ADCY9 (Adenylyl Cyclase 9)
Protein Structure and Regulation
The primary structure of adenylyl cyclase 9 (AC9) has two notable features when compared with its mammalian paralogues (Paterson et al. 2000). Firstly, the nonconserved cytoplasmic domains C1b and C2b (Fig. 2) are considerably longer, for example, in the case of C2b 112 amino acid residues for AC9 versus 16 for AC2. Secondly, there is a segment of amino acid residues in all adenylyl cyclases between the predicted 6th intramembrane helix and the relatively conserved C1a domain (pre-C1a), that is, highly variable between AC paralogues; in the case of AC9 it consists of 34 amino acid residues and contains a phosphorylation cluster (http://www.phosphosite.org/proteinAction.do?id=8311&showAllSites=true).
There is some controversy over the exact sequence of the COOH-terminal domain of human AC9. The first published sequence (Hacker et al. 1998) contains a double frame-shift mutation when compared with a sequence published later (Paterson et al. 2000). Genome sequencing suggests that the sequence of Paterson et al. (2000) is correct. However, it cannot be fully excluded that the sequence of Hacker et al. (1998) came from a cDNA library with a somatic mutation, or some form of as yet uncharacterized RNA modification gave rise to the double-frameshift version of human AC9 and was transcribed into cDNA.
Adenylyl cyclases are subject to three main classes of physiological regulation by (1) protein–protein interactions, cardinally heterotrimeric G proteins, (2) calcium ions, and (3) protein phosphorylation. Further forms of regulation are likely to emerge through the study of individual isoforms. For instance, database entries show that similar to other membrane proteins, AC9 is subject to ubiquitinylation and acetylation.
Protein–Protein Interactions: G proteins
All isoforms of AC, including AC9, are activated by the alpha subunit of the heterotrimeric stimulatory G protein Gs (Gsα) and are thus stimulated by a variety of heptahelical cell surface receptors (7-TMR) that couple to Gs. Note, however, that the effect of Gs on AC9 could be dependent on the cellular context. There are apparently no effects of G protein beta/gamma subunits or the alpha subunit of the inhibitory G protein Gi (Giα) on AC9 activity in membranes prepared from human embryonic kidney cells (HEK 293) overexpressing human AC9 or Sf9 cells infected with baculovirus encoding AC9 (Premont et al. 1996). In accordance, a putative Giα binding site sequence delineated in AC5 is absent from AC9. However, in intact AtT20 cells where the cAMP response to the activation of 7-TMR is predominantly through AC9 (Antoni et al. 1995), prominent inhibition of cAMP production by somatostatin acting through SST2/5 receptors in a pertussis toxin sensitive manner has been demonstrated, suggesting the involvement of Gi. Moreover, in HEK293 cells, stably overexpressing AC9 and the long form of the D2 dopamine receptor, the D2 agonist quinpirole inhibited isoproterenol evoked cAMP accumulation – an effect also blocked by pertussis toxin (Cumbay and Watts 2004). A possible caveat in the latter study is that the expression plasmid for human AC9 used (Hacker et al. 1998) does not code for the entire correct sequence of AC9 as the C-terminal region contains a double frame-shift mutation (Paterson et al. 2000). Taken together, the action of 7-TM coupled to Gi on AC9 is in need of further investigation.
More recent work has shed light on the physical association of AC9 with other components of cAMP signaling. Dessauer and co-workers concluded that the isoform-specific NH2-terminal domain of AC9 enables the enzyme to form a complex with the A-kinase anchoring protein (AKAP) yotiao, also known as AKAP 9 (Piggott et al. 2008; Li et al. 2012). AKAPs are scaffolding proteins that recruit several elements of cAMP signaling, thus giving rise to signalosomes that facilitate localized intracellular cAMP signaling with unique regulatory properties (microdomains) (Houslay 2010; Lefkimmiatis and Zaccolo 2014). In the case of the yotiao scaffold, further signaling proteins in proximity of AC9 may include the voltage-gated K+-channel subunit KCNQ1 (Kv7.1) that forms the IKs channel responsible for the repolarization of heart muscle cells (Osteen et al. 2010) and underlies various forms of “long-QT” syndrome (Dvir et al. 2014). This finding is potentially significant, as the other transmembrane ACs expressed by cardiomyocytes (AC5 and 6) do not form a complex with yotiao and KCNQ1 (Piggott et al. 2008; Li et al. 2012).
Ca2+ and Phosphorylation/Ubiquitinylation
Adenylyl cyclase 9 (AC9) was discovered on the basis of unique sensitivity of the agonist-evoked cAMP response to inhibitors of calcineurin in the AtT20 pituitary tumor cell line (Antoni et al. 1995; Antoni 2000). The molecular choreography of the regulation by calcineurin is not known, currently it cannot be excluded that it is indirect, that is, via a protein phosphatase cascade the main effector of which would be protein phosphatase 1. Pharmacological evidence indicates that activation of protein kinase C inhibits Gsα-mediated activation of AC9 (Cumbay and Watts 2004). Proteomic analysis has revealed at least 12 sites of phosphate incorporation in the isoform-specific segments (pre-C1a, C1b, and C2b) of human AC9 (http://www.phosphosite.org/proteinAction.do?id=8311&showAllSites=true). Some of these phosphorylations, especially those in the pre-C1a cluster as well as Ser1307 in the C2b domain, which is an amino acid residue unique to AC9 in primates, have been analyzed in detail in HEK293 cells (Simpson et al. 2006). Specifically, Ser 365 and Ser1307 are constitutively phosphorylated by cyclin-dependent protein kinase 5 in HEK293 cells. Stimulation by cAMP leads to a marked increase in the levels of phosphoSer374 and a reduction in phosphoSer1307. The functional impacts of these changes of phosphorylation await elucidation.
Tissue Distribution and Subcellular Targeting
AC9 mRNA is widely expressed in the body (Antoni et al. 1998). The brain, heart, skeletal muscle, endocrine organs, the aorta, and the prostate all express high levels of AC9 mRNA, whereas the spleen and thymus have low levels. Within the brain, all neurons appear to express AC9, with particularly high levels of protein in the hippocampus and the cingulate cortex (Antoni et al. 1998). The promoter region of the Adcy9 gene has not been analyzed in detail.
Physiological Role and Phenotypes
AC9 is by far the most abundant adenylyl cyclase in the brain, yet relatively little is known about its role in the CNS. AC9 mRNA levels in the hippocampal CA1 field and the dentate gyrus were reduced in aged mice (Mons et al. 2004). Moreover, AC9 mRNA levels in the hippocampus of young adult mice were significantly increased after training sessions in the Morris water maze (fixed hidden platform paradigm) and showed a significant correlation with the level of performance in this test of spatial memory (Mons et al. 2004). While these data indicate that higher AC9 levels are associated with superior cognitive performance, further evidence is required to substantiate this notion. The association of AC9 with KCNQ1 through yotiao (Piggott et al. 2008; Antoni 2012) indicates a role of AC9 in regulating the excitability of neurons and heart muscle cells. However, specific physiological paradigms involving AC9 in these systems are yet to emerge.
In the neuroendocrine system, AC9 appears to play an important role in glucocorticoid feedback regulation of the secretion of adrenocorticotropin by the anterior pituitary gland (Antoni 2012).
Although AC9 is not abundant in the immune system, the enzyme has been implicated in the response of macrophages to stimuli activating pattern recognition receptors (Alper et al. 2008).
A mutation in the AC9 coding sequence has been identified which is present in 30% of the North American Caucasian and Oriental population and leads to a substantial reduction of receptor-induced cAMP synthesis (Small et al. 2003). This may potentially contribute to vulnerability to certain polygenic disorders such as bronchial asthma. Intriguingly, a significant association between the same mutation and the familial occurrence of bipolar depression has been reported in a cohort of patients in Japan (Toyota et al. 2002). The mechanistic basis of the effect of this mutation (Ile772→Met) in the isoform-specific C1b domain is not known. However, it can be speculated that as the lysine residue in position 773 is a target for ubiquitinylation (http://www.phosphosite.org/siteAction.action?id=27175599), the mutation at position 772 may alter the efficiency of this process and thus modify the intracellular trafficking of AC9.
Yet further work has suggested the involvement of cAMP signaling in the development of resistance of melanoma to cells towards the cytotoxic effects of inhibitors of the RAF and MEK protein kinases. As AC9 is expressed in melanoma cells, it is a potential component of the cAMP resistance pathway (Johannessen et al. 2013).
Analysis of over 6000 patients from a clinical phase three study of the cholesterylester transfer protein inhibitor drug dalcetrapib led to the surprising finding that an intronic allele of the AC9 gene had a major influence on the clinical efficacy of the drug. The trial failed because of apparent lack of efficacy to reduce the arteriosclerotic thickening of blood vessel intima. A single SNP in ADCY9 was associated with cardiovascular events upon treatment with dalcetrapib: Genotype A/A at rs1967309 had a 39% reduction in CV events, compared with a 27% increase among patients with the G/G genotype (Tardif et al. 2015). The finding was hailed as a major advance towards personalized, pharmacogenetics-driven drug therapy. More recently, it was reported that cholesterol efflux of the macrophages of genotyped patients was modified by dalcetrapib in accordance with the clinical findings, that is, G/G rs1967309 patients had unaltered cholesterol efflux, whereas the cells from A/A rs1967309 patients had significantly enhanced cholesterol efflux. Based on further genetic analyses of polymorphisms in Adcy9, a generic role of the enzyme in the pathogenesis of cardiovascular diseases was proposed (Niesor et al. 2015).
Pharmacology and Therapeutic Potential
Adenylyl cyclases have been considered as therapeutic targets for a long time, but potent and selective drugs to modulate these enzymes have remained elusive (Brand et al. 2013). The paucity of data on the physiological role of AC9 is also reflected by the dearth of ideas concerning its potential in clinical therapy. Forskolin, a direct activator of ACs 1 through 8, has no or very little effect on AC9 heterologously expressed in HEK293 or SF9 cells. Moreover, soluble miniproteins constructed from AC9 C1 and C2 domains also fail to respond to this drug (Yan et al. 1998; Haunsø et al. 2003). Yan and co-workers have shown that mutation of a single amino acid, Tyr1082→Leu, will render AC9 sensitive to the stimulatory action of forskolin. Another unusual feature of AC9 is that it is largely resistant to inhibition by adenosine analogs (Haunsø et al. 2003), which are “dead-end” inhibitors of all other ACs (Brand et al. 2013). Calmidazolium is a very effective (Ki = 5 μM), albeit nonspecific inhibitor of AC9 (Haunsø et al. 2003).
Adenylyl cyclase 9 is a membrane bound adenylyl cyclase with unique regulatory properties. While the enzyme is prominently expressed in vital organs of the body such as the brain, the heart, and most endocrine glands, precious little is known about its physiological role and potential involvement in the pathogenesis of human diseases. More work with genetically modified animal models guided by recent intriguing human genetic studies is required to understand the biological role of AC9.
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