these regulatory molecules paved the way for integrating GC isoforms in signaling pathways or led to formulation of new signaling concepts (for historical aspects see Beavo and Brunton 2002; Kots et al. 2009; Sharma 2010; Kuhn 2016).
Guanylate Cyclase Forms
In addition GCs are also found in nonmammalian vertebrates like teleost fishes, in insects, nematodes, unicellular eukaryotic organisms, and bacteria (Baker and Kelly 2004; Ortiz et al. 2006; Rätscho et al. 2010). Some of these organisms express a larger variety of GCs than mammalians and are at least partially homologous to the GC forms found in mammalians.
Protein Structure and Topography
Instead of a kinase homology domain soluble GCs contain a heme-binding domain that is N-terminally located from the amphipathic dimerization domain (Fig. 1). The two heme-binding domains of one soluble GC heterodimer coordinate a heme prosthetic group (a porphyrin ring structure with a central ferrous ion (Fe 2+)) that is required for the enzyme to become activated by nitric oxide (see below).
The least homology among all membrane-bound GCs is found in the extracellular domain. This large domain consists of approximately 500 amino acids and harbors the ligand-binding region, in which the ligand can be either a hormone or an odorant molecule depending on the GC subtype. Sensory GCs that are expressed in the photoreceptor cells of the vertebrate retina are an exception of this rule. No external ligand is known for the extracellular domain of these GCs, and in the case of GCs expressed in rod photoreceptor cells the corresponding extracellular domain is located in the disk lumen.
The natriuretic peptide receptor GCs GC-A and GC-B show the most diverse tissue distribution (Lucas et al. 2000; Tamura et al. 2001). GC-A is expressed in adrenal gland, pituitary gland, adipose tissue, cerebellum, aorta, heart, kidney, liver, spleen, testis, colon, brain stem, retina, cochlea, ovary, thymus, and others. Expression of GC-B largely overlaps with that of GC-A, but a more specific expression pattern is found for GC-C, the intestinal receptor for guanylin and uroguanylin. This GC is mainly present in the colon and intestine but is also found in the kidney, testis, and liver. A similar expression profile (lung, kidney, skeletal muscle, and intestine) is found for GC-G. Different from these receptor-type GCs are the sensory GCs that display a very restricted expression profile. For example, the odorant receptor GC-D is found in a subset of sensory neurons of the olfactory neuroepithelium (Sharma and Duda 2010; Zufall and Munger 2010), and the retina-specific GC-E and GC-F (ROS-GC1 and ROS-GC2) are predominantly expressed in photoreceptor cells of the vertebrate retina (Koch et al. 2010). In addition, GC-E is also found in the pineal gland.
Soluble GC forms are widely distributed and are mainly present in lung, heart, liver, kidney, cerebellum, skeletal muscle, and retina, whereby expression of the β2 isoform seems to be more restricted.
Mode of Activation and Regulation
Soluble GCs are activated by a fundamentally different mechanism. Gaseous molecules like NO and CO bind to the heme prosthetic group that is held between the two subunits forming the heterodimeric GC (Fig. 2). Binding of NO to the Fe2+ in the porphyrin ring of the heme group triggers the breakage of histidine coordinating bond and leads in consequence to a conformational change. By this mechanism the GC activity can be stimulated by about 5000-fold (Garthwaite 2010).
Signaling Pathways and Physiological Responses Involving Guanylate Cyclases
Additional effects of cGMP that are mediated via natriuretic peptide signaling are cardiac hypertrophy and fat metabolism. The factor CNP that acts on GC-B stimulates bone growth and is involved in vascular remodeling.
The paracrine hormones guanylin and uroguanylin regulate the fluid and electrolyte homeostasis in intestinal epithelial cells by binding to the extracellular site of GC-C. A similar ligand-receptor interaction is observed with bacterial enterotoxins leading also to an increase of intracellular cGMP. The latter is a major cause of secretory diarrheal disease. Elevated levels of cGMP activate PKG type II, but can also inhibit a cAMP-specific PDE leading to an increase in cAMP levels and in consequence to an activation of protein kinase A (PKA). PKGII and PKA then phosphorylate and thereby activate the cystic fibrosis transmembrane regulator (CFTR) a chloride-ion channel in the intestinal brush border membrane (Fig. 3).
Sensory GCs are predominantly expressed in sensory cell types and are part of signaling pathways that have CNG-channels instead of PKGs as main downstream targets of cGMP. Among them GC-D is a receptor GC that can be activated by the ligand peptides guanylin and uroguanylin (see above). GC-D is present in a subpopulation of olfactory sensory neurons and not part of the predominant cAMP-signaling pathway in most olfactory sensory neurons. Intracellular signaling of GC-D is linked to Ca2+-signaling pathways as well, since Ca2+-binding proteins of the NCS protein subfamily were shown to regulate GC activity by binding to intracellular regions of GC-D. These NCS proteins are GCAP1, neurocalcin-δ, frequenin, and hippocalcin. The biological role of GC-D has been discussed in the context of chemosensory detection of the metabolic status (Sharma and Duda 2010; Zufall and Munger 2010).
Vision in vertebrate photoreceptor cells involves membrane-bound GCs that are regulated by intracellular Ca2+-binding proteins named GCAPs. No extracellular ligands are known so far. Mammalian rod and cone cells express GC-E and GC-F. They synthesize the intracellular messenger of visual excitation, cGMP, thereby keeping the CNG-channels open to allow influx of Na+ and Ca2+ from the extracellular medium in the dark adapted state of the cell. GCAPs are an important regulatory part of an intracellular feedback loop, since they activate photoreceptor GCs at low Ca2+concentration and inhibit them at higher Ca2+concentration leading to resynthesis of cGMP after illumination, when the cytoplasmic Ca2+concentration has dropped (Fig. 3). Photoreceptor GCs are key components of signal transduction in rod and cone cells by controlling the second messenger level. Thereby they are important for the cell’s recovery to the dark state after illumination and its adaptational properties (Koch et al. 2010).
Soluble GCs are the target of the versatile messenger molecule NO that is synthesized by nitric oxide synthase isoforms. NO, for example, is mediating smooth muscle relaxation and is involved in the central and peripheral nervous system and in the immune response of macrophages. NO can act on other targets than soluble GCs, but increase of cGMP production by NO leads in general to activation of PKG and subsequent phosphorylation of PKG targets.
The second messenger cGMP and its synthesizing enzymes (membrane and soluble GCs) have become essential parts of different signaling pathways that mediate important physiological functions. These include, for example, blood pressure regulation, kidney and smooth muscle function, olfaction, and vision. Components of the corresponding signaling pathways have been characterized at the molecular level, and the physiological impact of GCs has been investigated by a combination of biochemical, genetic, and physiological studies. For these systems a deeper mechanistic understanding at the structural level has also partially been achieved. Elucidating the structure-function relationships of GCs and their regulatory components will also guide future directions of pharmaceutical and therapeutical inventions, since several diseases correlate with dysfunctions of GC-signaling systems. These include, for example, hypertension, different forms of retinal degeneration, and colorectal cancer. Another challenge is to dissect other signaling pathways, in which cGMP has been implicated, but information on a participating GC in these pathways is missing. Examples are other sensory cell systems (e.g., sensing of tastants, heat, and pain) or the Wnt-frizzled signaling pathway in early embryonic development (Wang and Malbon 2004). Finally, some nonmammalian organisms express a larger variety of GC forms than mammalians. It will be a promising task to investigate the properties and the operation principles of these GC forms in an environmental and evolutionary context.
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