The availability of genome sequence information and a large number of protein structures has allowed the cataloging of genes into various families, based on their function and predicted biochemical activity. Intriguingly, a number of proteins harbor changes in the amino acid sequence at residues, that from structural elucidation, are critical for catalytic activity. Such proteins have been categorized as ‘pseudoenzymes’. Here, we review the role of the pseudokinase (or kinase-homology) domain in receptor guanylyl cyclases. These are multidomain single-pass, transmembrane proteins harboring an extracellular ligand-binding domain, and an intracellular domain composed of a kinase-homology domain that regulates the activity of the associated guanylyl cyclase domain. Mutations that lie in the kinase-homology domain of these receptors are associated with human disease, and either abolish or enhance cGMP production by these receptors to alter downstream signaling events. This raises the interesting possibility that one could identify molecules that bind to the pseudokinase domain and regulate the activities of these receptors, in order to alleviate symptoms in patients harboring these mutations.

Introduction

Almost all proteins in the cell are modified by protein phosphorylation, thereby enabling the modulation of various biological pathways such as transcription, metabolism and cellular fates [1]. To achieve this wide-spread control of cellular function, the human genome encodes numerous protein kinase-like genes (more than 500). Based on extensive biochemical and structural analyses of a number of protein kinases, three motifs have been identified to be important for kinase activity (Figure 1). These include, a lysine residue in the VAIK motif which interacts with the α and β phosphate of ATP, an aspartate residue in the HRD motif which acts as a base for phosphate transfer, and an aspartate residue in the DFG motif which forms interactions with the metal ion that, in turn, co-ordinates the β and γ phosphates of ATP. Changes in amino acid sequences in one or more of these motifs may result in loss of kinase activity [2]. Interestingly, nearly 10% of proteins present in the human kinome harbor alterations in residues present in these motifs, and can therefore be termed as pseudokinases [1].

Alignment of active kinases and the kinase homology domains of receptor guanylyl cyclases.

Figure 1.
Alignment of active kinases and the kinase homology domains of receptor guanylyl cyclases.

Sequences corresponding to the kinase domain in known active kinases and the pseudokinase domain present in receptor guanylyl cyclases were aligned with ClustalW2. Boxed residues are those found to be essential for kinase activity and include the ATP-binding residue (K in VAIK), metal binding residue (D in DFG) and the D required for phosphotransfer (HRD). All sequences shown are from human. SRC, src tyrosine kinase; EGFR, epidermal growth factor receptor tyrosine kinase; PRKACA, cAMP-dependent protein serine/threonine kinase; RAF, MAPKKK. GC-D and GC-G are pseudogenes in humans.

Figure 1.
Alignment of active kinases and the kinase homology domains of receptor guanylyl cyclases.

Sequences corresponding to the kinase domain in known active kinases and the pseudokinase domain present in receptor guanylyl cyclases were aligned with ClustalW2. Boxed residues are those found to be essential for kinase activity and include the ATP-binding residue (K in VAIK), metal binding residue (D in DFG) and the D required for phosphotransfer (HRD). All sequences shown are from human. SRC, src tyrosine kinase; EGFR, epidermal growth factor receptor tyrosine kinase; PRKACA, cAMP-dependent protein serine/threonine kinase; RAF, MAPKKK. GC-D and GC-G are pseudogenes in humans.

Pseudokinases have been shown to be involved in numerous cellular functions despite their lack of kinase activity, and mutations in a large number of characterized pseudokinases are associated with human disease [3]. The absence of catalytic activity in these domains has allowed them to function as allosteric regulators of the proteins in which they are found, or as scaffolds that participate in signal transduction cross-talk when present as independent proteins in the cell [4]. Despite the absence of critical residues for kinase activity in pseudokinases, many of them have been shown to bind ATP and metal ions, implying that the overall fold of these proteins adopts one seen in active protein kinases [4,5].

A few proteins are comprised of only a single pseudokinase domain. These autonomous pseudokinase proteins include Tribbles (TRB), STRAD and VRK3 [4]. The three isoforms of Tribble proteins harbor a pseudokinase domain lacking the DFG motif and display low affinity ATP binding in vitro [6].Tribbles regulate cell proliferation and differentiation by providing a binding platform for substrates which undergo E3-ligase-dependent ubiquitination and thus their stability [68], and serve as an example where a pseudokinase is involved in protein–protein interactions, a theme we will see occurs in receptor guanylyl cyclases (rGCs).

STRAD proteins lack the VAIK, DFG and HRD motifs in the pseudokinase domain but display high affinity ATP binding [9,10]. STRAD forms a heterotrimeric complex with MO25 and LKB1 [9]. MO25 functions as the scaffolding protein and STRAD's interaction with LKB1 results in activation of LKB1 and phosphorylation of AMPK. This serves as an example of a pseudokinase that can modulate the activity of a bonafide kinase (LKB1) by direct interaction and perhaps serves as a harbinger to interactions of pseudokinase domains with other domains present along with them in a single protein [10,11].

In many instances, a pseudokinase domain is present in a larger protein that consists of multiple domains, some with functional and/or catalytic activity. In these cases, the pseudokinase may act to allosterically regulate the activity of the protein in which it is found or interact with another protein in the cell to induce a signaling event. For example, HER3 belongs to the EGF-receptor family of tyrosine kinases and consists of a ligand-binding extracellular domain, an intracellular pseudokinase domain and a C-terminal domain. The pseudokinase domain of HER3 lacks the conserved catalytic aspartate residue but can bind ATP with high affinity [4,12]. Upon binding of the ligand neuregulin to HER3, HER3 heterodimerizes with HER1 or HER2. HER1 and HER2 are active EGF receptor kinases, and heterodimerization with HER3 leads to autophosphorylation and activation of these kinases [13,14]. Mutations in HER3 are associated with various human cancers and HER3 is overexpressed in ∼60% of breast cancer cases, indicating the critical role for this pseudokinase-domain containing receptor [15,16].

Another well-studied example of a pseudokinase domain is present in the Janus tyrosine kinases (JAK). JAKs harbor both a pseudokinase domain as well as an active kinase domain in a single polypeptide chain. The JH1 domain which has kinase activity is preceded by the JH2 domain which is a pseudokinase domain [17]. JH2 lacks the conserved aspartate in the HRD motif which is essential for phosphotransfer activity, but the domain can still bind ATP. JAKs undergo autophosphorylation and activation, resulting in phosphorylation of the cytokine receptor with which they are associated. The JH2 domain directly binds to JH1 and inhibits its kinase activity in the absence of the cytokine [1720]. A mutation (V617F) in the JH2 domain of JAK2 is linked to patients with polycythemia vera and essential thrombocythemia, as well as chronic idiopathic myelofibrosis [21]. This mutation leads to constitutively active JAK2 and cytokine-independent proliferation of cells. Structural studies of the JH2 mutant domain in Mg-ATP bound state suggest that this mutation may promote an altered conformation in the JH2 C-alpha helix which enables a stimulatory interaction with JH1 domain to activate it [22]. Therefore, this serves as an example where mutations in the pseudokinase domain can regulate the activity of an associated domain by direct interaction and inducing conformational changes.

Here, we provide an overview of the role of the pseudokinase (or kinase-homology domain; KHD) found in receptor guanylyl cyclases [23]. The biological roles of these receptors span a number of processes ranging from visual signal transduction to gut physiology [24]. We highlight common, as well as unique aspects, by which the pseudokinase domain in these receptors regulates cGMP production by the associated guanylyl cyclase domain.

Receptor guanylyl cyclases and cGMP-mediated signal transduction

Receptor guanylyl cyclases (rGCs) consist of an extracellular ligand-binding domain (ECD) followed by a single transmembrane domain, a juxtamembrane domain, a pseudokinase domain, and a C-terminal catalytic guanylyl cyclase domain [25] (Figure. 2). The first rGC was characterized and purified from the sperm of the sea urchin Arbacia punctulate [26] and is involved in chemoattraction to the egg that produces ligands for the sperm receptor [27]. Seven different mammalian rGCs have been identified (GC-A to GC-F) and known ligands include the atrial natriuretic peptide (important for vasodilation) and the gastrointestinal peptide hormones, guanylin and uroguanylin, that regulate fluid and ion secretion in the gut [24].

Domain organization of receptor guanylyl cyclases.

Figure 2.
Domain organization of receptor guanylyl cyclases.

Numbers inside filled symbols represent the approximate number of amino acids in the domain, and numbers in brackets indicate the approximate % similarity across human receptor guanylyl cyclases, with respect to the amino acid sequence of GC-A. GC-D and GC-G are pseudogenes in humans and therefore not shown.

Figure 2.
Domain organization of receptor guanylyl cyclases.

Numbers inside filled symbols represent the approximate number of amino acids in the domain, and numbers in brackets indicate the approximate % similarity across human receptor guanylyl cyclases, with respect to the amino acid sequence of GC-A. GC-D and GC-G are pseudogenes in humans and therefore not shown.

Receptor GCs are activated by ligand binding to the extracellular domain, or as in the retinal guanylyl cyclases, by binding to guanylyl cyclase-activating proteins (GCAPs), resulting in conformational changes leading to increased cGMP production by the guanylyl cyclase domain [24,25]. Downstream actions of cGMP include activation of protein kinases, regulation of phosphodiesterases and activation of cGMP-gated ion channels [25].

The pseudokinase domains of rGCs lack the catalytically important aspartate residue in the HRD motif [5] (Figure 1), but play important roles in ligand-mediated activation of these receptors and/or interaction with other proteins [28]. An analysis of the sequence conservation of the pseudokinase domains in rGCs and the C-terminal guanylyl cyclase domains present in these receptors indicated coevolution of these two domains, suggesting that the pseudokinase domain of one rGC might exclusively regulate its associated catalytic domain, in spite of the conserved domain organization in these receptors [23].

We will highlight the properties of individual receptor GCs below, emphasizing the role of the kinase-homology domain in transducing conformational changes that modulate cGMP production. Importantly, we will also describe human diseases attributed to mutations that map to the pseudokinase domain in these receptors, indicating the critical role this domain has in regulating receptor activity.

Guanylyl cyclase A (GC-A)

Natriuretic factors can be purified from the atrium or the brain and are therefore terms as atrial natriuretic peptide (ANP) [29] or brain natriuretic peptide (BNP), respectively [30]. They are small peptides containing a single disulfide bond in their sequence, creating a cyclic structure [31]. Receptor guanylyl cyclase A (GC-A) binds both ANP and BNP, but the affinity for ANP is ∼4-fold higher [32]. Binding of ANP results in activation of the receptor and enhanced cGMP production. The main role of GC-A is in maintenance of arterial blood pressure, intravascular volume balance and homeostasis. GC-A accomplishes this task by modulating the vasculature, kidney, adrenal and central nervous function [24,33].

The role of the pseudokinase domain in ligand-mediated regulation of GC-A is still moot. In one study, deletion of the pseudokinase domain in GC-A led to a constitutively active receptor (i.e. cGMP production in the absence of ANP or BNP) with no further response on ligand addition [34,35]. However, partial deletion of the kinase homology domain leads to complete inactivation of the receptor [35]. The crystal structure of the extracellular domain of GC-A revealed that ligand binding results in an intermolecular rotation across the dimer. It was proposed that a similar rotation is then transduced to the intracellular domain, thus achieving an active conformation of the cyclase domain, which needs to form a head-to-tail dimer in order to be catalytically active [36,37]. The role of the pseudokinase domain in such rotation and conformational changes is unclear, but ATP binding was thought to be essential for activation of GC-A [38].

The pseudokinase domain in GC-A contains a glycine-rich loop observed in active kinases and may form the ATP-regulatory motif, that allows ATP binding in the presence of ligand [38]. Mutation of this putative site failed to block activation of GC-A by ANP, but additional mutations in other residues conserved in protein kinases reduced, and in some cases abolished, ANP-mediated cGMP production in vitro [39]. However, these mutations also altered glycosylation and trafficking of the receptor, and therefore could have distorted the correct tertiary conformation of the receptor, resulting in reduced activity.

In another study, 8-azido-3′-biotinyl-ATP could be photoaffinity cross-linked to a construct of GC-A which was devoid of the catalytic domain [40]. Cross-linking was enhanced on prior incubation of membranes with ANP, indicating that the ligand-bound receptor allowed the receptor to adopt a conformation that facilitated ATP interaction.

The presence of ATP in biochemical assays was reported to be essential for ligand-mediated activation in an initial study [34], suggesting that ATP may bind to the pseudokinase domain. However, sufficiently high concentrations of GTP in biochemical assays allowed activation of GC-A since ATP reduced the Km for GTP, without altering the Vmax [41]. These results questioned whether ATP binding to the kinase-homology domain was essential for GC-A activation.

Specific serine and threonine residues in the N-terminus of the pseudokinase domain are phosphorylated in GC-A in the basal state [42]. Homologous desensitization of GC-A requires dephosphorylation of these residues, probably by exposing these sites to a phosphatase following ligand and ATP binding [31,38]. Therefore, the presence of ATP in earlier studies may have increased the basal phosphorylation of GC-A, thus augmenting guanylyl cyclase activity, and giving the impression that ATP binding to the kinase homology domain regulated the activity of GC-A [43]. Mutations in the kinase homology domain that reduced guanylyl cyclase activity did indeed reduce basal phosphorylation of mutant receptors [39].

In view of all these findings, and biochemical experiments carried out in the presence of physiological concentrations of GTP and ATP during assays, it was proposed that an allosteric ATP-binding site is actually present in the guanylyl cyclase domain of GC-A [43]. Because of high concentrations of ATP in the cell, this site would always be occupied by ATP. Ligand binding to the extracellular domain in this model allows the correct juxtapositioning of the catalytic domains, reduces the Km for GTP allowing cGMP generation and also increases the affinity of ATP for the allosteric site. The authors also propose radically that identical guanylyl cyclase domains actually form an asymmetric dimer, with one catalytic site (where GTP is convered to cGMP) and one allosteric site, where ATP binds [43].

In summary, there is evidence to suggest that ATP may bind to the pseudokinase domain and/or to the catalytic domain. The complex interplay between these two binding sites, if present, and the way they regulate the guanylyl cyclase domain will need structural elucidation of the intracellular domain of GC-A, which may be a challenging task.

Intriguingly, no mutations in GC-A, let alone the kinase-homology domain, have been associated with human disease. Variations (single nucleotide polymorphisms) in non-coding regions (5′-UTR, introns and 3′-UTR) have been reported and showed modest effects using reporter constructs [44]. Perhaps, the consequences of altered activity of GC-A are too deleterious to permit embryos harboring such mutations from surviving.

Guanylyl cyclase B (GC-B)

Guanylyl cyclase B (GC-B) is the receptor for a third natriuretic peptide, CNP [45]. GC-B and CNP are expressed in various tissues such as the bone, brain, heart, lung, blood vessels, kidney, uterus, ovaries and testis. Various roles for GC-B via autocrine/paracrine signaling include maintenance of blood pressure, cell cycle regulation, inhibition of oocyte maturation in mice and endochondral bone growth [31,46]. Knock-out mice for CNP or GC-B displayed slow skeletal growth post-birth, resulting in severe dwarfism [47].

The GC-B gene can be alternatively spliced, and one splice form generates a protein that lacks three of the six potential phosphorylation sites and the glycine-rich motif [48]. This variant failed to respond to CNP in terms of cGMP production but could heterodimerize with the wild-type receptor and act as a dominant negative. In a manner similar to GC-A, residues in the kinase- homology domain are phosphorylated in the basal, ligand-free state, and dephosphorylation is required for homologous or heterologous desensitization in the presence of ligand [49]. Therefore, the reduced activity of the splice variant could be a consequence of decreased phosphorylation and/or absence of ATP binding to the pseudokinase domain.

The important role of the kinase-homology domain in regulating GC-B activity was evident with the report of loss-of-function, homozygous mutations in GC-B, leading to skeletal dysplasia with acromesomelic dysplasia, type Maroteaux (AMDM; Table 1) [50,51]. AMDM is an autosomal recessive rare disease which results in slow skeletal growth post birth [52]. The mutations lay in the pseudokinase domain at residues Y708 and R776, that are conserved in orthologues from fish to mammals. These mutants were expressed on the cell surface but were inactive in vitro in terms of cGMP production [53]. Using molecular modeling, it was postulated that the Y708C and R776W mutations may result in improper folding of the pseudokinase domain, and therefore abolish ligand-mediated activation of GC-B, that was essential for proper skeletal formation [50].

Table 1
Mutations in receptor guanylyl cyclases that lie in the pseudokinase domain and are associated with human disease
Receptor Residue position Clinical features Status Biochemical properties Reference 
GC-B p.G630 frame shift mutation Acromesomelic Dysplasia, Type Maroteaux (AMDM) Homozygous; loss of function NA [50
p.L658F Homozygous; loss of function Similar ligand binding affinity
No ligand-mediated cGMP production 
[51
p.Y708C Homozygous; loss of function Can bind ANF and traffic to cell surface; not processed to fully glycosylated and phosphorylated forms [50,53
p.R776W Homozygous; loss of function Can bind ANF and traffic to cell surface; not processed to fully glycosylated and phosphorylated forms [50,53
p.W769K Homozygous; loss of function NA [50
p.R655C Extremely tall (height 232 cm at age of 53, BMI 34)
Mild scoliosis, secondary degenerative changes of the skeleton 
Heterozygous; gain of function Higher cGMP production in absence of ligand.
Increased cGMP production on ligand treatment 
[54
GC-C p.K507E Congenital sodium diarrhea Heterozygous; gain of function Higher cGMP production in absence and presence of ligand.
No change in affinity for the ligand, but increased sensitivity to ligand induced cGMP production 
[65
p.A670T/p.C928R Meconium ileus Compound heterozygous; loss of function NA [62
GC-E p.R540C Leber congenital amaurosis (LCA) Heterozygous NA [90
p.F589S Homozygous NA [88
p.D639Y Heterozygous Significantly lower GMP production [82
p.R768W Heterozygous Significantly lower GMP production [82
p.A710V Homozygous NA [91
Receptor Residue position Clinical features Status Biochemical properties Reference 
GC-B p.G630 frame shift mutation Acromesomelic Dysplasia, Type Maroteaux (AMDM) Homozygous; loss of function NA [50
p.L658F Homozygous; loss of function Similar ligand binding affinity
No ligand-mediated cGMP production 
[51
p.Y708C Homozygous; loss of function Can bind ANF and traffic to cell surface; not processed to fully glycosylated and phosphorylated forms [50,53
p.R776W Homozygous; loss of function Can bind ANF and traffic to cell surface; not processed to fully glycosylated and phosphorylated forms [50,53
p.W769K Homozygous; loss of function NA [50
p.R655C Extremely tall (height 232 cm at age of 53, BMI 34)
Mild scoliosis, secondary degenerative changes of the skeleton 
Heterozygous; gain of function Higher cGMP production in absence of ligand.
Increased cGMP production on ligand treatment 
[54
GC-C p.K507E Congenital sodium diarrhea Heterozygous; gain of function Higher cGMP production in absence and presence of ligand.
No change in affinity for the ligand, but increased sensitivity to ligand induced cGMP production 
[65
p.A670T/p.C928R Meconium ileus Compound heterozygous; loss of function NA [62
GC-E p.R540C Leber congenital amaurosis (LCA) Heterozygous NA [90
p.F589S Homozygous NA [88
p.D639Y Heterozygous Significantly lower GMP production [82
p.R768W Heterozygous Significantly lower GMP production [82
p.A710V Homozygous NA [91

A second novel missense mutation in the pseudokinase domain (L658F) was identified in heterozygous and homozygous states and led to short stature (Table 1). In vitro studies of the L658F mutation demonstrated that it acted as a dominant negative protein when co-expressed with wild-type GC-B, with no production of cGMP in the presence of ligand, in spite of similar ligand-binding affinity. The L658 residue is also highly conserved evolutionarily [51].

A heterozygous and hyperactivating mutation in the pseudokinase domain of GC-B was identified in a man of unusually high stature, but with few skeletal anomalies (Table 1). This mutation (R655C) was engineered in GC-B for in vitro characterization, and the mutant receptor demonstrated enhanced cGMP production on ligand stimulation as a homodimer, or as a heterodimer with wild-type GC-B [54]. Using homology modeling, the authors suggested that R655 was solvent exposed and may not interact with the ATP-binding pocket, leaving it unclear as to how this mutation may regulate the activity of the guanylyl cyclase domain [54].

Guanylyl cyclase C (GC-C)

Receptor guanylyl cyclase C (GC-C) is predominantly expressed in the apical epithelial cells of the intestine and was identified as the receptor for heat stable enterotoxin (ST), the causative agent of Enterotoxigenic Escherichia coli (ETEC)-mediated diarrhoea [55]. Guanylin and uroguanylin were identified as the endogenous ligands for GC-C [25]. Activation of receptor GC-C upon ligand binding results in production of cGMP from GTP which regulates intestinal epithelial cell fluid and ion secretion, cell proliferation and gut immune responses [55,56].

GC-C shares the overall domain organization seen in other rGCs (Figure 2). The phosphorylated residues in the N-terminal region of the kinase-homology domains of GC-A and GC-B are not conserved in GC-C, and no basal phosphorylation of the receptor has been detected till date [57]. The pseudokinase domain of GC-C lacks the conserved glycine-rich loop at the N-terminal region in kinases, which is important for ATP binding [58]. Nevertheless, the kinase-homology domain along with the linker region of GC-C displayed binding to ATP-agarose [59]. Interestingly, a construct comprising just the kinase-homology domain (without the linker) did not bind to ATP-agarose, indicating that additional residues outside the region that shows sequence similarity to protein kinases, are important for stabilizing a structure that can bind ATP [59].

Homology modeling followed by mutational and biochemical analysis, demonstrated that K516 is important for ATP binding and indispensable for ligand-mediated regulation of GC-C [58]. Conformational changes that occur in GC-C and the pseudokinase-linker region were identified using a monoclonal antibody that failed to bind to native GC-C in the presence of ATP [59]. The epitope of this antibody laid C-terminal to K516. Interestingly, mutation of K519 present in the epitope did not abrogate ATP binding to the pseudokinase domain, indicating the specificity of interaction of K516 with ATP [59].

The precise role of ATP in regulating GC-C activity awaits elucidation of the structure of this receptor. Ligand-mediated in vitro production of cGMP, using membranes expressing GC-C, is enhanced in the presence of ATP when magnesium ions are provided along with GTP as a substrate to measure guanylyl cyclase activity [60]. Using a variety of adenine nucleotide analogs, those not substituted at the 2-position activated GC-C, with ATPγS being the most potent [60]. However, nonionic detergent-mediated activation of GC-C is inhibited in the presence of ATP and Mg2+ GTP [61]. Finally, ATP inhibits guanylyl cyclase activity when measured using Mn2+ GTP as a substrate, presumably by binding to an allosteric site in the guanylyl cyclase domain [61], since this inhibition was also observed in the K516A mutant receptor. Clearly, much needs to be learned in mechanisms underlying the biochemical regulation of this receptor.

Disease-associated mutations in the pseudokinase domain of GC-C have been reported (Table 1). A Lebanese child presented with meconium ileus (defined as the inability of the newborn to pass the first stool), and a mutation in the pseudokinase domain (A670T) was found in a compound heterozygous form with a mutation in the guanylyl cyclase domain (C928R) [62]. The C928R mutation was also found in a homozygous condition in a sibling in the same family, and this child also presented with meconium ileus [62]. The authors suggested that the C928R mutation may inactivate the guanylyl cyclase domain. Inactivation of GC-C would result in lower levels of cGMP in the intestinal epithelial cell, and as a consequence, reduced phosphorylation and activity of the chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) [63,64]. Consequently, fluid efflux from the intestinal epithelial cell would be reduced, resulting in meconium ileus. Whether A670T present alone can compromise GC-C activity is not known at present, and warrants further studies, given that this residue in the pseudokinase domain is conserved evolutionarily [23]. Aberrant dimerization of mutant receptors, when present in a compound heterozygous state, may result in low production of cGMP and reduced intestinal fluid secretion.

An activating mutation in GC-C was identified in a child with congenital sodium diarrhea (Table 1) [65]. This secretory diarrhea is of intrauterine onset with high fecal loss of sodium. No congenital malformations were detected in the child, but parenteral nutrition needed to be provided till the age of 14 months. Through whole exome sequencing, a mutation was identified in the pseudokinase domain (K507E) of GC-C. The mutation resulted in hyperactivation of GC-C with ∼3-fold higher production of cGMP in the absence of ligand (i.e. high basal activity). The mutant receptor was further activated upon ligand stimulation and displayed ∼8-fold higher cGMP production in comparison with the wild-type receptor in the presence of ligand. Interestingly, the mutant receptor showed a lower EC50 to both exogenous and endogenous ligands, but no change in ligand-binding affinity was observed [65]. This suggests that the pseudokinase domain may also play a role in ‘inside-out’ regulation of the receptor, whereby the intracellular domain modulates conformational changes that occur on ligand-binding to the extracellular domain.

Hyperactivation of GC-C and high levels of intracellular cGMP result in increased chloride secretion by CFTR. Moreover, increased phosphorylation of the sodium–hydrogen exchanger, NHE3 [64,66] by cGMP-dependent protein kinase II (PKGII) results in inhibition of the sodium-hydrogen exchanger and consequently, higher levels of sodium in the intestinal lumen. Increased luminal chloride and sodium would enhance fluid secretion from the epithelial cell leading to the sodium diarrhea seen in this child [55,65].

Guanylyl cyclase D (GC-D)

Olfaction determines various behaviors in animals such as aggression, mating and feeding [67]. Olfaction is achieved by millions of chemosensory neurons which harbor canonical G-protein coupled receptors and cAMP-mediated Ca+ channel regulation, that transduce cues to generate electrical signals [68]. Cyclic GMP can also play a role in olfactory neurons [69,70]. GC-D was identified in a specialized sub-population of rodent olfactory neurons which lack the canonical cAMP pathway [71]. GC-D possesses a similar domain organization as other receptor GCs but can also be activated by bicarbonate and environmental cues such as temperature. Mice contain four anatomically defined organs for olfaction, namely the main olfactory epithelium, septal organ of Meara, Gruenberg ganglion and vomeronasal organ [71]. GC-D is expressed in the main olfactory epithelium and can be stimulated by uroguanylin and guanylin but not the bacterial heat-stable enterotoxin, ST [69,72]. This is somewhat surprising, given that the similarity in the extracellular ligand binding domains of GC-C and GC-D is of the order of 28%. GC-D may also play a role in CO2 sensing, since CO2 can be converted to carbonate by carbonic anhydrase isoform II (CAII) in the GC-D-containing neurons, and therefore activate GC-D by directly binding to the guanylyl cyclase domain of receptor [73,74].

In humans and primates, GC-D has acquired various inactivating mutations during evolution, and hence is a pseudogene [75]. No mutations in rodent GC-D have been reported, nor has the role of the pseudokinase domain in regulating guanylyl cyclase activity been elucidated. However, given the similar domain organization of this receptor, it can be predicted that the pseudokinase domain is critical for activation by ligands such as uroguanylin and guanylin, as seen in GC-C.

Retinal receptor guanylyl cyclases (GC-E and GC-F)

Approximately 30 years ago, the first ever cGMP-gated cation channel was identified in retinal photoreceptor cells [76]. This discovery was soon followed by the identification of a particulate rGC which produced cGMP in the presence of low concentrations of Ca2+ in the bovine rod outer segments (OS) [77]. Two rGCs, GC-E and GC-F, were identified in the photoreceptor cells, i.e. rods and cones and their regulation resulted in restoring the dark current through cyclic nucleotide-gated channels after excitation of the photoreceptor [78,79].

GC-E and GC-F have no identified ligands that could bind to the extracellular domain. However, they are activated by guanylyl cyclase activating proteins (GCAPs) [79]. GCAPs in the Mg2+ bound form directly bind to the pseudokinase domain and activate GC-E and GC-F resulting in ∼10-fold higher production of cGMP [80]. This is an example of activation of rGCs by modulation and binding of another protein to the pseudokinase domain. GCAP-mediated activation might be the only way of activating the guanylyl cyclase domain in GC-E and GC-F, since heterologous expression of truncated GC-E lacking the extracellular domain did not reduce guanylyl cyclase activity, and the truncated receptor was stimulated by GCAPs [81,82]. However, a mutation in the extracellular domain of GC-E in humans leads to congenital blindness. Therefore, the role of the extracellular domain in these receptors may be in compartmentalization and trafficking of receptors to the membrane [83] and/or affecting the conformation of the kinase-homology domain, allowing it to interact with GCAPs.

There are two isoforms of GCAP in mammals, GCAP1 and GCAP2 [84]. GCAPs are membrane-associated proteins and consist of four helix-loop-helix EF-hand motifs found in calcium-binding proteins. EF1 is probably the motif which binds to the pseudokinase domain and motifs EF2–EF4 contain binding sites for Ca2+ and Mg2+ [85]. Two GCAP protein binds to a homodimer of GC-E or GC-F [86]. Photoactivation results in hydrolysis of cGMP via the cGMP-specific phosphodiesterase, PDE6, resulting in closure of the cGMP-gated channel. This is followed by a decrease in intracellular Ca2+ levels and hyperpolarization of photoreceptor cells. The reduction in Ca2+ levels allows the exchange of Ca2+ with Mg2+ in GCAPs, which now bind and activate GC-E and GC-F, thus replenishing cGMP levels. This results in the opening of cyclic nucleotide-gated channels (CNGs) thereby restoring Ca2+ levels, depolarization of the photoreceptor and restoration of the ‘dark state’ [87].

Inactivating mutations in GC-E causes Leber congenital amaurosis (LCA; Table 1) [88]. LCA is marked by impaired vision and the disappearance of outer segments in rods and cones [89]. Mutations associated with LCA1 are found in the extracellular, pseudokinase and linker regions. While mutations in the ECD have mild effects [83], mutations in the pseudokinase domain, either frame shift or splice site mutations, would lead to the formation of a truncated product [90]. One missense mutation (R540C) in the kinase-homology domain GC-E may also inactivate GC-E, since the patient was found to suffer from LCA [90]. The F589S mutation is autosomal recessive and the authors speculated that this mutation resulted in instability of protein, due to the substitution of an aromatic non-polar amino acid by an uncharged polar amino acid [88]. A further two mutations (D639Y and R768W) in the pseudokinase domain were identified in a heterozygous condition, resulting in LCA. D639Y and R768W showed no cGMP production following heterologous expression in HEK293 cells. Moreover, GCAP1 did not bind to the mutant GC-E [82].

Recently, a mutation in the pseudokinase domain of GC-E was identified in a large inbred Bedouin Israeli tribe (Table 1). The mutation was in a conserved residue at amino acid position 710 (A710V). Molecular modeling studies revealed that the mutation may affect overall folding and conformational stability of the protein, rendering it inactive [91].

No human mutations leading to any disease in GC-F are reported till date. Nevertheless, the pseudokinase domain of retinal GCs plays a vital role in modulating the activity of the guanylyl cyclase domain by altering critical interaction with the GCAPs.

Guanylyl cyclase G (GC-G)

GC-G is expressed in the neurons of the Gruenberg ganglion in the anterior region of the murine nose [92,93]. These neurons are activated by distinct odors and cool temperatures. Activation of GC-G results in elevation of intracellular cGMP and opening of cyclic nucleotide-gated channels, increasing the influx of Ca2+ [94]. In vitro studies showed that homodimerization and activation of GC-G occurred at 15–20°C, and the receptor was poorly active at 37°C [95,96]. One member of the large rGC family found in Caenorhabditis elegans also appears to be thermosensitive. Deletion of the extracellular and the kinase-homology domain generated a protein which showed lower cGMP production in comparison with the wild-type protein, but still demonstrated significantly higher activity at lower temperatures, indicating that the dimerization motif resided in the linker region of this receptor [97]. GC-G is a pseudogene in humans.

Conclusion

If a gene is expressed in an organism, and conserved evolutionarily, it must have a function, even if amino acids that have been shown to be critical for activity of its orthologues are missing or divergent. Thus, ‘pseudoenzymes’ need to be studied individually to decipher their diverse roles in cellular physiology [3]. If a larger protein contains a domain that appears to be a ‘pseudo’, it is almost assured that this domain will regulate the activity of the protein in which it is present. In this review, we have summarized information available on the pseudokinase domain present in rGCs, emphasizing how this domain can allosterically control both ligand binding and guanylyl cyclase activities. While initial efforts were directed towards biochemically dissecting how the activity of rGCs are toggled between off and on states by the kinase-homology domain [31,58], the discovery of mutations associated with, or the cause, of human disease has emphasized how critically, yet subtly, the pseudokinase domain regulates receptor activity (Table 1).

What is lacking, however, is an appreciation of these receptors at the structural level. There is no structure available of the kinase-homology domain or the guanylyl cyclase domain in isolation, let alone the entire intracellular domain, of any rGC. We are therefore left to evaluate structures of individual domains by molecular modeling. Since sequence similarity between the kinase-homology domains of rGCs and protein kinases is of the order of ∼25% (Figure 1), the models that are built, at best, will be informative guesses. Nevertheless, it appears that the kinase-homology domains of rGCs are able to bind ATP, and hydrolysis of ATP is not required to regulate the guanylyl cyclase domain [40,58,59]. Binding of ATP, at least in the case of GC-C, requires the presence of the linker region along with the kinase-homology domain, presumably by allowing the kinase homology domain to adopt a structure capable of binding the nucleotide [59]. GTP cannot bind to the KHD [59], again emphasizing the specificity of interactions in the pseudokinase domain. Given the high level of sequence similarity of the linker region in rGCs [98,99], along with a conserved mechanism of regulation of the guanylyl cyclase domain by the linker [98], we predict that all receptors may show similar three- dimensional juxta positioning of the linker and kinase-homology domains.

A number of inhibitors of protein kinases have been identified and some are in clinical use [100]. Hypothesizing that molecules that bind protein kinases may interact with GC-C, the activity of the receptor was tested in vitro with a panel of inhibitors against tyrosine kinases [101]. Tyrphostin A25 was shown to inhibit the activity of GC-C, but by performing assays with the guanylyl cyclase domain of GC-C alone, we reported that many tyrosine kinase inhibitors are noncompetitive inhibitors of both adenylyl and guanylyl cyclases [101]. These results, however, do not rule out the possibility that tyrphostins also interact with the KHD. Indeed, the complexities of elucidating precise interactions in a multidomain protein in the absence of structural insight preclude precise understanding of molecular detail.

Retinal rGCs are regulated by protein binding to their kinase-homology domain. No interacting proteins that stably bind to other rGCs have been identified, though changes in phosphorylation of these receptors must involve transient interactions with kinases and phosphatases [39,102]. It is conceivable that small molecules may be identified that regulate, for example, binding of GCAPs to retinal guanylyl cyclases, providing another opportunity to control the activity of the guanylyl cyclase domain by enhancing or interfering with interaction with other cellular proteins.

In summary, rGCs play important roles in physiology, and mutations in these receptors are associated with severe human disease. The presence of a pseudokinase domain that regulates receptor activity provides an opportunity to identify molecules that bind to this domain and modulate signaling. Evolutionarily, ‘pseudokinases’ may have evolved from ATP-binding proteins [103] and kinase activity was later acquired. However, rGCs are found only in metazoans, and therefore the pseudokinase domain in these proteins may have evolved from proteins that originally possessed phosphotransfer activity. Intriguingly, a protein from mimivirus contains a putative active kinase domain associated with a cyclase domain [23]. Since cyclic nucleotides and protein phosphorylation participate in prokaryotic signaling, it seems that their ‘coming together’ has facilitated the evolution of variants around two themes, allowing diversification in the repertoire of these enzymes and their regulation.

Abbreviations

     
  • ANP

    atrial natriuretic peptide

  •  
  • BNP

    brain natriuretic peptide

  •  
  • CFTR

    cystic fibrosis transmembrane conductance regulator

  •  
  • ECD

    extracellular ligand-binding domain

  •  
  • GC-A

    guanylyl cyclase A

  •  
  • GCAP

    guanylyl cyclase activating proteins

  •  
  • GC-B

    guanylyl cyclase B

  •  
  • GC-C

    guanylyl cyclase C/heat-stable enterotoxin receptor

  •  
  • GC-D

    guanylyl cyclase D

  •  
  • GC-E

    guanylyl cyclase E/retinal guanylyl cyclase 1

  •  
  • GC-F

    guanylyl cyclase F/retinal guanylyl cyclase 2

  •  
  • GC-G

    guanylyl cyclase G

  •  
  • rGCs

    receptor guanylyl cyclases

Author Contribution

V.M. and R.G. prepared the first draft. S.S.V. compiled the manuscript and finalized the review.

Funding

S.S.V. is supported by the Department of Biotechnology (BT/PR15216/COE/34/02/2017). S.S.V. is a J.C. Bose National Fellow (SB/S2/JCB-18/2013), and is also a recipient of a Royal Society Grant for Research Professors, UK (IC160080).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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