Alpha1 sGC splice forms as potential regulators of human sGC activity

44 Pages·2008·2.11 MB·English

JBC Papers in Press. Published on April 1, 2008 as Manuscript M710269200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M710269200
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ALPHA1 sGC SPLICE FORMS AS POTENTIAL REGULATORS OF HUMAN sGC ACTIVITY
Iraida G. Sharina*, Filip Jelen, Elena P. Bogatenkova, Anthony Thomas, Emil Martin and Ferid
Murad*
From : The Brown Foundation Institute of Molecular Medicine, University of Texas Houston Medical
School, Houston , Texas 77030;
Running title: novel splice forms of soluble guanylyl cyclase
* To whom correspondence should be addressed at The Brown Foundation Institute of Molecular
Medicine, University of Texas Houston Medical School, Houston , Texas 77030, USA. Tel: (713) 500-
2480; fax: [phone]; e-mail: [email protected] or [email protected]
Soluble Guanylyl Cyclase (sGC), a key Since the studies of the late 1970’s-early
protein in the NO/cGMP signaling pathway, is 1980’s which underlined the obligatory role of the
an obligatory heterodimeric protein composed endothelium in mediating acetylcholine-induced
of one α and one β subunit. The α /β sGC vasodilatation, nitric oxide (NO) has been
1 1 D
heterodimer is the predominant form expressed recognized as an endogenous nitrovasodilator that o
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in various tissues and is regarded as the major mediates the local regulation of basal arterial tone nlo
a
isoform mediating NO-dependent effects such (1-4). Many of the physiological functions of NO de
d
as vasodilation. We have identified three new α1 in the cardiovascular, neuronal, gastrointestinal fro
m
sGC protein variants generated by alternative and other systems are mediated through its h
splicing. The 363 residue N1-α sGC splice primary receptor, soluble guanylyl cyclase (sGC). ttp
variant contains the regulatory 1domain, but The heme-containing sGC heterodimer converts ://w
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lacks the catalytic domain. The shorter N2-α1 guanosine triphosphate into the secondary .jb
sGC maintains 126 N-terminal residues and messenger guanosine 3’:5’-cyclic monophosphate c.o
gains additional 17 unique residues. The C-α1 (cGMP). The sGC activity increases more than brg/
sGC variant lacks 240 N-terminal amino acids, 200 fold in response to NO (5,6). High y g
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but maintains a part of the regulatory domain concentrations of cGMP produced by activated es
and the entire catalytic domain. Q-PCR of N1- sGC modulate functions of numerous enzymes, t on
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α , N2-α sGC mRNA levels together with RT- such as cyclic nucleotide phosphodiesterases, an
1 1 u
a
PCR analysis for C-α1 sGC demonstrated that cGMP-gated ion channels, and cGMP-dependent ry 2
the expression of the α1 sGC splice forms vary protein kinases (PGKs). Recently, the vital , 2
0
in different human tissues indicative of tissue- importance of sGC for mammalian physiology 1
9
specific regulation. Functional analysis of the was directly confirmed by generation of the sGC
N1-α sGC demonstrated that this protein has a knockout mice (7-9). The absence of sGC protein
1
dominant negative effect on the activity of sGC resulted in a significant increase in blood pressure,
when co-expressed with α /β heterodimer. The complete loss of NO-dependant aortic relaxation
1 1
C-α sGC variant heterodimerizes with the β and inhibition of platelet aggregation in knockout
1 1
subunit and produces a fully functional NO- animals, which died prematurely at the age of 4
and BAY41-2272-sensitive enzyme. We also weeks due to severe gastrointestinal disorders (7).
found that despite identical susceptibility to
inhibition by ODQ, intracellular levels of the 54 Four sGC isoforms, products of four
kDa C-α band did not change in response to genes, have been identified so far: α , α , β and β .
1 1 2 1 2
ODQ treatments, while the level of 83 kDa α Only α /β and α /β heterodimers are activated by
1 1 1 2 1
band was significantly affected by ODQ. These NO (10). The α /β sGC is the most abundant
1 1
studies suggest that modulation of the level and isoform and is distributed ubiquitously in
diversity of splice forms may represent novel mammalian tissues with the highest levels of
mechanisms modulating the function of sGC in mRNA in brain, lung, heart, kidney, spleen and
different human tissues. muscle (11). Vascular smooth muscle and
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
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endothelial cells express predominantly α and β MATERIALS AND METHODS.
1 1
subunits (12). The functional importance of α /β
1 1
sGC was demonstrated by the significantly Reagents. DMEM/F12 medium was from
decreased relaxing effects of major vasodilators Gibco; Grace medium and FBS were from Sigma.
(acetylcholine, NO, YC-1 and BAY 41-2272) in NO donor DEA-NO was from Calbiochem. The 5-
the α sGC knockout mice of both genders (9). cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-
1
pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine
sGC function is affected not only by NO, (BAY41-2272) activator was a generous gift from
but also by regulation of the expression of sGC J. P. Stasch (Bayer, Wuppertal, Germany). [α-
subunits at transcriptional and post-transcriptional 32P]GTP was from NEN. ODQ (1H-
levels. The steady state mRNA levels of α and β [1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one) was
1 1
subunits decrease with hypertension, aging and from Sigma-Aldrich (USA). Polyclonal anti-α
1
vary during embryonic development (13). The sGC antibodies were raised against the peptide
expression of sGC subunits is regulated by FTPRSREELPPNFP of the human α subunit,
1
estrogen (14), cAMP-elevating compounds while anti-β antibodies were raised against the
1
(15,16), cytokines (NGF, LPS, IL-1β) (17) and SRKNTGTEETKQDDD peptide of the human β
1
NO donors (18). Subcellular localization of sGC subunit.
and its activity can also be affected in proliferating D
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tissue (19) by protein interactions and Primers, RNA and RT-PCR. All primers n
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phosphorylation (13). In mammals, the alternative were custom synthesized by Integrated DNA ad
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splicing for the α2 subunit generates a dominant Technologies (Coralville, IA). To subclone N1-α1 d fro
negative variant (20). Splice forms for β and β sGC we used the upstream primer PR1 5’- m
1 2 h
subunits have been also demonstrated (21-23). 318CAACACCATGTTCTGCACGAAGC-3’ and ttp
Recently, a shortened α1 sGC transcript, which the downstream primer PR2 5’- ://w
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lacks the predicted translation site in exon 4, has 1411GCTTTCATATTCAAGATAGTATTATG-3’ w
been found and its expression was correlated with (numbering according to sequence with Acc. N .jbc
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lower sGC activity in several cell lines (24). CR618242). To subclone the N2-α sGC we used rg
However, splice variants of α sGC have not been the upstream primer 1 PR3 5’- by/
1 g
described previously. 192CAACACCATGTTCTGCACGAAGC-3’ and ue
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the downstream primer PR4 5’- t o
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Here we report the isolation and 611GTATCACTCTCTTTGTGTAATCC-3’ (Acc. Ja
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characterization of three new α1 sGC splice forms N BC012627). To detect the deletion of exon 4 in ary
encoding N- and C-terminal truncated proteins. C-α1 sGC we used the upstream primer PR5 5’- 2, 2
We demonstrate that the N-terminal truncated C-α1 120GCTAGAGATCCGGAAGCACA-3’ and the 019
splice form heterodimerizes with the β sGC downstream primer 5’-
1
subunit to create an active NO-sensitive enzyme 317TTGCAAATACTCTCTGCCAAA-3’ (Acc. N
both in Sf9 and human neuroblastoma BE2 cells. AK226125). To detect the deletion of in the exon
Moreover, this splice variant is more resistant to 7 of C*-α sGC we used the upstream primer PR7
1
ODQ-induced protein degradation than the wild 5’-561GAA CGG CTG AAT GTT GCA CTT
type sGC. N1-α sGC splice form lacking the C- GAG-3’ and the downstream primer PR8 5’-
1
terminal catalytic domain has a dominant negative 922GTA GGG CTG ATT CAC AAA CTC G-3’
effect when co-expressed with α /β sGC in Sf9 or (Acc. N BX649180). Total RNA from BE2 cells
1 1
BE2 cells. The functional role of N2-α sGC splice was isolated using RiboPure kit (Ambion, TX).
1
variant is yet to be determined. Q-PCR and semi- The panel of total RNA from human tissues was
quantitative RT-PCR analyses of different human purchased from Ambion (FirstChoice Human
tissues demonstrate tissue-specific expression of Total RNA Survey Panel, Lot 08608142). 5 μg of
the identified splice forms. Together our data total RNA was used for RT reactions, which was
suggest that alternative splicing of the α sGC performed with a mixture of oligo(dT) and
1
subunit may be a novel mechanism that regulates Random Hexamer primers using SuperScript III
sGC function and activation in some human RT (Invitrogen) according to manufacturer’s
tissues. protocol. PCR reactions with PfuUltra DNA
3
polymerase (Stratagene) were performed for 35 workstation (Tecan US, Research Triangle Park,
cycles at the T of 55oC. PCR products were NC); PCR master mixes were pipetted utilizing a
a
separated on agarose gel, purified using QIAEX II Biomek 2000 robotic workstation (Beckman,
gel extraction kit (Qiagen) and sequenced using Fullerton, CA). Each assembled plate was then
PCR primers. All sequencing was performed by covered with optically clear film (Applied
Nucleic Acid Core Facility at Medical School of Biosystems, Foster City, CA) and run in the 7700
University of Texas in Houston. or 7900 real-time instrument using the following
cycling conditions: 95°C, 1 min; followed by 40
Real-Time Quantitative RT-PCR. Real- cycles of 95°C, 12 sec and 60°C, 30 sec. The
time quantitative RT-PCR (RT-qPCR) was resulting data were analyzed using SDS 1.9.1
performed utilizing the 7700 or 7900 Sequence (7700) or SDS 2.3 (7900) software (Applied
Detector instrument (Applied Biosystems, Foster Biosystems, Foster City, CA) with ROX as the
City, CA) (25,26). Specific quantitative assays for reference dye.
α1 sGC, N1-α1 sGC and N2-α1 sGC were Synthetic DNA oligos used as standards
developed using Primer Express software version (sDNA) encompassed the entire 5’ – 3’ amplicon
1.0 for Macintosh (Applied Biosystems) or for the assay (Invitrogen, Carlsbad, CA). Each
Beacon Designer (Premier Biosoft) or oligo standard was diluted in 100 ng/µl yeast or E.
RealTimeDesign (Biosearch Technologies) based coli tRNA-H O (Invitrogen, Carlsbad, CA or D
2 o
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on sequences from Genbank. The assays are listed Roche Diagnostics, Indianapolis, IN) and span a 5- n
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in Table S3. cDNA was synthesized in 10 µl (96- log range in 10-fold decrements starting at 0.8 ad
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wtheel la pdldaitteio) no ro 5f µ6l µ(3l 4o8r- w3e lµl lp/wlaetell) RtoTta lm vaosltuemr em bixy pthga/tr eianc tviiotnro. Ittr ahnassc rbibeeedn RshNoAw na mfopr lisceovne rsatla nadssaarydss d from
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consisting of: 400 nM assay-specific reverse (sRNA) and sDNA standards have the same PCR ttp
primer, 500 μM deoxynucleotides, Superscript II efficiency when the reactions are performed as ://w
w
buffer and 10 U Superscript II reverse described above (G.L. Shipley, personal w
transcriptase (Invitrogen, Carlsbad, CA), to a 96- communication). .jbc
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well plate (ISC Bioexpress, Kaysville, UT) or 384- Due to the inherent inaccuracies in rg
well plate (Applied Biosystems, Foster City, CA) quantifying total RNA by absorbance, the amount by/
g
and followed by a 4 µl or 2 µl volume of sample of RNA added to an RT-PCR from each sample ue
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(25 ng/µl), respectively. Each sample was was more accurately determined by measuring a t o
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determined in triplicate plus a control without housekeeping transcript level in each sample. The Ja
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reverse transcriptase to access DNA contamination final data were normalized to 36B4 (500-fold a
ry
levels. Each plate also contained an assay-specific dilution of sample in tRNA-H O). 2
2 , 2
sDNA (synthetic amplicon oligo) standard 0
1
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spanning a 5-log template concentration range and Cell culture. BE2 human neuroblastoma
a no template control. Each plate was covered with cell line (American Type Culture Collection) was
Biofilm A (Bio-Rad, Hercules, CA) and incubated cultured in 1:1 mixture of DMEM/F12K media
in a PTC-100 (96) or DYAD (384) thermocycler supplemented with 10% FBS, 0.1 mM MEM
(Bio-Rad, Hercules, CA) for 30 minutes at 50°C nonessential amino acids, penicillin-streptomycin
followed by 72˚C for 10 min. Subsequently, 40 μl mixture (50 units/ml and 50 µg/ml), 10 mM Hepes
or 20 µl of a PCR master mix (400 nM forward (pH 7.4), 1 mM sodium pyruvate, 2 mM L-
and reverse primers (IDT, Coralville, IA), 100 nM glutamine (all from Gibco) and maintained at 37°C
fluorogenic probe (Biosearch Technologies, and 5% CO . For in vivo ODQ treatments 80%
2
Novato, CA), 5 mM MgCl , and 200 μM confluent neuroblastoma cell cultures were treated
2
deoxynucleotides, PCR buffer, 150 nM SuperROX with 20 μM ODQ for up to 24 hours. To prepare
dye (Biosearch Technologies, Novato, CA) and lysates the cells were collected by trypsinolysis,
1.25 U Taq polymerase (Invitrogen, Carlsbad, CA) washed twice with PBS, resuspended in 40 mM
was added directly to each well of the cDNA plate. TEA (pH 7.4) containing protease inhibitor
RT master mixes and all RNA samples were cocktail (Roche) and disrupted by sonication. The
pipetted by a Tecan Genesis RSP 100 robotic lysates were centrifuged at 15,000 x g for 30 min
to prepare the cleared supernatant fractions, which
4
were used for western blotting, Co-Immunoprecipitation. BE2, BE-CαF
immunoprecipitation or activity measurements. and BE-N1αF cells collected from confluent 10
cm culture dish, were washed twice with PBS,
Generation of BE2 Stable Transfectant resuspended in 500 μl of PBS containing protease
Lines. Coding sequences of N1-, N2- and C-type inhibitor cocktail, disrupted by sonication, spun
α sGC variants obtained from BE2 total RNA as down at 15,000 rpm for 30 min at 4oC and
1
described above were first cloned into pCR-Blunt supernatants were collected. Then polyclonal anti-
vector (Invitrogen). For N1- and N2-type α forms, β -sGC antibodies or pre-washed anti-FLAG M2
1 1
the coding sequence of the FLAG-peptide was affinity resin (Sigma) were added and tumbled for
inserted by PCR in front of the stop codon and 1.5 h or overnight, repectively, at 40C. The lysate
recloned into pCR-Blunt. The coding regions of mixture with anti-β -sGC antibodies was then
1
N1-, N2- and C-type α1 sGC variants were then combined with 100 μl of prewashed Protein A-
isolated by restriction with NsiI/XbaI enzymes and agarose beads (Upstate) and further incubated for
subcloned into PstI/XbaI sites under the control of 1.5 h. The Protein A-agarose beads were washed
CMV promoter of the mammalian expression three times with 40 mM TEA, 200 mM NaCl, 1%
vector pMGH2 (Invivogen). These pMG-CαF, NP40, pH 7.4 and bound proteins eluted by boiling
pMG-N1αF and pMG-N2αF plasmids were in 100 μl of Lamelli buffer. The anti-FLAG M2
transfected into BE2 cells by Lipofectamine affinity resin was washed three times with ice cold Do
reagent (Invitrogen) according to manufacturer’s TBS buffer and bound proteins were eluted with wnlo
protocol. 48 hours post-transfection, the cells were high salt buffer or with buffer containing the ad
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plated on 96 well plates at 2500 cells/ml density FLAG peptide, according to manufacturer’s d fro
and selected by 380 μg/ml of Hygromycin m
instructions. Western blot was probed for the α1 h
(Sigma). Two weeks later, individual hygromycin- and β subunits of sGC using polyclonal anti-α - ttp
resistant colonies were collected and expanded on 1 1 ://w
100-mm2 tissue-culture dishes. The clones stably sGC and monoclonal anti-β1-sGC antibodies. ww
transfected by N1- or N2-type α1 sGC were Expression in Sf9 cells. To express the α .jbc.o
identified by anti-FLAG western blotting of 1 rg
lysates prepared from the hygromycin-resistant splice forms in Sf9 cells, the coding regions of N1, by/
N2 or C-α sGC were subcloned into the pVL1392 g
csGulCtu rwese. reT hied ecnltoifnieesd sutasbinlyg eaxnptir-eαss insGg CC -wtyepset eαrn1 transfer ve1ctor. The baculoviruses producing the uest o
blotting. 1 α1 splice forms were generated by recombination n Ja
with BaculoGold DNA using manufacturer’s nu
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protocol (BD Biosciences). To obtain sGC enzyme ry
Western blot analysis. Protein samples containing the splice form α subunits Sf9 cells at 2, 2
were resolved on 8 or 12 % SDS-polyacrylamide 1.8 x 106 cells/ml were1 infected with the 019
gels and transferred to methanol-activated
baculoviruses expressing full length β sGC
polyvinylidene difluoride membranes. After 1
subunit and the corresponding splice form at
blocking the membranes, the following antibodies
multiplicity of infection of 2.
were used for detection: anti-FLAG M2
monoclonal antibody (Sigma) at 1:500 dilution;
Assay of sGC Activity. Soluble guanylyl
anti-α sGC polyclonal antibody generated in our
1 cyclase activity in lysates of BE2 or Sf9 cells were
laboratory at 1:2000 dilution; anti-β sGC
1 assayed by formation of [32P]cGMP from [α-
polyclonal antibodies generated in our laboratory
32P]GTP at 37°C as described previously (27). The
at 1:3000 dilution; anti β-actin antibody (Santa
concentration of DMSO used as a vehicle for
Cruz) at 1:1000 dilution. Secondary horseradish
BAY41-2272 did not exceed 0.1% and alone had
peroxidase-conjugated antibodies (Sigma) were
no effect on sGC activity.
used at 1:5000 (anti-rabbit) and 1:10000 (anti-
mouse) dilutions. Protein bands were visualized by
enhanced chemiluminescence (ECL Plus, Statistical Analysis. All data are presented
Amersham Pharmacia Biosciences). as mean ± standard error or standard deviation.
Statistical comparisons between groups were
performed by Student’s t-test. Nonlinear
5
regression and calculations of EC50 and IC50 AK226125, C-α in Fig. 1A), the alternative splice
1
were performed using Graph Pad Prism 3.0 acceptor in intron 3 generates a 179 bp deletion in
software (GraphPad Software). exon 4, eliminating the translation start site. The
open reading frame (ORF) starts at the alternative
RESULTS. methionine located in exon 7. In another splice
variant (accession number BX649180, C*-α in
1
Identification of α sGC alternative splice Fig. 1A), the alternative acceptor site results in a
1
variants in the NCBI database. A previous report 140 bp deletion and a premature stop codon in
demonstrated the existence of alternative splicing exon 7. The alternative start codon in exon 7
for the α1 sGC in human tissues (24). We used the restores the ORF. Thus, both C-α1 and C*-α1 splice
α1 sGC cDNA (accession number Y15723) to mRNA species encode the same C-α1 sGC protein.
screen a human RNA database available on the
Human Genome web page (NCBI) for additional Alternative splicing of α sGC in human
1
splice variants of sGC. This in silico analysis BE2 neuroblastoma. We have previously
identified twelve unique α sGC cDNA sequences demonstrated that human BE2 neuroblastoma cell
1
cloned from different human tissues (Table S1). line expresses high levels of functional sGC
Their comparison with Human Genome sequences enzyme (28). Using the information on the
revealed that they are all generated by alternative structure of α1 spliced mRNA, we designed pairs Do
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splicing from the α sGC gene. of primers to amplify the fragments specific for n
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N1-α1, N2-α1, C-α1 and C*-α1 splice forms (Fig. ade
Seven of these sequences encode full size 1A). As depicted in the Supplemental Figure S1, d fro
α sGC protein, while five of them encode RT-PCR analysis showed that BE2 cells expresses m
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strpulnicciantge d oαn1e p rooft eitnhse (Tidaebnltei fSie1d). Dαu es GtoC a ltmerRnaNtiAves athlle fsopulnicde -αs1p espcilficice fmraRgNmAen, te xwcaesp tp uCr*if-iαe1d. Eanadch t hoef ttp://w
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(accession numbers CR618242, CR614534) lost identity of the splice form confirmed by w
the non-coding exon 2 and the coding exons 8 sequencing. .jbc
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through 10, but acquired additional 131 bp at the rg
b/
end of exon 7. This mRNA encodes a protein that Expression of α1 sGC alternative splice y g
maintained 363 N-terminal amino acids of α sGC, variants in different human tissues. We next ue
but lost the catalytic domain because of a s1plice- investigated the tissue-specific expression of the α1 st on
generated frameshift (Fig. 1A and 1B). Three new sGC splice variants. We designed a Q-PCR assay Jan
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splice-specific amino acid residues and a for N1 and N2-α1 sGC splice forms based on their ary
premature stop codon were acquired (Table S2). unique sequences inserted by alternative splicing 2, 2
We named this splice variant N1-α sGC (Fig.1). (Table S3). We quantified the abundance of the 01
1 9
fulllength and spliced mRNA in various human
In N2-α1 sGC mRNA (accession number tissues. As expected, full length α1 sGC mRNA
BC012627), splicing eliminates exons 7 and 8 and was detected in RNA of all tested tissues. N1-α1
introduces additional 54 base pairs and a sGC was observed at detectable levels in all
premature stop codon in exon 9. The N2- α human organs, except bladder, testis, thyroid,
1
protein retains only the first 126 residues of the α1 placenta and skeletal muscle (Fig. 2). N2- α1 sGC
sequence, but acquires additional 17 aa at the C- was present in all tissues, but at significantly lower
terminus (Fig. 1A and B, Table S2). levels than α1 sGC.
The third identified α sGC splice variant, The absence of sequences specific only for
1
termed C-α1 sGC, lost 240 N-terminal amino C- α1 or C*-α1 mRNA and insignificant difference
acids, but maintained part of the regulatory and the in size from the full length sGC transcript (Table
complete catalytic domains (Fig. 1B). S1) precluded us from using Q-PCR or Northern
Interestingly, the same C-α protein is encoded by blotting to estimate their levels and tissue
1
two differently spliced species of mRNA (Fig. distribution. Therefore, we used a semi-
1A). In one sequence (accession number quantitative RT-PCR method using primers
6
designed to detect the deletions specific for both We also selected BE2 stable lines
C- and C*- α sGC RNA species. The identity of expressing the C-α sGC splice variant tagged with
1 1
amplified fragments was confirmed by the FLAG epitope. BE2-CαF line was selected
sequencing. Full size α sGC transcript was using antibodies raised against a sequence at the
1
detected in all tissues together with C-α sGC, C-terminal end of the human α sGC subunit,
1 1
albeit at different ratios (Fig. S2). However, C*-α which recognizes both the short C-type α sGC (54
1 1
sGC mRNA was not detected in esophagus, heart, kDa) and the full length (83 kDa) α subunit (Fig.
1
kidney, liver, lung, bladder, brain, cervix and 3A). We found that the DEA-NO or DEA-
colon (Fig. S3). NO/BAY41-2272-stimulated cGMP synthesis in
the lysates of the BE2-CαF clone was not
Effect of expression of α sGC alternative significantly different from parental BE2 cells. On
1
splice variants on sGC activity. Next we tested the other hand, amount of α -sGC protein in BE2-
1
whether α sGC splice variants possess any CαF was lower then in BE2 cells (Fig. 3A). It
1
catalytic activity or whether they affect the appears that expression of C-α variant can
1
function of full length α /β sGC in BE2 cells. We compensate for decreased levels of full size α -
1 1 1
selected several stable clones expressing N1-α or sGC subunit through a formation of active
1
N2-α sGC, which were tagged with the FLAG heterodimer.
1
epitope at the C-terminal end. The stable clone D
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expressing 41 kDa N1-α sGC protein was Functional characterization of C-α /β n
1 1 1 lo
identified using anti-FLAG antibodies (BE2-N1αF heterodimer. Since the activity in BE2-CαF cells ad
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in Fig. 3A). We measured the rate of cGMP suggested that C-α1 splice form can compensate d fro
production in the lysates of these cells in response for α function we characterized the properties of m
1 h
to the NO donor, DEA-NO or DEA-NO in the the C-α1/β1 heterodimer. We co-expressed the C- ttp
presence of the heme-dependent allosteric α1 variant with β1-sGC in Sf9 cells. Sf9 lysates ://w
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regulator BAY 41-2272. As demonstrated in Fig. expressing the C-α /β -sGC displayed a robust w
3B, the BE2-N1αF clone showed a significant guanylyl cyclase act1ivit1y. We found no difference .jbc
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dcoecmrepaasries oonf wsGithC -pdaerpeenntadle nBt Ec2G cMelPls .s yTnhtihse sliosw ienr ebneztwymeeens itnh er esapcotnivsea titoon v aorifo uαs 1/cβo1n caenndtr atCio-nαs1 /oβf1 byrg/
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sGC activity cannot be attributed to a decreased DEA-NO, while the effect of BAY41-2272 was ue
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expression of sGC, since Western blotting showed decreased by about 20% (Fig. 4A and B). We also t o
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no significant decrease in the levels of endogenous found no difference in EC50 values for DEA-NO Ja
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full length α and β subunits (Fig. 3B). The lower or BAY41-2272 between C-α /β and α /β a
1 1 1 1 1 1 ry
cGMP synthesis was observed in all tested BE- enzymes (125±12.2 vs. 108±7.1 nM and 7.8±0.08 2
, 2
N1αF clones (results are not shown), suggesting vs. 7±0.10 μM, respectively). Moreover, both α β 0
1 1 19
that the N1-α sGC splice form acts as an inhibitor and C-α /β heterodimers demonstrated similar
1 1 1
of sGC function. This conclusion was confirmed sensitivity to selective sGC inhibitor ODQ, (Fig.
when the N1-α splice form was co-expressed 4C). Estimated ODQ IC50 was almost identical -
1
together with the full length α and β subunits in 185±5 nM for α /β and 193±11 nM for C-α /β .
1 1 1 1 1 1
Sf9 cells. The addition of increasing amounts of
virus producing N1-α sGC protein correlated with Interaction of the C-α splice variant with β
1 1 1
the decrease of DEA-NO, BAY41-2272- and subunit. To confirm the interaction of the C-α
1
DEA-NO/BAY41-dependent α /β sGC activity sGC with the β sGC subunit we tested for co-
1 1 1
(Fig. S4). The lysates from stable clones immunoprecipitation of the C-α sGC with the β
1 1
overexpressing N2-α sGC did not show sGC subunit from the BE2-CαF clone. As shown
1
significant changes of the NO and/or BAY41- in Figure 5A, polyclonal antibodies raised against
2272- dependent cGMP synthesis. We did not the C-terminus of the β subunit precipitated both
1
observe significant changes of the NO/BAY41- α and C-α variants. This immunoprecipitation
1 1
dependant cGMP production in lysates of the lines correlates with the depletion of the C-α and α
1 1
overexpressing N2-α sGC splice form (results are signals from the post-IP lysates (Fig. 5A).
1
not shown). Moreover, similar intensity of the co-precipitated
signals suggests that both α and C-α
1 1
7
heterodimerize with β equally well. Alternatively, regulating the function of several members of the
1
immunoprecipitation of C-α sGC with anti-FLAG cGMP signaling pathway. For example, a splice-
1
M2 affinity gel precipitated the β sGC subunit dependent deletion of 90-100 N-terminal residues
1
from the lysate of BE2-CαF stable clones, but not of cGMP-dependent protein kinase I (PKGI)
from BE2 lysates (Fig. 5A). The amount of β sGC produces two splice variant isoforms, Iα and Iβ,
1
was higher in the eluates treated with the FLAG which have different tissue distribution and target
peptide, consistent with a FLAG-specific elution. specificity (31). Recent studies also demonstrated
that PKGI splice isoforms respond differently to
Despite a clear inhibitory effect of the N1- activation by hydrogen peroxide (32). Guanylyl
α splice form (Fig. 3B and S4), we were not able Cyclase B (GC-B) is another example with two
1
to observe the co-precipitation of the N1-α sGC truncated splice forms. It was proposed that these
1
with β subunit (Fig S5). splice forms regulate the function of the full length
1
subunit and show different tissue distributions
Level of C-α splice form is not affected by (33).
1
ODQ treatment. A recent report indicated that
heme-deficient sGC or sGC with an oxidized Several reports suggested that splicing
heme prosthetic group is more prone to may also be a method of sGC regulation. For
degradation, which may contribute to endothelial example, the α2i sGC splice variant, carrying an Do
w
dysfunction (29). In those studies, treatment of insertion in the catalytic domain was detected in n
lo
intact cells with sGC inhibitor ODQ decreased the several human tissues (20). The α2i sGC splice ade
level of sGC protein due to ubiquitin-dependent variant has a dominant negative function. In d fro
degradation. Thus, we tested whether the active C- addition, the expression of two mRNA species of m
h
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w
amount of full length α band decreased after BE2- immortalized B-lymphocyte cell lines (24). These w
CαF cells were treated1 for 24 hours with 20 μM α1 sGC splice forms, however, were never isolated .jbc.o
ODQ. The C-α splice band, however, was not or characterized. rg
1 b/
affected by the same treatment. A time-course y
g
study showed that the intensity of the α band In this report, we characterized several ue
1 s
decreased shortly after ODQ administration in all newly identified splice forms of the α subunit of t o
1 n
tested cells, while the level of C-α even slightly human soluble guanylyl cyclase. Analyzing the Ja
1 n
u
increased with time (Fig. 6B). These data suggest abundant data available in the NCBI database, we ary
that the C-α1 subunit protein is more stable to identified a large number of individual α1 sGC 2, 2
intracellular processes occurring after oxidation of transcripts. It is notable that the diversity of 01
9
sGC heme. uncovered α sGC transcripts (12 cDNAs, see
1
Table S1) is much larger than for the β sGC (5
1
cDNAs). This diversity does not appear to be
unique for humans. Analysis of the mouse genome
DISCUSSION database also identified the N1-α1 splice variant
(Acc. N AK031305). Conservation of this
particular splice form point to a functional
Alternative splicing frequently occurs in
importance of the N1-α1 variant in mammals.
eukaryotic genes and provides an important
Most of these transcripts are produced by
mechanism for tissue-specific and developmental
regulation of gene expression. About 15% of sequence rearrangements in 5’ and 3’ untranslated
regions, which are usually associated with altered
mammalian gene mutations associated with
post-transcriptional regulation of gene expression.
pathological conditions affect RNA splicing
For example, binding of RNA stabilizing protein
signals (30).
HuR to the 3’UTR of α sGC plays an important
1
role in post-transcriptional regulation of sGC
A number of previous reports demonstrate
expression in response to cyclic nucleotides and
that alternative splicing is an essential mechanism
during aging in rats (15,34,35). Thus, the diversity
8
of the 5’and 3’UTR sequences, most likely, displays dominant negative properties. BE2 cells
reflects multiple selective mechanism(s) regulating overexpressing N1-α sGC have a significantly
1
the stability of α sGC transcripts in response to decreased NO- and NO/BAY42-2272-induced
1
various extracellular and intracellular stimuli in cGMP production despite the unchanged level of
different cell types or at different developmental endogenous α /β sGC (Fig. 3). Moreover, when
1 1
stages. co-expressed in Sf9 cells with α /β heterodimer,
1 1
N1-α reduced NO- and BAY41-2272-dependent
1
We concentrated our studies on four RNA sGC activity in direct correlation with the
splice variants which encode truncated α sGC due expression level of N1-α protein (Fig. S4). We
1 1
to deletions at the N- or C-termini of the protein were not able to detect any direct
(Table S1, Fig. 1). We found that human heterodimerization of the N1-α protein with the β
1 1
neuroblastoma BE2 cells express three (N1-α , subunit, even when both proteins were co-
1
N2-α , C-α ) out of four alternatively spliced expressed in large quantities in Sf9 cells (Fig. S5).
1 1
RNAs (Fig. S1). Quantitative PCR analysis of the This suggests that the dominant negative effect of
N1-α and N2-α splice mRNAs demonstrates that N1-α is not due to a direct competition with the
1 1 1
the majority of human tissues express more than full length α for the binding with β subunit, but is
1 1
one splice variant, but the levels and the relative rather indirect. Recent deletion mapping showed
ratio of the α and the N1-α and N2-α splice that the segment spanning residue 363-372 of the D
1 1 1 o
w
forms is tissue specific (Fig. 2 and Table S4). For α1 subunit is important for the formation of α1/β1 nlo
example, N2-α1 is found in all tested tissues, while heterodimer (36). Since the N1-α1 splice protein ade
tNes1t-eαs1, sthGyCro iisd ,n polta dceentetcat eadn di ns keesloeptahla gmuus,s cbllea d(Fdeigr,. pcoernhsatiptus,t eist tihnete frifresrte s3 6w3 itrhe sitdhue eh oeft etrhoed iαm1 esruizbautnioitn, d from
h
2). Similarly, C-α1 sGC is detectable in all tested function of the adjacent 363-372 segment of the ttp
human tissues, although the relative ratio was full length α1 subunit. ://w
w
tissue specific (Fig. S2), while C*-α sGC was w
detected only in some tissues (Fig. 1S3). Taken Direct regulation of guanylyl cyclases by .jbc
.o
together, these data indicate that the expression of dominant negative splice variants has been rg
α sGC splice forms is independently regulated. It proposed before. For example, the α sGC splice by/
1 2i g
should be noted, though, that the levels of N1-, variant blocks the formation of a functional α1β1 ues
N2-, C- and C*- α splice mRNAs are on average sGC heterodimer (20), while the truncated GC-B t o
1 n
lower than the full size α sGC. Interestingly, the splice variants hinder the formation of active full Ja
1 n
u
semi-quantitative PCR analysis suggests that in size GC-B homodimers (33). Considering the wide a
ry
human adipose tissue the level C-α and C*-α distribution of the N-α splice form in different 2
1 1 1 , 2
mRNA may be collectively comparable with the human tissues (Fig. 2 and Table S1), modulation 0
1
9
level of α sGC mRNA. Since the comparative of N1-α sGC protein expression may also be a
1 1
analysis reported here is based on RNAs extracted regulatory mechanism controlling the amount of
from entire organs, it might not represent the true active sGC heterodimer.
abundance of individual spliced sGC transcript at
the cellular level. Cellular and subcellular In contrast to N1-α , N2-α sGC had no
1 1
localizations of individual α sGC splice variants effect on sGC activity in cell lysates when over-
1
in human tissues remain to be determined. expressed in BE2 cells or co-expressed with α /β
1 1
Localization may significantly alter the functions in Sf9 cells. However, it cannot be excluded that
of these splice forms beyond the properties this splice variant may serve another function
reported here for BE2 neuroblastoma cells. besides directly affecting sGC activity.
In this report, we identified and Our studies also demonstrate that the C-α
1
characterized N1-α and N2-α sGC splice proteins sGC isoform clearly forms a fully functional sGC
1 1
lacking the catalytic domain (Fig.1). Although by heterodimer with β sGC subunit. The activity of
1
themselves or in combination with β subunit these the recombinant C-α /β enzyme expressed in Sf9
1 1 1
truncated splice α proteins do not have any cells was undistinguishable from the α /β sGC, at
1 1 1
catalytic activity (data not shown), one of them
9
least in regards to the degree of activation by NO while the level of C-α protein does not decrease.
1
donor and allosteric activator BAY 41-2272 and These data suggest that C-α lacks the structural
1
inhibition by ODQ (Fig. 4). The preservation of cues contributing to decreased levels of α after
1
the sGC activity in the BE-CαF stable line (Fig. 3) ODQ treatment. The functional studies presented
and co-immunoprecipitation of C- α sGC and β here prove that the C-α /β heterodimer can fully
1 1 1 1
sGC subunits (Fig. 5) all support the conclusion compensate for sGC activity. In light of reported
that C-α and α subunits are interchangeable. previously decreased level of α subunit in aged or
1 1 1
Also, these results suggest that the lack of 240 N- diseased vessels (41-44), the expression of a more
terminal amino acid residues by α sGC subunit stable C-α may be a specific protective adaptation
1 1
does affect the heterodimerization and enzyme to these conditions. Thus, it is possible that the
activity. This observation does not support positive vasodilatory effects of BAY58-2667 in
previous findings that the region between residues diseased blood vessels observed previously (29)
61-128 of α is mandatory for heterodimerization are mediated by the more stable C-α /β
1 1 1
(37). The properties of this naturally occurring heterodimer. Future studies will show if the C-α
1
splice variant, however, are in agreement with splice form may be, under certain circumstances,
earlier reports which show that a significant the main or the sole type of α subunit.
1
portion of the N-terminal region of the α sGC
1
subunit can be deleted without affecting NO- Although the functional studies suggest D
o
w
sensitivity and heterodimerization (36,38,39). that the N-terminal fragment missing in the C-α n
1 lo
subunit is not important for the activity of sGC, ad
e
The existence of α1-positive bands other this region is preserved in evolution in the α1 d fro
than the 83 kDa full length α sGC subunit was subunits of all vertebrates. It is possible that this m
1 h
mentioned in several previous reports. While region, often referred to as the regulatory region ttp
screening selected human tissues with anti-α1 (37,38), is responsible for the integration of ://w
w
antibodies, Zabel and colleagues observed in NO/cGMP signaling with other regulatory w
cortex, cerebellum and lungs a band similar in size pathways. In this case, despite similar catalytic .jbc
.o
w(4i0th). Itnhtee r~e5st4in gklDya, thCe- αa1n tdibeosdcrieibse uds eidn wtheirse rraeipsoerdt pmroodpuelratiteesd, α1/dβi1f faenrden Ctl-yα 1/β1b yh eterNodOim-inedrse pmeanyd ebnet byrg/
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against the same epitope as in current studies. mechanisms, e.g. protein modifications or protein- ue
s
Moreover, additional bands were also detected in protein interactions. t o
n
crude lysates from human amygdale (41). Ja
n
u
Presence of several bands with electrophoretic cGMP-dependent kinase is one of the ary
mobility similar to C-α1 indicates that either C-α1 major effectors of cGMP generated by sGC in 2, 2
form may undergo additional tissue-specific response to NO. Increased cGMP levels in 01
9
processing and/or modifications, or that additional, neuronal cells leads to a PKGI-dependent
yet unidentified, α sGC splice forms exist. phosphorylation of the Splicing Factor 1 (SF1),
1
which functions at early stages of pre-mRNA
Recent studies suggested that some splicing and splice site recognition (45). Thus, it is
conditions of endothelial dysfunction may be appealing that sGC-dependent cGMP production
associated with the accumulation of oxidized and may regulate sGC activity by affecting the
heme-free sGC leading to a poor response to NO spliceosome assembly and/or switching between
(29). The same report showed that in ODQ-treated different splice sites during pre-mRNA processing.
cells sGC is subjected to ubiquitination and This, in turn, may affect the diversity of expressed
subsequent degradation. Although our current α sGC splice proteins modulating the function of
1
studies showed that C-α /β and α /β heterodimers the sGC heterodimer.
1 1 1 1
have identical sensitivity to ODQ with a similar
IC (Fig. 4), C-α and α sGC subunits show an In summary, our present study identifies
50 1 1
opposite response to ODQ-induced degradation and characterizes several new splice variants of
(Fig. 5). Western blotting confirms that the 83 kDa the human α subunit of sGC. The splicing of α
1 1
α sGC band disappears after exposure to ODQ, mRNA seems to be ubiquitous and follows the
1
10
domain organization of the α sGC subunit. Our significant effects on nitric oxide and cyclic GMP
1
findings point to new mechanisms of modulation signaling and perhaps pathophysiology.
of sGC function and activity that may have

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