Functional characterization of alternatively spliced human SERCA3 transcripts

Esteban Poch1, Stephen Leach2, Susan Snape2, Tasha Cacic2, David H. MacLennan3, and Jonathan Lytton1,2

1 Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; 2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1; and 3 Banting and Best Department for Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

molecular cloning; calcium-adenosinetriphosphatase; Jurkat T cells; kidney

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

INTRACELLULAR ORGANELLES play a critical role in the regulation of free intracellular Ca2+ concentration. Ca2+ is stored in the sarcoplasmic reticulum in striated muscle, but the endoplasmic reticulum is the major Ca2+ storage and regulation site in smooth muscle and nonmuscle tissues. Functional experiments suggest that at least two distinct Ca2+ storage pools exist in smooth muscle and nonmuscle cells, one sensitive to the second messenger inositol-1,4,5-trisphosphate and the other sensitive to caffeine and Ca2+. The release of Ca2+ from these stores following cell stimulation activates Ca2+ signaling pathways, and return of Ca2+ to the stores terminates signaling (5, 8, 26).

Ca2+ uptake into the cellular stores is mediated via an ATP-dependent Ca2+ pump. Molecular cloning has defined a family of sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pumps that are encoded by three separate genes. SERCA1 is exclusively expressed in fast-twitch skeletal muscles as two alternatively spliced transcripts, SERCA1a and SERCA1b, that are developmentally regulated (6). Mutations in the SERCA1 gene, ATP2A1, have been linked to Brody's disease, a rare inherited disorder of skeletal muscle function (23). SERCA2, originally cloned from neonatal muscle, has been demonstrated to be expressed in both adult slow-twitch skeletal muscle and cardiac muscle (6). Like SERCA1, SERCA2 is alternatively spliced in the region encoding the carboxy terminus and produces two protein products. SERCA2a terminates in the sequence -Ala-Ile-Leu-Glu and is the major species expressed in striated muscle. In the protein encoded by SERCA2b, the four terminal amino acids of SERCA2a are replaced by an extended sequence of 49 amino acids. SERCA2b is expressed in smooth muscle and most nonmuscle tissues and appears to represent a generic endoplasmic reticulum form of Ca2+-ATPase (11, 19, 22, 34).

A third gene product, SERCA3, has been identified that is expressed in a variety of nonmuscle tissues, mostly in lymphoid cells and platelets, endothelial cells, intestinal epithelial cells, and cerebellar Purkinje neurons (1, 3, 34, 36, 37). SERCA3 encodes a protein ~75% identical to either SERCA1 or SERCA2, the differences being clustered at the amino terminus, in regions of the nucleotide-binding domain, and at the carboxy terminus (7). By contrast, ~85% identity exists between SERCA1a and SERCA2a. Analysis of different tissues has indicated that SERCA3 is always coexpressed along with SERCA2b, usually considered to be the housekeeping isoform (34, 36). It is not known, however, whether these SERCAs are located in physically and/or functionally distinct intracellular Ca2+ pools. Expression studies in COS cells have demonstrated that rat SERCA3 exhibits functional properties distinct from the other members of the SERCA family. These include a lower apparent affinity for Ca2+, an altered pH optimum, and a higher apparent affinity for vanadate inhibition, all of which are proposed to arise from an alteration of the conformational equilibrium of the enzyme (21).

Endothelial cells from mice lacking functional SERCA3 have a reduced Ca2+ transient and nitric oxide production in response to acetylcholine stimulation, compared with their wild-type counterparts (18). Alterations in the expression of SERCA3 have also been implicated in T cell activation, as well as in the pathogenesis of both hypertension and diabetes (14, 17, 27, 32).

The molecular cloning of human SERCA3 was described in a recent report (9). Several critical questions, however, remain unanswered. First, no functional data on human SERCA3 were reported. As rat SERCA3 has unique properties, it is important to establish whether these are also present in the human enzyme. Second, the subcellular localization of human SERCA3 has not been established. Finally, the monoclonal antibody PL/IM430 did not recognize the cloned human SERCA3 in previous studies (9). This is a critical issue, since PL/IM430 has been used extensively to characterize SERCA protein expression (3, 24, 37). In the present paper, we report the molecular cloning of alternatively spliced human SERCA3 transcripts that differ at their 3' ends and characterize the expression and function of the encoded protein products. In addition, direct evidence is provided that PL/IM430 does, indeed, recognize human SERCA3.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

All molecular biology procedures were conducted essentially according to published protocols (2, 28) unless otherwise indicated. Chemicals were of the highest available molecular biology grade from Fisher, BDH, or Sigma or as listed.

Cloning and expression constructs. A human kidney cDNA library was screened at low stringency with fragments of rabbit SERCA1 and SERCA2 clones as described previously (19). A single clone, HK3, was obtained that had high homology to rat SERCA3 (7). A Jurkat T cell cDNA library (Stratagene) was screened at high stringency as described previously (35) with two probes from HK3 corresponding to nucleotide positions 1600-1952 and 2311-2738 (these and all subsequent cDNA coordinates use the A of the AUG initiator codon as +1). This screen resulted in the isolation of 14 separate clones, all partial length. One of these Jurkat T cell clones contained a poly(A) tail, five contained the initiating methionine, and the remainder were partial length at each end. The 5'-end Jurkat T cell clone, J12.1, was combined separately with three different 3'-end clones (Jurkat T cell clones J7.2 and J23 and human kidney clone HK3) using a unique central Sph I site (1793-1798) to yield full-length constructs used for expression experiments: huS3-I, huS3-II, and huS3-IV, respectively (see Fig. 1). A variant of huS3-II was also constructed using a unique Kpn I site (1156-1161) to combine clones J12.1 and J23. The two huS3-II constructs differed by four silent substitutions and had identical function when expressed in HEK-293 cells. Constructs huS3-I and -II (Kpn I variant) were sequenced in their entirety, whereas construct huS3-IV was sequenced downstream from the Sph I site only. Construct huS3-I was identical to the published human SERCA3 cDNA sequence (Ref. 9; GenBank no. Z69881), except for an additional 118 nucleotides of 5' untranslated region and a C in place of a T at nucleotide 2103. Construct huS3-II differed from construct huS3-I as follows: T for G at 1272, T for C at 1302, C for T at 1359, T for C at 1518, A for G at 2017, T for C at 2103, C for T at 2400, G for A at 2451, deletion of nucleotides 2981-2983, and A for G at 3014. Construct huS3-II is truncated at 3076. As illustrated (see Fig. 1), many of these substitutions are silent, but three result in changes to the amino acid sequence. Construct huS3-IV, obtained from a human kidney clone, shares with construct huS3-II all of the differences from construct huS3-I downstream from the Sph I site (1793-1798), except for the change of A for G at 2017. In addition, construct huS3-IV has a 101-nucleotide exon inserted following nucleotide 2980, a deletion of 594 nucleotides in the 3' untranslated region from 3027 to 3620, and a G for C at 3630. As a result of the 101-nucleotide exon insertion, the reading frame is altered near the carboxy terminus, and the final 6 amino acids are replaced by 36 relatively hydrophilic amino acids (see Fig. 1). Construct huS3-V was derived from construct huS3-I by deletion of the region from a Sma I site at 3152-3157 to the poly(A) tail.

All constructs were subcloned as single EcoR I fragments into the mammalian expression vector pMT2 (15).

Nucleic acid sequencing. Clones and subcloned fragments of cDNA ligated into pBluescript II (Stratagene) were sequenced either with the Sequenase 2.0 kit (United States Biochemical) and deoxyadenosine 5'-[alpha -35S]thiotriphosphate or with the AmpliTaq FS kit from Perkin-Elmer. Fluorescent dye-terminator labeled sequencing reactions were analyzed at the University of Calgary DNA Services Facility on an Applied Biosystems model 373 automated sequencer.

Northern blot hybridization analysis. Multiple-tissue Northern blots, each lane containing 2 µg of poly(A)+ RNA from various human tissues, were purchased from Clontech Laboratories. The blots were hybridized at high stringency with a digoxigenin-labeled antisense riboprobe corresponding to nucleotides 1600-1952 of the human SERCA3 clone, HK3, essentially according to the directions of the label manufacturer (Boehringer Mannheim) and as previously described (35).

PCR amplification. The expression of alternatively spliced SERCA3 transcripts was analyzed using PCR and RT-PCR. First-strand cDNA was obtained from oligo(dT)-primed Jurkat T cell total RNA using Superscript II RT (Life Technologies). Control reactions were run in parallel but in the absence of the enzyme. cDNA from human tissues was obtained from Clontech as Marathon-Ready kits. PCR was conducted across the region of alternative splicing (see Fig. 3B). Amplifications used the combinations of primer b (sense; CCAGGTGACCCCACTGAG) and primer d (antisense; GCCAGGCTTTCCCGACTTG), resulting in products whose length is diagnostic for the pattern of exon usage, or a nested scheme using primer a (sense; CCACCATGGCCTTGTCCG) and primer d followed by primer a and primer c (antisense; CTGGGGTCCAAGAGGTGG; located within the sequence of the extra 101-nucleotide exon of clone HK3), with which the presence of a product is diagnostic for the inclusion of the extra 101-nucleotide exon present in clone HK3.

Functional expression in cultured cells. HEK-293 cells were maintained in DMEM and transfected using Qiagen-purified plasmids and a standard calcium phosphate precipitation protocol. Microsome preparation, immunoblotting, 45CaCl2 uptake, and Ca2+-dependent ATPase assays were all performed essentially as described previously (21, 31). In brief, 2 days after transfection, cells were harvested, swollen in hypotonic medium, and disrupted with a Dounce homogenizer, and a crude, postmitochondrial, particulate fraction ("microsomes") was isolated by differential centrifugation. For immunoblotting, 10-µg samples of microsomes were separated on 7% polyacrylamide-SDS gels and electrotransferred to nitrocellulose membranes. The membranes were blocked in PBS containing 5% nonfat dried milk and 0.1% Tween 20 and then incubated for 2 h at room temperature with primary antibodies diluted in PBS containing 1% milk and 0.1% Tween 20. Polyclonal antibody C4, raised against recombinant rabbit SERCA2, has been described previously (19, 21) and was used at 1:500 dilution. Monoclonal antibody PL/IM430 (Research Diagnostics), raised against platelet internal membranes (12), was used at 2 µg/ml. Polyclonal antibody PA1-910, directed against the rat SERCA3 peptide Val-Thr-Asp-Ala-Arg-Glu-Arg-Tyr-Gly-Pro-Asn-39 (the equivalent human SERCA sequence is Val-Thr-Gly-Ala-Arg-Glu-Arg-Tyr-Gly-Pro-Asn-39), was a generous gift from Phillip Schwartz (Affinity BioReagents) and was used at 1:100 dilution. After incubation with primary antibody, the blots were washed in PBS-0.1% Tween 20, incubated in horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories), washed again, and developed by enhanced chemiluminescence using the Pierce Supersignal substrates. Ca2+ uptake was assayed at room temperature by adding microsomes to (in mM) 120 KCl, 3 ATP, 3 MgCl2, 0.5 EGTA, 0.45 45CaCl2 (~1 µCi/ml), 5 potassium oxalate, and 25 MOPS-KOH (pH 7.0). Reactions were terminated by dilution into ice-cold 0.15 M KCl-1 mM LaCl3 and filtered through 0.3-µm nitrocellulose filters (Millipore). Ca2+-ATPase measurements were performed using an enzyme-coupled method in a Beckman DU-640 spectrophotometer at 30°C. Microsomes were diluted into (in mM) 120 KCl, 2 MgCl2, 1 ATP, 1.5 phosphoenolpyruvate, 1 dithiothreitol, and 25 MOPS-KOH (pH 7.0) containing ~10 U/ml each of pyruvate kinase and lactate dehydrogenase, 0.32 mM NADH, and 2 µM ionophore A-23187. They were then divided into cuvettes containing various concentrations of free Ca2+ generated using an EGTA buffer, taking into account temperature, ionic strength, and pH, essentially as described (13). The decrease in absorbance at 340 nm with time was recorded.

The data for the Ca2+-dependence of ATPase activity were fitted using an equation describing a three-component kinetic model that assumes two independent contributions to the increase in activity (corresponding to 2 ATPases with different apparent Ca2+ affinities: the endogenous HEK-293 cell SERCA2b and the transfected enzyme) and a third component due to Ca2+ inhibition of activity of the enzyme ("back" inhibition, presumably at the luminal release site)
<IT>V</IT> = <FR><NU>A[Ca]<SUP><IT>n</IT></SUP></NU><DE>(<IT>K </IT><SUP><IT>n</IT></SUP><SUB>A</SUB> + [Ca]<SUP><IT>n</IT></SUP>)</DE></FR> + <FR><NU>B[Ca]<SUP><IT>n</IT></SUP></NU><DE>(<IT>K </IT><SUP><IT>n</IT></SUP><SUB>B</SUB> + [Ca]<SUP><IT>n</IT></SUP>)</DE></FR> − <FR><NU>(A + B) [Ca]</NU><DE>(<IT>K</IT><SUB>I</SUB> + [Ca])</DE></FR>
where A and B are the activities (as %) of endogenous and transfected enzymes, respectively, KA, KB, and KI are constants for endogenous and transfected activity and for back inhibition, respectively, n is the Hill coefficient, and [Ca2+] is free Ca2+ concentration. Note that if the decrease in ATPase activity for free Ca2+ concentration >20 µM is ignored, then all the data except for those of construct huS3-I can be adequately fit using a single cooperative Michaelis-Menten term. This is in agreement with the observation that only huS3-I activity is contaminated with a significant level of endogenous SERCA activity. Nevertheless, for consistency, each data set was fitted using the three-component equation. Curve fitting was performed using a quasi-Newton (Davidon-Fletcher-Powell algorithm) least-squares minimization regression method with MacCurveFit from Kevin Raner Software (http://www.home.aone.net.au/krs).

Several assumptions were incorporated in the generation of curves (see Fig. 5A). The fits suggest that the true maximum activity is closer to 105% than 100%, since when activity reaches a measured "100%" value, there is already a small amount of back inhibition. The values for A (endogenous enzyme) and B (transfected enzyme) were set on the basis of the observed increase in activity over control (see Fig. 5B). These values were A = 15% and B = 90% for rat SERCA1a, A = 25% and B = 80% for human SERCA3-I, and A = 5% and B = 100% for the remaining SERCA3 fits. In all cases n was set at 1.5, which corresponds to partial cooperativity between the two Ca2+ binding sites, as seen typically in previous reports (21, 30). The derived KA value (for endogenous SERCA2b) was 2 × 10-7 M, the KB values were 6 × 10-7 M for SERCA1 and 1.8 × 10-6 M for all SERCA3 constructs, and the KI values were 5.8 ×10-4 M for SERCA3 and 1.8 × 10-4 M for SERCA1. Note that these values correspond closely to previously measured values (21).

Immunofluorescence. For immunofluorescence measurements, HEK-293 cells were grown on protamine-coated glass coverslips. Jurkat T cells were cultured in RPMI 1640 medium and were allowed to settle onto similarly coated coverslips. The cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with monoclonal antibodies IID8 (Affinity BioReagents) or PL/IM430 (Research Diagnostics), followed by a rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories), essentially as described previously (21). Control experiments, either omitting the primary antibody or in untransfected HEK-293 cells (in the case of PL/IM430), resulted in a fluorescence signal not different from the background. The IID8 antibody detects the endogenous SERCA2b enzyme in untransfected HEK-293 cells, which results in a fluorescence signal at least 10-fold lower than in transfected cells, consistent with activity measurements (data not shown). Images were captured and digitally deconvoluted using Vaytek Micro-tome software in the University of Calgary Microscopy and Imaging Facility.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characterization of cDNAs encoding human SERCA3. In initial studies, we isolated a single partial-length clone, HK3, encoding human SERCA3 from a human kidney cDNA library. Exhaustive further screening of the kidney library revealed only shorter clones. Because human Jurkat T cells were reported to express SERCA3 (37), probes derived from HK3 were used to screen a human Jurkat T cell cDNA library. Fourteen distinct overlapping clones encoding the cDNA of human SERCA3 were obtained. Four of these extended past the initiating methionine at the 5' end, whereas five extended past the carboxy terminus of the protein, and the remainder were partial length at both ends. Notably, only HK3 and one clone from the Jurkat library (J7.2) contained a poly(A) tail. The 5'-end clone, J12.1, was combined using a central Sph I site with the three 3'-end clones, J7.2, J23, and HK3, creating three full-length constructs, huS3-I, huS3-II, and huS3-IV, respectively (construct huS3-III, derived from a partially processed mRNA, encoded a truncated protein that was nonfunctional and was not analyzed further). A schematic of the clones, and the sequence of their encoded proteins, is shown in Fig. 1.


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Fig. 1.   Human sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA)-3 clones. A: diagram of clones used to prepare full-length human SERCA3 expression constructs (bottom) and schematic of full-length transcript (top). Top: open area, SERCA3 coding region; shaded area, position of extra sequence present in HK3 clone. Positions of restriction sites used for construction of full-length expression clones are indicated. Bottom: J series of clones was isolated from a Jurkat T cell cDNA library, and HK3 was isolated from a human kidney library. Open areas aligned with vertical bars, segments missing in a given clone with respect to others. B: deduced amino acid sequence for huS3-II (clones J12.1 and J23 combined at either Kpn I or Sph I sites) is shown in single-letter code. Differences between this sequence and that of huS3-I (clones J12.1 and J7.2 combined at Sph I site) and huS3-IV (clones J12.1 and HK3 combined at Sph I site) are shown below huS3-II sequence. Positions of 10 hydrophobic transmembrane spans are underlined and labeled (M1-M10). See MATERIALS AND METHODS for further details. These sequences have been deposited in GenBank database (nos. AF068220 and AF068221).

Construct huS3-I is 4671 nucleotides in length and contains a poly(A) tail. This molecule is identical to the published human SERCA3 cDNA sequence (Ref. 9; GenBank no. Z69881) except for an additional 118 nucleotides of 5' untranslated region and a silent nucleotide change, T in place of C at position 2017. Construct huS3-II, on the other hand, contains several nucleotide substitutions that result in three amino acid changes, Thr for Ala-673, Met for Ile-817, and the deletion of Glu-994. In comparison to huS3-I, huS3-II is truncated at its 3' end, whereas construct huS3-IV, derived from the HK3 (human kidney) clone, is very similar to huS3-I, including a poly(A) tail at the same site. huS3-IV contains essentially all of the changes found in huS3-II (see MATERIALS AND METHODS) and, in addition, possesses an insertion of 101 nucleotides following position 2980 and a deletion of 594 nucleotides from the 3' untranslated region. The extra 101-nucleotide sequence of huS3-IV can be found in the human SERCA3 gene (GenBank no. Z69880) and presumably represents an alternatively spliced exon. This results in a shift of the reading frame near the carboxy terminus of the protein, so that the final 5 or 6 amino acids are replaced by an extended, relatively hydrophilic, 36-amino acid sequence, as depicted in Fig. 1.

Northern blot hybridization analysis. The human SERCA3 mRNA tissue distribution was analyzed by Northern blot hybridization with a coding region probe, as illustrated in Fig. 2. A major ~5-kb transcript was detected, with the highest level of expression in thymus, colon, pancreas, and spleen. Intermediate levels of expression were observed in prostate, small intestine, and leukocytes, and the lowest level of expression was observed in lung, testis, kidney, brain, placenta, ovary, and liver. The probe detected a 3.8-kb mRNA in skeletal muscle, likely representing cross-hybridization with very high levels of SERCA1 mRNA. A 4.4-kb transcript was also detected in both skeletal muscle and heart, but not in the other tissues, that most likely represents cross-hybridization with high levels of SERCA2 mRNA. These strong cross-hybridization signals in heart and skeletal muscle made it impossible to determine whether SERCA3 was also present in these tissues. Additional bands at ~6.5, 8, and 9 kb were evident in thymus mRNA, and, at longer exposures, bands of 3.8 and 4.4 kb were also visible. All of these bands were observed at longer exposure in mRNA from spleen, leukocytes, and intestine but were absent from pancreas. Because the appearance of the minor bands correlated with the intensity of the major SERCA3 species at 5 kb (except in pancreas) and not with the reported pattern of expression of the other SERCA isoforms (34), it is likely that these bands correspond to alternatively spliced SERCA3 species.


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Fig. 2.   Distribution of SERCA3 transcripts in human tissues. A multiple-tissue Northern blot containing 2 µg poly(A)+ mRNA/lane from indicated tissues was hybridized at high stringency with a SERCA3 coding region riboprobe. Prominent bands in skeletal muscle and heart of 3.8 and 4.4 kb represent cross-hybridization with the very large amount of SERCA1 and SERCA2 transcripts present in these tissues. Note that smearing of signal in leukocyte lane suggests that this sample was partially degraded. See MATERIALS AND METHODS for further details.

Alternative splicing of SERCA3 transcripts. On the basis of the differences observed among our various human SERCA3 clones and the pattern observed by Northern blot analysis, we examined the structure of SERCA3 transcripts by PCR using primers that flank the two sites of deletion/insertion (see Fig. 1). As illustrated in Fig. 3A, four ethidium bromide-stained product bands are observed for Jurkat T cell RNA and, less clearly, for thymus and bone marrow samples. These fragments correspond in size to all four possible combinations of inclusion and exclusion of these two regions, as illustrated in Fig. 3B (see also Fig. 1, for comparison). The major band of 850 nucleotides, denoted band 2, observed in all lanes except the control, corresponds in size to the product expected from a transcript with the structure of huS3-I (clone J7.2). A faint band at 951 nucleotides, band 1, observed in bone marrow, thymus, and Jurkat T cell lanes, is consistent with the product expected from an huS3-I-like transcript also containing the 101 extra nucleotides found in huS3-IV (clone HK3). Band 3, evident at 357 nucleotides in Jurkat T cell, thymus, bone marrow, and kidney lanes, corresponds in size to the expected product from a transcript with the structure of huS3-IV. Finally, band 4, at 256 nucleotides, is consistent with the expected product from a transcript similar to huS3-IV but lacking the 101-nucleotide exon. The identity of bands 2 and 4 was confirmed by restriction enzyme digestion with Sma I and Rsa I (data not shown). A similar analysis on bands 1 and 3 was not performed due to the limited amount of pure material we could isolate.


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Fig. 3.   A: alternative splicing of human SERCA3 transcripts. cDNA obtained from Jurkat T cell RNA by reverse transcription (Jurkat RT) with (+) or without (-) enzyme or purchased from Clontech (heart, brain, kidney, bone marrow, and thymus) was amplified either with primers b and d (top) or using a nested reaction with primers a and d, followed by primers a and c (bottom). Ethidium bromide-stained gels of products are shown; size markers, in nucleotides, are indicated at left. Bands discussed in text (bands 1-5) are indicated by numbers at right. B: schematic of SERCA3 cDNA, with position and polarity of primers indicated by arrowheads. Structure of numbered bands is also shown in an expanded view, with vertical bars denoting splice sites and bridged gaps indicating regions absent as a consequence of alternative splicing. See MATERIALS AND METHODS for further explanation.

To confirm that the 101-nucleotide exon of huS3-IV was present in some SERCA3 transcripts, we performed a nested PCR reaction, first with primers a and d (flanking the sites of alternative splicing) and then with primers a and c; primer c lies within the 101-nucleotide exon. As illustrated in Fig. 3A, bottom, a product (band 5) was observed in reactions from all tissues except heart (which, based on the intensity of the bands in Fig. 3A, top, expressed only very low levels of SERCA3). The identity of band 5 was confirmed by the predicted pattern of products obtained following digestion with the restriction enzymes Stu I and Nco I (data not shown). Together, these data suggest that several tissues express transcripts that contain the extra 101-nucleotide exon of clone HK3 and encode a SERCA3 protein with an extended carboxy terminus (see Fig. 1).

Expression of human SERCA3 constructs in HEK-293 cells. Human SERCA3 constructs, huS3-I, huS3-II, and huS3-IV, were expressed by transient transfection in HEK-293 cells. As illustrated in Fig. 4A, ATP-dependent and thapsigargin-sensitive Ca2+ uptake by microsomes from cells transfected with huS3-II was observed. The magnitude of uptake was similar to that observed for microsomes from rat SERCA3- and SERCA1a-transfected cells and was at least 20-fold greater than that of control-transfected microsomes. By contrast, Ca2+ uptake by microsomes from huS3-I-transfected cells was almost 10-fold lower than huS3-II uptake but was still ~3-fold greater than the control value. The difference in activity between huS3-I and huS3-II was confirmed by measuring Ca2+-dependent ATPase activity, as shown in Fig. 5.


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Fig. 4.   Functional expression of human SERCA3 isoforms in HEK-293 cells. A: oxalate-dependent 45Ca2+-uptake into microsomes isolated from HEK-293 cells transfected with constructs encoding control (vector plasmid only; ×), rat SERCA1a (open circle ), rat SERCA3 (triangle ), human SERCA3-I (black-diamond ), or human SERCA3-II (black-triangle). Uptake into thapsigargin-treated (0.2 µM) microsomes from human SERCA3-II transfected cells is also shown (black-down-triangle ). Note that these data were obtained from different microsome preparations than those used for B or Fig. 5. B: immunoblot of 10-µg samples of microsomes isolated from cells transfected with indicated constructs. Blots were incubated with either polyclonal anti-SERCA antibody C4, monoclonal antibody PL/IM430 raised against human platelet membranes, or Affinity BioReagents polyclonal anti-SERCA3 antibody 910. Approximate positions of prestained size markers (New England Biolabs) are shown at left. See MATERIALS AND METHODS for more details.


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Fig. 5.   Ca2+-dependent ATPase activity of human SERCA3 isoforms. A: Ca2+ dose-response curves generated with EGTA buffer are shown for microsomes from HEK-293 cells transfected with constructs encoding rat SERCA1a (open circle ), rat SERCA3 (triangle ), human SERCA3-I (black-diamond ), human SERCA3-II (black-triangle), or human SERCA3-IV (sideways triangles). As described in MATERIALS AND METHODS, curves correspond to a multi-component cooperative model that accounts for activity of endogenous SERCA2b [concentration producing half-maximal effect (K1/2) = 0.2 µM] and of transfected enzyme. In all cases, Hill coefficient was set to 1.5. K1/2 for rat SERCA1a = 0.6 µM, and K1/2 for all SERCA3 constructs = 1.8 µM. Error bars are SE for 3-5 determinations; SE smaller than symbols are not visible. B: maximal ATPase rates, at a free Ca2+ concentration of ~5 µM, are shown for microsomes isolated in parallel from HEK-293 cells transfected with indicated constructs. In all cases, error bars are SE for 3-15 determinations on at least 3 different microsomal preparations. Note that data were derived from different preparations of microsomes than those used for Ca2+ uptake experiments of Fig. 4.

The level of SERCA protein expression was evaluated by immunoblotting using the polyclonal antibody C4, which recognizes all of the known SERCA gene products (21), the monoclonal antibody PL/IM430, thought to recognize human but not rat SERCA3 (4), and a new anti-peptide SERCA3 antibody, PA1-910 (Fig. 4B). Strong SERCA protein expression from all the transfected clones was revealed by C4 antibody staining. PL/IM430 recognized exclusively the expressed human SERCA3 proteins. The 910 antibody selectively recognized both rat and human SERCA3, albeit with different sensitivity. This is most likely due to the amino acid difference between human SERCA3 and the rat peptide used to raise the antibody (see MATERIALS AND METHODS). Based on these immunoblot data, it is clear that the level of expression of huS3-I was not significantly different from huS3-II, despite the 10-fold difference in their activities.

Although the difference in the deduced protein sequence of huS3-I and huS3-II is restricted to 3 amino acids, huS3-I possesses 1471 nucleotides of extra 3' untranslated sequence. Because the structure of our clones also suggested that this region was subject to alternative splicing, we were concerned that the long untranslated region might undergo aberrant splicing in the transfected HEK-293 cells, leading to an artificially altered protein. Thus we created the construct huS3-V, in which all but 81 nucleotides of the extra sequence were removed. The huS3-V protein was expressed at a level similar to huS3-II (Fig. 4B) but had very low ATPase activity, comparable to huS3-I (Fig. 5B). Analysis by immunofluorescence of the subcellular distribution of huS3-I and huS3-II expressed in transfected HEK-293 cells indicated that these molecules were present in the same compartment, identified as the endoplasmic reticulum (Fig. 6). Thus the difference in activity between the proteins encoded by huS3-I and huS3-II can be explained neither by the long 3' untranslated region nor by expression of the proteins in different subcellular compartments.


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Fig. 6.   Subcellular localization of SERCA isoforms in HEK-293 cells and Jurkat T cells. Digitally deconvoluted images obtained from planes at middle (Mid) and close to bottom (Bottom) of cell are shown for Jurkat T cells (2 rows at bottom) and for HEK-293 cells (2 rows at top) transfected with constructs encoding human SERCA2b (huS2B), human SERCA3-I (huS3-I), or human SERCA3-II (huS3-II). Protein expression was observed on Triton-permeabilized paraformaldehyde-fixed cells using monoclonal antibody IID8 (recognizes SERCA2; left) or PL/IM430 (recognizes SERCA3; middle and right) and a rhodamine-conjugated secondary antibody. See MATERIALS AND METHODS for further details.

The alternatively spliced SERCA3 construct, huS3-IV, encodes a protein with a carboxy-terminal extension of 30 amino acids. Expression of huS3-IV in HEK-293 cells results in a protein of slightly larger size (Fig. 4B), possessing activity essentially indistinguishable from that of huS3-II with respect to both magnitude and apparent Ca2+ affinity (Fig. 5). Indeed, as illustrated in Fig. 5, when the component of activity due to endogenous HEK-293 cell SERCA2b was accounted for, we calculated that all of the human SERCA3 constructs coded for proteins whose Ca2+ dependence matched that observed for rat SERCA3 [concentration producing half-maximal effect (K1/2) = 1.8 µM] but was significantly different from the Ca2+ dependence of rat SERCA1a (K1/2 = 0.6 µM).

Subcellular localization of human SERCA3 in Jurkat T cells and in transfected HEK-293 cells. Recent experiments suggested that SERCA3 is associated with a functionally distinct Ca2+ storage compartment in endothelial cells (18). Little is known, however, regarding the subcellular distribution of SERCA3, especially in comparison to SERCA2b, with which it is always coexpressed (34, 36, 37). We examined the intracellular location of SERCA2b and SERCA3, both in Jurkat T cells endogenously expressing these two gene products and in transfected HEK-293 cells. Figure 6 shows immunofluorescence micrographs of planes through the middle of and close to the bottom of selected transfected HEK-293 cells (2 rows at top) and Jurkat T cells (2 rows at bottom). The distributions of SERCA2b and SERCA3 were indistinguishable, both for the transfected HEK-293 cell protein and for the endogenous Jurkat T cell protein. Staining of the nuclear envelope and a prominent reticular pattern throughout the cytoplasm can be appreciated, consistent with an endoplasmic reticulum distribution for both proteins.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have isolated and characterized several cDNA clones encoding alternatively spliced transcripts of human SERCA3. Robust SERCA3 expression was restricted to a distinct set of tissues, including lymphoid tissues, intestinal epithelia, pancreas, and prostate. The most abundant SERCA3 species expressed corresponds in exon structure to clone huS3-I. A second alternatively spliced species, huS3-IV, which was identified from a human kidney library, possesses an extra 101-nucleotide exon near the carboxy terminus of the protein, as well as a 594 nucleotide deletion from the 3' untranslated region. As a consequence of the extra exon, the encoded protein has an extended carboxy-terminal tail of 30 amino acids. Transcripts for this form were present in low abundance in most tissues but, based on the data of Fig. 3A, appear to represent a higher proportion of total SERCA3 transcripts in kidney (although overall SERCA3 mRNA expression is still quite low in this tissue).

It is noteworthy that all three known SERCA genes have now been shown to be alternatively spliced at a very similar position, resulting in transcripts that code for protein products that differ in their carboxy-terminal sequences. The mechanism used to generate the alternatively spliced products is different for the different genes, however. As demonstrated in this paper, the SERCA3 gene, ATP2A3, utilizes a cassette-type exon mechanism. When this exon is absent, the transcript codes for a protein that terminates in the sequence Glu-Glu-Met-Ser-Gln-Lys. When it is present, the encoded protein ends in a long hydrophilic sequence of 36 amino acids. In the SERCA1 gene, ATP2A1, a similar cassette-type mechanism operates. In that case, an exon that codes for a carboxy-terminal Gly residue in adult muscle is excised in neonatal muscle, resulting in a protein that terminates in the highly charged sequence Asp-Pro-Glu-Asp-Glu-Arg-Arg-Lys (6). In the SERCA2 gene, ATP2A2, on the other hand, either a donor splice site is used, in which case the encoded protein ends in Pro-Ala-Ile-Leu-Glu, or the splice site remains cryptic, in which case the carboxy terminus of the protein is extended by a hydrophobic sequence of 49 amino acids (19).

The monoclonal antibody PL/IM430, raised to highly purified platelet intracellular membranes (12), had been shown to recognize a 97-kDa protein, presumed to be a Ca2+ pump, in platelet membranes as well as in various cell lines (4, 37). In the last few years, however, there has been controversy concerning whether PL/IM430 recognizes human SERCA3 or a novel Ca2+ pump (16). Indeed, a recent paper even suggested that cloned human SERCA3, when expressed in COS-1 cells, was not recognized by PL/IM430 (9). This result has cast doubt on earlier data describing the expression pattern of human SERCA3, which was documented largely with PL/IM430. Our data showing expression of full-length clones in HEK-293 cells, however, clearly demonstrate that the antibody PL/IM430 recognizes the human SERCA3 protein, which as predicted runs with a mobility corresponding to ~97 kDa on SDS-PAGE gels. It is unclear exactly why the previous investigators were unable to see immunoreactivity and we were, but this may reflect the levels of protein expression in the different systems.

Our studies in HEK-293 cells are the first to characterize the function of cloned human SERCA3. The protein displayed a maximal capacity for Ca2+ transport similar to that of other SERCA enzymes but an apparent affinity for Ca2+ that is ~0.5 pCa unit lower than that of SERCA1 or SERCA2. We were unable to document any functional difference between the two alternatively spliced human SERCA3 isoforms. In contrast, the two protein products that arise from alternatively spliced SERCA2 gene, which also differ at their carboxy termini, display differences in maximal transport capacity and apparent affinity for Ca2+ (21, 33).

Functional expression of human SERCA3 reported here has confirmed the unusual pattern of tissue distribution and the different biochemical characteristics previously described for rat SERCA3 (7, 21, 34). It seems likely that these properties are related to the unique biological function of SERCA3. Recent studies on mice that lack functional SERCA3 have indicated that this isoform is associated with a Ca2+ pool responsible for hormone-regulated nitric oxide production from endothelial cells (18). Our immunofluorescence localization data on both transfected HEK-293 cells and Jurkat T cells, however, did not reveal any distinct spatial difference between the patterns of SERCA3 and SERCA2b expression. Instead, both isoforms appeared to be present on the nuclear envelope and throughout the cytoplasmic network of endoplasmic reticulum membranes.

We did, however, find that two different human SERCA3 clones, both derived from the same Jurkat T cell cDNA library, differed dramatically in their function. The construct huS3-I displayed a 10-fold lower Ca2+ transport capacity and Ca2+-ATPase activity than construct huS3-II. This may explain why previous investigators (who isolated a clone essentially identical to huS3-I) were unable to obtain functional expression of human SERCA3 (9). huS3-I and huS3-II differ in only three amino acids: comparing huS3-I with huS3-II, Ala for Thr-673, Ile for Met-817, and the inclusion of Glu-994. The latter change occurs at the position of alternative splicing that creates the huS3-IV isoform and is, presumably, a consequence of two potential splice acceptor sites three nucleotides apart. Among our different partial-length Jurkat T cell cDNA clones, four had the Glu residue present and two had it deleted. In any event, because the substitution of this Glu residue and the subsequent five amino acids with the much longer sequence of huS3-IV had no measurable influence on function, we think it unlikely that the presence or absence of the single Glu residue at this site influenced function.

All SERCA proteins except huS3-II have an Ala in place of Thr-673. This includes rat SERCA3 and huS3-IV, both of which have activity similar to that of huS3-II. Thus it seems unlikely that the replacement of Thr-673 by Ala was the cause of the functional difference found between huS3-I and huS3-II. Met-817, which lies in the relatively long cytoplasmic loop between transmembrane spans 6 and 7 (M6-M7 loop), is conserved among all SERCA proteins except for huS3-I and the previously reported human SERCA3 sequence (9). It is likely, then, that the relatively conservative substitution of Met-817 with Ile is responsible for the observed 10-fold reduction in SERCA activity without a change in apparent Ca2+ affinity. Two recent publications have also implicated the functional importance of the M6-M7 loop for both SERCA and H+-K+-ATPase activity (10, 29). Site-directed mutagenesis studies will be required to prove the importance of Met-817 in SERCA activity.

The underlying cause of the structural differences between huS3-I and huS3-II is presently unclear. Such differences could have arisen from cloning artifacts or from mutations accruing in the Jurkat cell SERCA3 gene with time during culture of these immortalized cells. It is tempting to speculate, however, that the differences between the two clones represent allelic variations that might influence SERCA3 function in different individuals. This hypothesis deserves investigation.

In summary, our molecular cloning data demonstrate the existence of two human SERCA3 proteins differing at the carboxy terminus as a consequence of alternative splicing. Although these differences appear not to affect SERCA3 function, a single-amino acid change in the region between putative transmembrane regions 6 and 7 may explain the dramatic differences in function between huS3-I and huS3-II. In addition, we provide direct evidence that the monoclonal antibody PL/IM430 does recognize human SERCA3 and show that SERCA3 and SERCA2b have indistinguishable patterns of subcellular distribution both in transfected HEK-293 cells and when expressed endogenously in Jurkat T cells. These studies lay the groundwork for characterization of the physiological role of human SERCA3 and investigations into the association between SERCA3 expression and human disease.

    ACKNOWLEDGEMENTS

We thank W. Michael Schoel for assistance with digital deconvolution of fluorescence images, Phillip Schwartz (Affinity BioReagents, Golden, CO) for the gift of antisera, and Randal J. Kaufman (Genetics Institute, Boston, MA) for the gift of the expression vector pMT2.

    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42879 and by a grant from the Alberta Heritage Foundation for Medical Research (to J. Lytton), by Grant MT3399 from the Medical Research Council of Canada (to D. H. MacLennan), and by grants from the Catalan Council of Universities and Research and the Hospital Clínic of Barcelona (to E. Poch).

J. Lytton is an Established Investigator of the American Heart Association and a Scholar of the Alberta Heritage Foundation for Medical Research.

Parts of this work have been presented in abstract form (20, 25).

Present address of E. Poch: Servei de Nefrologia, Hospital Clínic, Institut d'Investigacions Biomediques August Pi i Sunyer, Universitat de Barcelona, Villarroel 170, 08036 Barcelona, Spain.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. Lytton, Dept. of Biochemistry and Molecular Biology, University of Calgary Health Science Centre, Rm. 2518, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1.

Received 17 June 1998; accepted in final form 19 August 1998.

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Results
Discussion
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Am J Physiol Cell Physiol 275(6):C1449-C1458
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