Molecular Variants of Soluble Guanylyl Cyclase Affecting

Home , Cyclase, GUCY1A2, GUCY1A3, GUCY1B3, NOS1, Soluble guanylyl cyclase

Advance Publication by-J-STAGE Circulation Journal REVIEW Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Molecular Variants of Soluble Guanylyl Cyclase Affecting Cardiovascular Risk Jana Wobst; Philipp Moritz Rumpf, MD; Tan An Dang; Maria Segura-Puimedon, PhD; Jeanette Erdmann, PhD; Heribert Schunkert, MD

Soluble guanylyl cyclase (sGC) is the physiological receptor for nitric oxide (NO) and NO-releasing drugs, and is a key enzyme in several cardiovascular signaling pathways. Its activation induces the synthesis of the second messenger cGMP. cGMP regulates the activity of various downstream proteins, including cGMP-dependent protein kinase G, cGMP-dependent phosphodiesterases and cyclic nucleotide gated ion channels leading to vascular relaxation, inhibition of platelet aggregation, and modified neurotransmission. Diminished sGC function contributes to a number of disorders, including cardiovascular diseases. Knowledge of its regulation is a prerequisite for understanding the pathophysiology of deficient sGC signaling. In this review we consolidate the available information on sGC signaling, including the molecular biology and genetics of sGC transcription, translation and function, including the effect of rare variants, and present possible new targets for the development of personalized medicine in vascular diseases.

Key Words: Cardiovascular disease; Cyclic guanosine-3’,5’-monophosphate (cGMP); Molecular variants; Nitric oxide (NO); Soluble guanylyl cyclase (sGC)

he major components of the nitric oxide (NO)/cyclic varied and include vascular smooth muscle cell (VSMC) relax- guanosine-3’,5’-monophosphate (cGMP) pathway ation,11 inhibited platelet aggregation12 and modified neuro- T were identified in the late 1980s.1,2 NO is biosynthe- transmission,13 for example. sized endogenously through sequential oxidation of the amino acid L-arginine by an enzyme family called the NO synthases (NOS).3 Three isoforms of NOS with different tissue distribu- sGC Subunits tions are known: neuronal NOS (nNOS/NOS1), inducible NOS sGC is composed of 2 subunits: α and β.14 In 1981 Gerzer et in macrophages (iNOS/NOS2) and endothelial NOS (eNOS/ al showed that sGC contains a heme in form of ferroprotopor- NOS3).4 In the cardiovascular system, eNOS is the main source phyrin IX.15 However, it was unknown for a long time whether of NO production.5,6 The formation of NO by eNOS is increased this prosthetic heme group is sandwiched between the α and β by various stimuli such as platelet-derived factors, shear stress, subunits or whether it exclusively binds to the β subunit. In acetylcholine, and cytokines.7 1997, Zhao and Marletta demonstrated the ferrous heme to be NO mediates its functions through its primary receptor: sol- ligated to the N-terminal part of the β subunit at His105.16 uble guanylyl cyclase (sGC). Together with the adenylate In humans, 2 types of each subunit exist: α1 and α2 for the cyclases, sGC belongs to the class III purine nucleotidyl cyclase α subunit and β1 and β2 for the β subunit. These 4 proteins are family.8 Binding of NO to the heme moiety of sGC induces encoded by 4 distinct genes: GUCY1A3 (α1), GUCY1A2 (α2); the transition from basal to activated sGC. Activated sGC GUCY1B3 (β1), and GUCY1B2 (β2). The genes for human α1 converts guanosine-5’-triphosphate (GTP) to cGMP and pyro- and β1 have been mapped to chromosome 4,17 and those encod- phosphate (PPi).9 PPi is emerging as a major factor in prevent- ing α2 and β2 to chromosomes 1118 and 13,19 respectively ing vascular calcification.10 cGMP acts as a ubiquitous second (Table 1). Dimerization of the enzyme is a prerequisite for its messenger in intracellular signaling cascades, which serves to catalytic activity.20 Both α subunits give rise to a functional regulate the activity of a number of downstream proteins, enzyme when coexpressed with β1 (ie, both α1/β1 and α2/β1 including cGMP-dependent protein kinase G (PKG), cGMP- heterodimers are activated by NO).21 Although heterodimers dependent phosphodiesterases (PDE) and cyclic nucleotide seem to be the preferred form in cells, the features that deter- gated ion channels. The signals propagated through cGMP are mine this preference, and any possible role for homodimers,

Received January 13, 2015; accepted January 14, 2015; released online February 6, 2015 Department of Cardiovascular Diseases, German Heart Center Munich, Technical University Munich, Munich (J.W., P.M.R., T.A.D., H.S.); Institute for Integrative and Experimental Genomics, University of Lübeck, Lübeck (M.S.-P., J.E.); German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, Lübeck (J.E.); and German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich (H.S.), Germany Mailing address: Heribert Schunkert, Professor Dr, MD, Klinik für Herz- und Kreislauferkrankungen, Deutsches Herzzentrum München, Technische Universität München, Lazarettstr. 36, 80636 Munich, Germany. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-15-0025 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by-J-STAGE WOBST J et al.

Table 1. Overview of Human Soluble Guanylyl Cyclase Subunit Isoforms Chromosomal Protein size Gene Transcript variant Exons Isoform location (aa)

GUCY1A3 4q31.1-q31.2 GUCY1A3-Tr1 11 α1-IsoA 690 GUCY1A3-Tr2 10 GUCY1A3-Tr3 10 GUCY1A3-Tr4 8

GUCY1A3-Tr5 10 α1-IsoB 455

GUCY1A3-Tr7 9 α1-IsoD 624

GUCY1A3-Tr8 10 α1-IsoA 690

GUCY1A2 11q21-q22 GUCY1A3-Tr1 9 α2i 763

GUCY1A3-Tr2 8 α2 732

GUCY1B3 4q31.3-q33 GUCY1B3-Tr1 15 β1-Iso1 641

GUCY1B3-Tr2 14 β1-Iso2 619

GUCY1B3-Tr3 15 β1-Iso3 599

GUCY1B3-Tr4 16 β1-Iso4 594

GUCY1B3-Tr5 14 β1-Iso5 586

GUCY1B3-Tr6 13 β1-Iso6 551 GUCY1B2 13q14.3 – 17 – 617

are not fully defined. Koglin et al could show thatβ 2 does not which likely affects mRNA stability. exhibit cyclase activity when expressed with either α1 or α2 but In mammals, a splice variant of α2 (α2i) generates a dominant that β2 is active in the absence of an α subunit.22 This implies negative variant when forming a dimer with β1 because α2i con- that the β2 protein can function as a homodimer ex vivo. How- tains an in-frame insertion of 31 amino acids within the cata- ever, the physiological role of the β2 subunit in cGMP signal- lytic domain.37 Concerning β2, the NCBI nucleotide database ing remains elusive. By contrast, Zabel et al were able to just provides information on a single human β2 transcript overexpress α1/α1 and β1/β1 homodimers in Sf9 cells, both (Table 1). Because at least 3 different β2 transcripts have been being catalytically inactive.23 observed in humans38–40 and different β2 isoforms have been cloned from rats,41 there is evidence that β2 also exists as different isoforms in humans. Alternative Splicing Recently, Martin et al36 conducted a study of the role of alter- Diminished expression and function of sGC contributes to the native splicing of GUCY1A3 and GUCY1B3 sGC in healthy pathogenesis of several cardiovascular disorders such as coro- and diseased human vascular tissue. As splicing regulation of nary artery disease (CAD), atherosclerosis and hypertension.24 sGC and its biological role in vascular tissue has not been Most recently, the chromosomal locus harboring GUCY1A3 previously examined in vivo, they were the first to show splic- (α1) and GUCY1B3 (β1) was shown to contain genetic variants ing diminishing sGC function. Using a semiquantitative with genome-wide significant association to CAD and hyper- reverse transcriptase PCR approach, they uncovered various tension.25–27 Thus, the GUCY1A3/GUCY1B3 locus adds to the GUCY1A3 and GUCY1B3 splice variants in human aorta. growing list of those contributing to CAD and myocardial Quantifying the total levels of GUCY1A3 and GUCY1B3 sGC infarction (MI) risk.28,29 transcripts using quantitative PCR revealed a 3.2- and 2.3-fold The expression of sGC subunits is modulated at different increase in, respectively, GUCY1A3 and GUCY1B3 mRNA in levels, including inhibition of transcription,30,31 destabilization aortas with aneurysms compared with healthy control aortas. of mRNA,32,33 and protein degradation.34 The role of alterna- Interestingly, the aortas with aneurysms demonstrated decreased tive splicing in this process still needs to be uncovered. Precise sGC activity that correlated with increased expression of dys- understanding of sGC splicing regulation could serve as a tar- functional sGC splice variants, because the composition of the get for new therapeutic interventions and help to personalize splice forms in the aortas with aneurysms differed from that in sGC-targeting therapies in the treatment of vascular disease. control aortas (Figure 1). As demonstrated by several studies, human α1, α2 and β1 exist GUCY1A3-Tr1 coding for α1-IsoA and GUCY1A3-Tr7 cod- as different isoforms because of alternatively spliced transcript ing for α1-IsoD were higher in diseased aortas. α1-IsoD lacks variants35–37 (Table 1). Besides several predicted sequences, 66 C-terminal amino acids and has impaired enzymatic activ- NCBI nucleotide database research reveals a total of 7 alter- ity. In contrast, the level GUCY1A3-Tr5 coding for α1-IsoB natively spliced human transcript variants for GUCY1A3, 2 for was lower in the aneurysm samples. α1-IsoB is 235 amino GUCY1A2 and 6 for GUCY1B3. In the case of GUCY1A2 (α2 acids shorter at the N-terminus and oxidation resistant.42 The and α2i) and GUCY1B3 (β1-Iso1 to Iso6), each transcript vari- immunoprecipitation studies and activity evaluation presented ant codes for 1 unique isoform. However, the 7 alternatively by Martin et al clearly demonstrated that α1-IsoB forms a func- spliced variants of GUCY1A3 only code for 3 different iso- tional heterodimer with β1 subunit in aorta in vivo. Concerning forms: canonical full-length 690 aa α1-IsoA (GUCY1A3-Tr1 the expression of β1 isoforms, they found GUCY1B3-Tr1, to Tr4 and GUCY1A3-Tr8), 455 aa α1-IsoB (N-term∆235aa; GUCY1B3-Tr4 and GUCY1B3-Tr6 being more highly expressed GUCY1A3-Tr5) and 624 aa α1-IsoD (C-term∆66aa; GUCY1A3- in the aneurysm group than in controls. β1-Iso1 contains a 22 Tr7). GUCY1A3-Tr1 to Tr4 coding for the identical α1-IsoA amino acid insertion in the N-terminal regulatory heme-NO/ differ from each other only in their 5’- and 3’-UTR sequences, oxygen domain (H-NOX) domain, so heme-NO binding might Advance Publication by-J-STAGE Soluble Guanylyl Cyclase

Figure 1. Expression of different transcripts coding for sGC α1 and β1 in aneurysm and control aortas. Splice variants visualized on agarose-gels after RT-PCR. (A) GUCY1A3-Tr1 and GUCY1A3-Tr7 were higher in diseased aortas, GUCY1A3-Tr7 almost exclusively; GUCY1A3-Tr5 was more abundant in controls. (B) All GUCY1B3 transcripts shown were predominantly expressed in aortas with aneurysm. RT-PCR, reverse transcriptase polymerase chain reaction; sGC, soluble guanylyl cyclase. (Reprinted with permisson from Martin E, et al.36 Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm.)

be negatively affected. β1-Iso6 with a large 68 amino acid tested the effect of different truncations at the N-terminus of deletion in the H-NOX domain would also have impaired α1 and β1 on dimerization and found the amino acids 61–462 heme function. The β1-Iso4 not only carries the same 68-resi- in α1 to build up the shortest possible fragment exhibiting due deletion, but also a 43 amino acid insertion close to the wild-type-like dimerization. In a follow-up study in 2011, catalytic domain. Kraehling et al showed that that the translation initiation site Comparison of sGC activity measurement of aortic lysates in GUCY1A3-Tr5 at position 259 is dominant over ATG at posi- from control samples that predominantly expressed either tion 236 in the human sequence.42 Therefore, translation of α1-IsoA or α1-IsoB proteins showed α1-IsoB compensating for GUCY1A3-Tr5 should lead to a subunit with a N-term∆258aa low levels of canonical α1-IsoA. In short, NO-induced sGC activ- and not N-term∆235aa deletion. Our working group also inves- ity was comparable, regardless of whether α1-IsoA or α1-IsoB tigated the translation initiation at both ATG236 and ATG259 of was predominant. By contrast, the sGC heterodimer contain- α1-IsoB but in a different way. Kraehling et al generated their ing α1-IsoD exhibited a diminished activation. Martin et al construct coding for α1-IsoB-ATG259 by exchange of a nucle- evaluated the enzymatic properties of the recombinant α1-IsoD otide in the triplet coding for ATG236.42 Our coding sequence subunit in Cos7 cells. Despite a detectable basal level of cGMP- for α1-IsoB-ATG259 directly started with the triplet coding for forming activity confirming the formation of a catalytically ATG259, lacking the 69 “non-coding“ nucleotides upstream. active heterodimer, stimulation with NO only increased the When transfecting HEK293E cells with both the sequences cGMP amount marginally in contrast to α1-IsoA/β1 sGC. The coding for α1-IsoB-ATG236 and α1-IsoB-ATG259 the corre- diminished function of α1-IsoD could arise from the close prox- sponding proteins resulted in bands of the same size on west- imity of the 66 amino acid deletion to the catalytic domain. ern blotting (unpublished data). Therefore, the hypothesis of The functionality of α1-IsoB was already shown in 2003 by ATG259 being dominant over ATG236 was confirmed. Koglin and Behrends.43 They analyzed N-terminal deletion mutants of the human 1 subunit after co-expression with the α Higher-Order Domain Arrangement in sGC human β1 subunit. They observed that deleting the first 258 amino acids of the α1 subunit exerted an effect on neither Each sGC subunit is a multi-domain protein comprising 4 func- sensitivity to NO nor heme binding. Unfortunately this finding tionally different parts: (1) H-NOX, (2) a Per/Arnt/Sim-like is inconsistent with Wagner et al reported in 2005.44 They domain (PAS), (3) an α-helical region capable of forming coiled- Advance Publication by-J-STAGE WOBST J et al.

Figure 2. sGC domain organization and X-ray crystallographic models. Each subunit contains 4 modular domains; α1 domains are shown in shades of gray, and β1 domains are shown in color. The H-NOX domain of the β1 subunit contains the heme cofactor, shown in red. H-NOX, heme-NO/oxygen domain; PAS, Per/Arnt/Sim-like domain; sGC, soluble guanylyl cyclase. (Reprinted with permission from Campbell MG, et al.46 Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase.)

coils involved in dimerization (CC), and (4) a C-terminal cata- conformation. In addition, Campbell et al46 described a second lytic domain (CAT) where the GTP binding and conversion direct allosteric control mechanism through interaction between takes place45 (Figure 2). These 4 specified domains form 2 H-NOX and the PAS domain, as previously observed by oth- rigid units within the sGC: the smaller unit comprises the ers.56,57 Campbell et al showed that these 2 domains form a dimeric catalytic domain, and the larger one is built from the tight cluster, sharing large surfaces of interactions, and allowing clustering of the PAS and H-NOX domains. The helical domains each H-NOX domain to interact with both the α1 and β1 PAS form a dimeric parallel coiled-coil that flexibly connects the 2 domains, allowing small-scale changes in the H-NOX domain modules.46 to be quickly recognized by the adjacent PAS. Contrary to Crystal structures of the independent domains have already Busker et al’s observations, Campbell et al did not describe a been reported.47–50 Recently, Campbell et al were the first to dramatic conformational change between the NO-bound and show the 3D structure of Rattus norvegicus sGC holoenzyme unbound states, which led them to assume that ligand binding using negative-stain electron microscopy.46 Still, no high-reso- only induces small-scale intradomain conformational changes. lution 3D structure of the complete human holoenzyme is available to date. Determining the structure of full-length sGC is a prerequisite to understanding its function and for the design and Tissue Distribution of α1/β1 and α2/β1 Heterodimers improvement of therapeutics for treatment of related diseases. Budworth et al investigated the localization of the subunits in humans and found α1 and β1 to be expressed in most tissues. The α2 subunit is found in fewer tissues, but is highly Signal Transmission in sGC expressed in the brain, lung, colon, heart, spleen, uterus, and Analogous to the transmembrane guanylyl cyclases, where bind- placenta.58 Pharmacological and biochemical kinetic studies ing of ANP is transmitted across the transmembrane helices conducted by Russwurm et al demonstrated that the naturally leading to an active conformation of the 2 intracellular domains,51 occurring sGC isoforms, α1/β1 and α2/β1, exhibit similar sen- it was assumed that binding of NO to the N-terminal H-NOX sitivities to NO in vitro.21 Further studies by Bellingham and domain is transmitted to the C-terminal CAT across the coiled- Evans59 showed that the differential biological effects of the 2 coil domain.52 This linear transmission model disagrees with forms are based on their localization. Although α1/β1 sGC is the findings from Winger et al,53 who showed that the isolated primarily localized in the cytosol, thus producing an unfo- H-NOX domain can directly interact with the isolated cata- cussed source of cGMP, α2/β1 has a tendency to localize at the lytic region of sGC. Consistent with those results, Haase et al membrane, providing a localized pool of cGMP at this site.59 demonstrated the N-termini of sGC being in close proximity Bellingham and Evans measured the functional properties of to the C-termini using fluorescence resonance energy transfer α2/β1 by utilizing the NO-dependent activation of the ion channel (FRET).54 In a very recent study, Busker et al55 studied the cystic fibrosis transmembrane conductance regulator (CFTR), conformational change of full-length sGC under NO-stimulated which occurs by phosphorylation via the membrane-bound conditions. As sGC contains 5 tryptophane residues distributed PKGII isoform. They found that cGMP generated by α2/β1 acti- evenly over all 4 functional domains, Busker et al used these vates CFTR far more effectively than the cytoplasmically located as donors for FRET. The substrate analog 2’-Mant-3’-dGTP α1/β1, despite near identical catalytic properties. This suggests was used as acceptor, making it possible to identify movements α2/β1 to be of general importance in mediating the membrane of the functional domains relative to the substrate-binding cata- effects of NO and a potentially important selective drug target. lytic region. Their FRET signals indicated Trp-22 and Trp-466 However, the 150-kDa α1/β1 heterodimer is regarded as the were in close proximity to the catalytic domain upon activa- most physiologically relevant isoform and therefore the most tion of NO, which means that activation of sGC by binding of extensively studied one. The functional importance of α1/β1 NO to the β1 H-NOX domain is transmitted to the catalytic sGC was demonstrated by the significantly decreased relaxing domain both through the α1 coiled-coil domain and by direct effects of major vasodilators such as acetylcholine, NO, YC-1 interdomain interaction between the H-NOX and catalytic and BAY 41-2272 in α1 sGC knockout mice.60 domain forcing the catalytic domain into the NO-activated Advance Publication by-J-STAGE Soluble Guanylyl Cyclase

Table 2. Functional Mutations in the Soluble Guanylyl Cyclase α1 Subunit Mutation Where identified p.Leu163Phefs*24 MI family p.Gly537Arg MI family p.Lys53Glu 252 young MI cases p.Thr64Ala 252 young MI cases p.Thr229Met 252 young MI cases p.Ser478Gly 252 young MI cases p.Val587Ile 252 young MI cases MI, myocardial infarction.

sGC Activation and Maintenance of cGMP The formation of the NO-heme complex is responsible for an up to 250-fold increase in the GTP cyclase activity rate of the enzyme.61 NO binds to the heme of sGC forming an unstable 6-coordinate complex, which rapidly converts into a 5-coordinate complex because of disruption of the coordinating bond between His105 and the heme.62 Once NO dissociates from sGC, basal cGMP production is restored,45 which ensures sGC activity is quickly up- and downregulated. However, not only is sGC activity itself regulated but also the amount of cGMP is controlled by certain PDEs that break the phosphodiester bond within cGMP, hydrolyzing it to GMP.63 In total, 11 different types of isoenzymes, each with several isoforms, exist. Whereas some PDEs are said to be cGMP-selective, because Figure 3. Reduced expression and enzymatic activity of the of their 100-fold substrate preference for cGMP over adenosine- identified sGC mutants in HEK293 cells. (A) GUCY1A3 3’,5’-monophosphate (cAMP), others are specific for hydro- mutants were expressed together with GUCY1B3 in HEK293E cells. α1-sGC protein was strongly reduced in mutants (num- lyzing cAMP, and some PDEs can hydrolyze both cAMP and bers in bars are independent transfections performed in cGMP.64 For cGMP cleavage, PDE5 is considered to be the duplicates). (B) Accordingly, NO-induced concentration- most important player in humans, with regard to its catalytic dependent cGMP formation was significantly attenuated after affinity being in the physiologically range and PDE5 being transfection with mutant proteins. AU, arbitrary units; GSNO, expressed in most peripheral tissues (ie, VSMC, plateletes, S-nitrosoglutathione; sGC, soluble guanylyl cyclase; WT, wild 66 heart65). type. (Reprinted with permisson from Erdmann J, et al. Dys- functional nitric oxide signalling increases risk of myocardial Growing evidence indicates that imbalance of intracellular infarction.) cGMP levels from dysregulation of either sGC or PDE5 plays a role in the risk for CAD and MI.

Functional Mutations in sGC α1 Subunit Summary Recently, various mutations in the coding sequence of GUCY1A3 In this review we highlighted the fundamentals of sGC from were found by our group to be associated with CAD/MI.66 The it numerous forms of appearance at the mRNA level through starting point was a family with 32 members of whom 22 had its diversity in expression to the functional aspects. In the vas- CAD under the age of 60 years. Exome-sequencing in 3 dis- culature, the most important source of NO is eNOS. NO again tantly related family members with MI revealed a digenic is the main stimulus for sGC to transform GTP into the ubiq- mutation in GUCY1A3 and CCT7. CCT7 codes for CCTη, uitous second messenger cGMP, which regulates many down- which acts as a chaperonin folding the α and β subunits.67 stream proteins finally influencing vessel tonus and platelet Both mutations were absent in over 3,000 controls and in over aggregation, for example. Decreased cGMP production plays 3,000 unrelated MI cases. However, analysis of exome-sequenc- a decisive role in the pathogenesis of several disorders, including data of 252 young MI cases uncovered another 5 rare ing cardiovascular diseases. In their recent study, Martin et al missense variants associated with MI.66 Moreover, sequencing were the first to nail down the relationship between splice vari- GUCY1A3 in 48 patients from another 22 additional extended ants of GUCY1A3 and GUCY1B3 and a phenotype in human MI families revealed p.Gly537Arg, a rare mutation cosegre- aortas. Moreover, a recent genetic study revealed that alterna- gating with the disease in the family. These overall 7 rare vari- tive splice variants might henceforth play an important role as ants of GUCY1A3 are currently under investigation by our targets in the treatment of cardiovascular diseases. working group in consideration of levels of protein expression, dimerization and enzyme activity (Table 2). Preliminary data Acknowledgments have already revealed reduced protein expression, as well as The study was supported by the German Federal Ministry of Education reduced sGC activity by measuring NO-induced cGMP in and Research (BMBF) in the context of the e:Med program (e:AtheroSysMed) p.Leu163Phefs*24 and p.Gly537Arg mutants compared with and the FP7 European Union project CVgenes@target (261123). Further the wild type (Figure 3).66 grants were received by the Fondation Leducq (CADgenomics: Under- Advance Publication by-J-STAGE WOBST J et al. standing Coronary Artery Disease Genes, 12CVD02) and the Deutsche of a second subunit. J Biol Chem 2001; 276: 30737 – 30743. Forschungsgemeinschaft (DFG SFB 1123). 23. Zabel U, Hausler C, Weeger M, Schmidt HHHW. Homodimerization of soluble guanylyl cyclase subunits: Dimerization analysis using a glutathiones-transferase affinity tag. J Biol Chem 1999; 274: 18149 – Disclosures 18152. 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