Guanylyl Cyclase Receptors
In 1971 when Sutherland received the Nobel Prize for the discovery of the second messenger cAMP, the functions of cGMP were still a mystery, even though cGMP had been isolated from rat urine in 1963, and an enzyme that cleaved the phosphodiester bond was discovered the following year. The levels of cGMP are regulated by synthesis by guanylyl cyclases (GCs), extrusion from the cell, and/or degradation by phosphodiesterases (Kots et al. 2009).
Soluble Guanylyl Cyclases
The soluble guanylyl cyclases (sGC) are heterodimers of two dissimilar subunits, alpha and beta. There are four types of alpha subunits, namely, α1, α2, α3, and α2 i, and three types of beta subunits, namely, β1, β2, and β3. The ubiquitously expressed α1/ß1 dimer is the predominant complex, with highest levels of mRNA in brain, lung, heart, kidney, spleen, and muscle (Russwurm and Koesling 2002; Mergia et al. 2009). All the heterodimers are active, albeit to varying extents, except those containing α2 i. The α2 i is a splice variant of α2 and provides a mechanism for regulating the activity of the α2/β1 form of sGC by acting as a dominant negative protein in cells. However, active homodimers in lower organisms have also been reported (Morton et al. 2005).
The crystal structure of the sGC from the green algae Chlamydomonas reinhardtii has been solved (Winger et al. 2008) and the structure of the catalytic domain of human sGC has been solved (Allerston et al. 2013). There is also a report on the crystal structure of Cya2, a prokaryotic guanylyl cyclase from cyanobacterium Synechocystis PCC6803 (Rauch et al. 2008).
The sGCs are ubiquitously expressed and are responsible for smooth muscle relaxation, neurotransmission, and inhibition of platelet migration (Kots et al. 2009) and can be stimulated by free radicals, nitrovasodilators, and similar molecules. Nitric oxide (NO), the major ligand for sGC, binds to the N-terminal heme-binding H-NOX domain. In the absence of NO, the iron atom of the heme moiety bonds with a histidine residue present in the protein, keeping the cyclase domain inactivate. NO binding to the heme moiety leads to the disruption of this interaction, altering the conformation of the enzyme, thereby resulting in its activation. NO binds to ferrous (Fe2+) heme iron in the H-NOX domain of the resting sGC to activate it (Stone and Marletta 1996; Zhao et al. 1999); however, oxidized ferric (Fe3+) heme iron strongly attenuates the enzymatic response to NO (Zhao et al. 2000; Schrammel et al. 1996). A number of conserved cysteine residues in the alpha and beta subunits have been identified, and covalent modification of three cysteine residues (two in the alpha subunit and one in beta subunit) by S-nitrosation or S-nitrosylation has also been shown to affect sGC desentisization to NO stimulation (Beuve et al. 2016). Protoporphyrin IX can bind to and activate sGCs independent of heme or NO (Lucas et al. 2000). Carbon monoxide also regulates sGCs in a manner similar to that of NO, although its affinity for the enzyme is much lower than that of NO. ATP allosterically inhibits the activity of the soluble guanylyl cyclase (Derbyshire et al. 2009).
The C-terminal cyclase domains in each subunit are connected to the N-terminus by a linker region which contributes to the dimerization of sGC. Since the α-subunits of sGCs lack the critical residues required for catalytic activity, they heterodimerize with the β-subunits to give rise to a functional active site (Lucas et al. 2000; Perkins 2006).
Membrane-Associated Guanylyl Cyclases
Seven classes of membrane-associated guanylyl cyclases have been characterized. They are GC-A, GC-B, GC-C, GC-D, and GC-G, and the retinal GCs, GC-E, and GC-F. All the receptor guanylyl cyclases share a common domain organization with a single-pass transmembrane region. They have an extra cellular domain (ECD), the amino acid sequence of which is poorly conserved across the different classes, thereby imparting ligand specificity. This domain is followed by a transmembrane domain and an intracellular domain (ICD). The ICD is comprised of a juxtamembrane domain followed by a regulatory kinase homology domain (KHD) which binds ATP. Following the KHD is a linker region which connects to the guanylyl cyclase domain. Ligand binding to the extracellular domains results in receptor activation and increased production of cGMP in the cell. Classical membrane-bound GCs are often devoid of kinase activity (Potter 2011a), perhaps because of the absence of a glutamate residue in the KHD domain that is important for the phosphotransfer reaction (Potter 2005). However, a novel class of membrane-associated GC-linked receptor kinases (currently four members), possessing both intrinsic kinase and GC activity, with the GC catalytic center being encapsulated within an active kinase domain, was unearthed using homology-guided bioinformatic data mining tools designed from annotated amino acid residues in the GC catalytic centers of lower eukaryotes. (Kwezi et al. 2007; Kwezi et al. 2011; Meier et al. 2010; Qi et al. 2010).
The Natriuretic Peptide Family Receptors
These comprise three receptors, namely, Natriuretic Peptide Receptors A, B, and C. The ligands for these receptors are natriuretic peptides – atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).
Guanylyl Cyclase A/Natriuretic Peptide Receptor A (GC-A/NPR-A)
The mammalian cDNA encoding guanylyl cyclase A (NPR-A) was identified using the gene sequence of the sea urchin particulate guanylyl cyclase receptor as a probe to screen a cDNA library (Kots et al. 2009). NPR-A preferentially binds ANP and BNP. NPR-A is found in the heart, kidney, adrenal cortex, and vasculature.
Vasodilation and natriuresis are the most commonly studied aspects of signaling via NPR-A. GC-A prevents hypertrophy of the heart by relaxing the vascular musculature, thereby reducing the cardiac output. In kidneys, NPR-A assists in the secretion of sodium and reduction of aldosterone secretion by the adrenal cortex, thus reducing blood pressure (Martel et al. 2010).
Transcription of NPR-A is regulated by Sp1. NPR-A has been shown to downregulate its own expression by a feedback inhibition loop via a cGMP response element. Alternative splice variants of NPR-A can heterodimerize (Martel et al. 2010). The juxtamembrane domain in NPR-A has been shown to modulate the proteolytic cleavage of the ECD, thereby regulating its activity (Huo et al. 1999). Posttranslational modifications which include glycosylation are implicated in the folding of NPR-A and its trafficking to the surface of the cells. The receptor is highly phosphorylated on eight serine and threonine residues within the KHD. When activated by its ligands, NPR-A is deposphorylated, which then attenuates the activation by the ligand, leading to desensitization and signal downregulation (Koller et al. 1993). Five of the eight total phosphorylation sites are conserved in GC-A and GC-B (Potter 2011). Chronic exposure to high concentrations of their ligands (homologous desensitization) or exposure to growth hormones such as angiotensin II (ANG II) and endothelin (heterologous desensitization) are reported to lead to attenuation of the receptor (Potter 2011). Imbalance in circulating levels of natriuretic peptides and expression of NPR-A receptor is seen in hypertensive mice, making this receptor a potential target for gene therapy (Martel et al. 2010).
Guanylyl Cyclase B/Natriuretic Peptide Receptor B (GC-B/NPR-B)
Using the sequence of NPR-A, NPR-B was cloned from rat brain cDNA and human placenta. NPR-B is primarily located in the pituitary gland, adrenal gland, ovary, endothelium, vascular smooth muscle, and kidney (Schulz 2005). NPR-B binds CNP with highest affinity amongst the natriuretic peptides. Apart from the relaxation of the heart and lowering of blood pressure, NPR-B signaling also relaxes the smooth muscle of the oviduct, colon, and trachea. In the brain, CNP and NPR-B play a role in gonadotropin secretion. Expression of NPR-B in the ovary and testis may be responsible for development of mature ovarian follicles and regulation of sperm transport by relaxing the seminiferous tubules. NPR-B also promotes bone development and causes skeletal overgrowth on overexpression (Schulz 2005).
Like NPR-A, phosphorylation of the serine and threonine is required for peptide activation, and prolonged exposure with the natriuretic peptide (homologous desensitization) as well as platelet-derived growth factor, fibroblast growth factor, or serum or acute exposure to phorbol esters or calcium-elevating agents (heterologous desensitization) leads to dephosphorylation and inactivation of the receptor (Potter 2011; Dickey et al. 2010). The lack of an intact KHD in NPR-B causes stunted skeletal growth in humans, and splice variants of NPR-B lacking the KHD or intracellular domains are inactive and can act as dominant negatives, hindering bone development (Hachiya et al. 2007). Homozygous loss-of-function mutations in GC-B have been identified to cause acromesomelic dysplasia, a rare form of short-limbed dwarfism in human patients (Bartels et al. 2004).
Only one other protein has been identified which binds all the natriuretic peptides, the NPR-C receptor. This receptor has no guanylyl cyclase activity as it lacks the intracellular domain. NPR-C is expressed in the heart, lung, vascular smooth muscle cells, kidney glomeruli, adrenal gland, brain cortex, and pituitary gland (Martel et al. 2010). The primary function of the NPR-C receptor was thought to be sequestration and metabolic clearance of natriuretic peptides and it was therefore designated as the clearance receptor. The 37 amino acid long cytoplasmic tail of NPR-C has been shown to mediate inhibition of adenylyl cyclase activity on ligand binding to the receptor (Anand-Srivastava 2005).
Guanylyl Cyclase C (GC-C)
Guanylyl cyclase C (GC-C) is the third member of the family of receptor guanylyl cyclases that was identified. It was identified as the receptor for the bacterial heat-stable enterotoxin peptides produced by pathogenic strains of Escherichia coli (Schulz et al. 1990). GC-C is predominantly expressed in the intestinal tract.
Endogenous ligands for GC-C are guanylin and uroguanylin and have been purified from intestinal mucosa and urine. They are cysteine-rich peptides, similar to the stable toxin peptide, but, they have a 10–100-fold lower affinity for GC-C. It is believed that they regulate intestinal fluid and ion transport. Activation of GC-C, following binding of its ligands, results in increased synthesis cGMP. Accumulation of intracellular cGMP activates cyclic nucleotide-dependent protein kinases, PKA and PKG, leading to the phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) and increased chloride secretion through CFTR. Ion efflux leads to increased fluid secretion from the cell, resulting in a watery diarrhea. This manifests as traveler’s diarrhea in adults which is self-limiting but may be fatal in infants (Basu et al. 2010). Recently, missense mutation, in them were identified. Individuals harboring this mutation show mild or severe forms of congenital or familiar secretory diarrhea (Fiskerstrand et al. 2012; Muller et al. 2015). These mutations resulted in hyperresponsiveness to ligand stimulation, with subsequent enhanced intracellular accumulation of cGMP. Also, homozygous inhibitory mutations in GUCY2C, attenuating or abolishing GC-C activity, are also reported, leading to neonatal meconium ileus and obstruction of the small intestine by thick mucoid stools in two unrelated Bedouin children (Romi et al. 2012).
GC-C prexists as an oligomer in the absence of ligand. GC-C is differentially glycosylated, and glycosylation is important for the binding of ligands to GC-C, as well as trafficking of GC-C to the cells membrane (Arshad et al. 2013; Hasegawa et al. 1999). GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Phosphorylation of a serine residue in the C-terminus by protein kinase C results in potentiation of ligand-stimulated activity in vivo and in vitro. Prolonged exposure of GC-C to its ligands leads to cell-specific desensitization of the receptor, by mechanisms that are not clearly understood at present. Intestine and kidney-enriched PDZ protein (IKEPP) and c-src are the only proteins to be identified that interact with GC-C and regulate its activity (Basu et al. 2010).
The involvement of intestinal (uro)guanylin/GC-C pathway in regulation of epithelial functions other than ion transport have also been identified. Loss in the gene expression of the paracrine hormones guanylin and uroguanylin has been reported in colorectal carcinogenesis (Wilson et al. 2014). Increased intestinal tumor initiation and growth on mutation in Apc or upon exposure to the carcinogen azoxymethane in GC-C-deficient mice (Li et al. 2007) was observed. The ligands for GC-C have been shown to have a cytostatic effect on intestinal cells and thereby act as a barrier to tumorogenesis. Also, binding of c-src via the SH2 region to phosphorylated GC-C contributes to enhanced intestinal cell proliferation and dedifferentiation in cultured intestinal tumor cell lines (Basu et al. 2009). Several downstream signaling pathways have been identified to modulate GC-C-mediated colonic cell cytostasis, such as inhibition of oncogenic Akt signaling (Lin et al. 2010), reduced expression of cell cycle drivers including β-catenin, cyclin D1, pRb, increased expression of cell cycle inhibitors including p27 (Blomain et al. 2013), and the upregulation of p21 resulting in cellular cytostasis and senescence (Basu et al. 2014).
GC-C is highly expressed in the intestine and is evolutionarily preserved. The GC-C knockout mouse have been reported to show hyperphagy resulting in obesity (Valentino et al. 2011). However, in another recent study reported that GC-C knockout mice have normal body weight, adiposity, and glucose tolerance (Begg et al. 2014). The mice were however resistant to stable toxin-mediated diarrhea. GC-C knockout mice were also reported to exhibit hyperactivity and attention deficit disorders (Gong et al. 2011).
Guanylyl Cyclase D (GC-D)
Canonical olfactory signaling occurs when cyclic nucleotide gated (CNG) ion channel are activated by cAMP in the olfactory neurons. When it was discovered that odorants triggered cGMP production, which activated a cGMP-gated ion channel, the olfactory guanylyl cyclase GC-D was identified. This gene is found to be well conserved in rodents and canines, but is a pseudogene in a number of primate species, including the human (GC-E). A very small subset of the olfactory sensory neurons (OSN) of mice expresses GC-D, and these neurons terminate in necklace glomeruli (Zufall and Munger 2010). Nonprimates detect more olfactory cues than primates, and may even detect pheromones via GC-D (Young et al. 2007). Recent reports suggest that uroguanylin and/or guanylin can activate GC-D.
GC-D can also be activated by bicarbonate ions which are produced by carbonic anhydrase specifically expressed in the GC-D subset of OSNs. Reports have shown that rodents can be trained to avoid CO2 concentrations as low at 0.5%. It is speculated that this is a means to detect other animals in the vicinity. There is also evidence that guanylyl cyclase activating protein (GCAP1) could also modulate a calcium-dependent activation of GC-D (Zufall and Munger 2010).
Retinal Guanylyl Cyclases: Guanylyl Cyclase E/F (GC-E/F)
In the mid-1990s, membrane-bound receptor guanylyl cyclases were identified in bovine photoreceptors. A gene encoding a particulate guanylyl cyclase was isolated from retinal cDNA based on its similarity to previously characterized GCs (Shyjan et al. 1992). This gene is referred to as retGC1 or retGC2 in humans or GC-E/GC-F in other mammals. RetGC2 is exclusively expressed in the retina while expression of retGC1 is seen in the pineal gland and retina. The mRNA of retGC1 has been detected in the olfactory bulb of fish, cochlear nerve and organ of Corti (Hunt et al. 2010).
GC-E and GC-F are important in retinal phototransduction and regulate the opening and closure of cGMP-gated ion channels. These channels are open in the dark and close on perception of light. The loss of either retGC results in compromised visual signal transduction suggesting that both retGCs are necessary for normal phototransduction.
The retGCs are orphan receptors as no ligands for the ECD have been discovered till date. These receptors are regulated by proteins which associate with their intracellular domains. Guanylate cyclase activating proteins (GCAPs) are always found associated with the retGCs. In resting depolarized cells, GCAPs are bound by Ca2+ ions which prevent activation of retGC. Upon light stimulus, the cGMP-specific phosphodiesterase, PDE6, found in photoreceptor cells is activated and degrades cGMP present in the cell, leading to closure of the CNG, thereby decreasing Ca2+ levels in the now hyperpolarized cell. The Ca2+ ions dissociate from GCAP, permitting them to activate retGC. Cyclic GMP levels are now elevated resulting in the reopening of the ion channels and reversion of the cell to a depolarized state.
Despite the importance of both retGCs for visual signal transduction in mice, visual impairments studied so far in humans map only to the retGC1 locus. Recessive mutations in retGC1 are a major cause of Leber congenital amaurosis (LCA). Retinitis pigmentosa also is caused by a mutation in GC1. Other mutations in retGC1 are responsible for 35% of all rod-cone dystrophies (Hunt et al. 2010).
Guanylyl Cyclase G (GC-G)
The GC-G is an orphan receptor and transcripts have been found in mouse testis, kidney, and in the Grueneberg ganglion (Lin et al. 2008). This receptor may play a role in thermo-sensation, a process which has been shown to be via cGMP signaling in C. elegans (Zufall and Munger 2010). GC-G receptor is absent in humans.
Almost 50 years after the discovery of cGMP, its function in vision, olfaction, vasodilatation, and fluid ion homeostasis has been established. A number of guanylyl cyclases have been identified and characterized but questions still remain as to the ligands and exact functions of some of the receptors. The complex domain organization of these receptor guanylyl cyclases indicate that they may be regulated in multiple ways and be involved in crosstalk with other signaling pathways, and studies along these lines will be of interest in future.
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