Characterization and expression of plasma membrane Ca2+ ATPase (PMCA3) in the crayfish Procambarus clarkii antennal gland during molting
Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA
* Author for correspondence (e-mail: michele.wheatly{at}wright.edu)
Accepted 18 May 2004
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Summary |
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Key words: calcium transport, plasma membrane calcium ATPase, PMCA, crayfish, Procambarus clarkii, antennal gland, gill, axial abdominal muscle, cardiac muscle
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Introduction |
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The natural molting cycle of a freshwater crayfish, Procambarus
clarkii, has emerged as an ideal non-mammalian model to study
Ca2+ homeostasis and the genes encoding the Ca2+
handling proteins (Wheatly,
1999). As arthropods, crustaceans possess an external calcified
cuticle that is periodically shed, enabling growth to occur. These episodes
are preceded in premolt by reabsorption of Ca2+ from the existing
cuticle and deposition in storage sites (often regions of the digestive
tract). Following ecdysis, there is intense pressure in postmolt to
remineralize the new cuticle primarily with Ca2+ absorbed from the
external water. After calcification is completed, the animal returns to
intermolt, a period during which net Ca2+ flux is minimal. The
beauty of this model system is that net Ca2+ flux alternates from
Ca2+ balance (intermolt) to net loss (premolt) and then to
significant net uptake (postmolt), offering an ideal model to examine the
temporal and spatial regulation of genes coding for Ca2+ handling
proteins. Among crustaceans, the crayfish exhibits highly developed strategies
for Ca2+ homeostasis that have enabled it to evolve in freshwater,
a highly inhospitable environment with respect to Ca2+ availability
(levels typically below 1 mmol l-1 compared with 10 mmol
l-1 in seawater). Specifically, the antennal gland (kidney analog)
produces a dilute urine, contributing significantly to the organism's ability
to maintain its hemolymph Ca2+ hyperionic to the external
environment. This ability is relatively rare in the animal kingdom. In
postmolt, the gills effect massive net Ca2+ influx from low
external levels using active influx mechanisms.
While our lab has had a long-term interest in several Ca2+
import/export proteins, the most interesting and elusive of these is the
high-affinity plasma membrane Ca2+ ATPase (PMCA) that moves
Ca2+ against its electrochemical gradient from the cytosol into the
hemolymph using hydrolysis of ATP. This protein is critical to routine
maintenance of IC Ca2+ concentration and may have an enhanced role
in transcellular Ca2+ influx. Physiological examination of
ATP-dependent uptake into inside-out basolateral vesicles
(Wheatly et al., 1999)
suggests that postmolt Ca2+ influx at the antennal gland would
necessitate proliferation of Ca2+ pumps, while gills appear to be
engineered with an overcapacity to pump Ca2+.
Initial attempts to clone Ca2+ pumps in crustaceans were focused
on a related export protein, the sarco/endoplasmic reticulum Ca2+
ATPase (SERCA), which sequesters cytosolic Ca2+ into the SER. In
crayfish, SERCA expression is highest in intermolt and decreases in pre- and
postmolt in both Ca2+-transporting epithelia (hepatopancreas; Y.G.
and M.G.W., unpublished observations) and non-Ca2+-transporting
tissues alike (axial and cardiac muscle;
Zhang et al., 2000;
Chen et al., 2002
). However,
in the mineralizing anterior sternal epithelium (ASE) of the terrestrial
isopod Porcellio scaber, upregulation of SERCA was associated with
Ca2+-transporting stages (late premolt and intramolt), an effect
that was not apparent in nervous tissue
(Hagedorn and Ziegler, 2002
;
Hagedorn et al., 2003
).
PMCAs and SERCAs belong to the family of P-type ATPases characterized by
the formation of a covalently phosphorylated obligatory intermediate that
arises from the transfer of the phosphate of ATP to a specific
aspartate residue at the catalytic site of the polypeptide during the reaction
cycle; both are integral membrane proteins of 1000 amino acid residues with
three cytoplasmic domains joined to a set of 10 transmembrane (TM)
helices by a narrow pentahelical stalk of
helices. PMCAs are
distinguished from other P-type ATPases by their higher molecular mass (135
kDa) and the presence of a C-terminal regulatory region containing a
calmodulin (CaM) binding site as well as other regulatory domains. Originally
discovered in erythrocyte membranes
(Schatzmann, 1966
), PMCAs were
subsequently shown to be ubiquitous mechanisms for high-affinity
Ca2+ extrusion across membranes of eukaryotic cells. Primary
structure was first cloned in rat and human
(Shull and Greeb, 1988
;
Verma et al., 1988
). Early
progress was hampered by the low abundance of these proteins but, to date,
there is a comprehensive literature on enzymatic properties, biochemical
regulation, gross functional domain structure and primary amino acid
sequences, although largely restricted to vertebrate species (Carafoli,
1991
,
1994
;
Carafoli and Stauffer, 1994
;
Monteith and Roufogalis, 1995
;
Lehotsky, 1995
;
Penniston and Enyedi, 1998
;
Guerini et al., 1999
,
2000
). In vertebrates,
research has focused primarily on excitable tissues, although some studies
have involved intestinal absorptive epithelia
(Borke et al., 1990
;
Howard et al., 1994
) and
mammary secretory epithelium (Reinhardt
and Horst, 1999
).
Mammalian PMCAs are encoded by four non-allelic genes located on different
chromosomes, and additional isoform variants (as many as 30 in total) are
generated via alternative RNA splicing of the primary gene
transcripts at two major regulatory sites, one adjacent to the amino-terminal
phospholipid responsive region and another within the carboxyl-terminal CaM
binding domain (Olson et al.,
1991; Brandt et al.,
1992
; Latif et al.,
1993
; Wang et al.,
1994
). The four PMCA genes appear to be very closely
related in their exon-intron structure
(Burk and Shull, 1992
;
Hilfiker et al., 1993
;
Kuzmin et al., 1994
). General
consensus in vertebrates is that PMCAs 1 and 4 are found in virtually
all tissues and appear to be `housekeeping' isoforms whereas PMCA 2
and 3 are subject to temporal and spatial tissue- and cell-specific regulation
and, as such, may inform the functional adaptation to the physiological need
of preserving multiple isoforms over many years of evolution
(Burk and Shull, 1992
;
Carafoli and Stauffer, 1994
).
The substantial differences among PMCA isoforms are in their
regulation by kinases, proteases and the Ca2+-binding protein CaM
(Borke et at., 1990
;
Carafoli, 1991
;
Axelsen and Palmgren, 1998
;
Bourinet et al., 1999
).
PMCA3 was the isoform selected for complete characterization based on preliminary studies in our lab and the fact that it has emerged from mammalian studies as a candidate isoform for tissue-specific and developmental regulation and alternative splicing patterns. The freshwater crayfish molting cycle can offer unique insights into the spatial and temporal regulation of PMCAs during unidirectional Ca2+ influx. In the present study, we set out to clone and characterize PMCA3 in crayfish tissues and to compare tissue-specific expression as a function of the molting cycle in both Ca2+-transporting epithelia (gill/antennal gland) and non-Ca2+-transporting tissues (axial and cardiac muscle). We hypothesized that PMCA3 would be upregulated in Ca2+-transporting epithelia in pre- and postmolt compared with intermolt and that levels in non-transporting epithelia would be unchanged.
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Materials and methods |
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Isolation of total RNA and mRNA
After dissection, tissues were frozen immediately in liquid nitrogen and
stored at -80°C. Total RNA was isolated by utilizing Trizol reagent
(Invitrogen, Carlsbad, CA, USA), as specified by the manufacturer. Briefly,
0.5 g of tissue was finely ground in liquid N2 and lysed by adding
3 ml of Trizol reagent. The lysates were allowed to incubate at RT for 5 min.
Then, 1.2 ml chloroform was added followed by vigorous vortexing for 15 s.
Samples were then incubated for 5 min at RT and centrifuged for 15 min at 13
362 g. Following removal of the aqueous phase and addition of
1.5 ml of isopropanol, samples were placed at -80°C overnight and then
centrifuged for 15 min at 13 362 g. The RNA pellets were
washed with 1.5 ml 75% ethanol, sedimented for 5 min at 7516 g
and air-dried for 10 min before being dissolved in diethyl pyrocarbonate
(DEPC)-treated water and stored at -80°C. Messenger RNA was separated from
total RNA using an oligodT cellulose column (Stratagene, La Jolla, CA, USA).
RNA or mRNA was quantified spectrophotometrically at wavelengths of 260 and
280 nm.
Cloning of crayfish antennal gland (kidney) PMCA3
We elected to clone the PMCA3 initially from antennal gland
because physiological studies had indicated that it exhibited higher rates of
intermolt unidirectional Ca2+ influx than any other epithelial
tissue (Wheatly, 1999).
First-strand cDNA was reverse transcribed from 400 ng of mRNA from antennal
gland using the SuperScript II RNase H-reverse transcriptase (Gibco BRL,
Gaithersburg, MD, USA) with oligo(dT)12-18 as primer. Based on four
published PMCA3 sequences from human (GenBank accession nos US57971
and U60414; Brown et al.,
1996
), mouse (AKO32322;
Carninci et al., 2000
) and rat
(J05087; Greeb and Shull,
1989
), two primers were designed:
5'-GGGCAAYGCCACAGCCATCT-3' (sense) and
5'-CCCCACATGACYGCCTTGACR-3' (antisense). Primer location
corresponded to nucleotides 1518-2652 in human, 1686-2820 in mouse and
2045-3179 in rat. These primers targeted a fragment of approximately 1134 bp
located between transmembrane regions TM4 and TM5 of the PMCA3 gene
in human, mouse and rat. Polymerase chain reactions (PCR; total volume 50
µl) included 2 µl of first-strand cDNA from postmolt antennal gland, 20
mmol l-1 Tris HCl (pH 8.4), 50 mmol l-1 KCl, 1.5 mmol
l-1 MgCl2, 0.2 mmol l-1 dNTP mix, 0.1-0.2
µmol l-1 of each primer and 2.5 units of Taq DNA polymerase
(Gibco BRL). RT-PCR cycles were conducted at 94°C for 3 min followed by 30
cycles of 94°C for 30 s, 58.5°C for 1 min, 72°C for 1 min and a
final extension at 72°C for 10 min. Negative controls in which reactions
contained no template cDNA were included. RT-PCR products were analyzed by
electrophoresis on a 1.0% agarose gel with 0.5 µg ml-1 of
ethidium bromide in 1x TAE buffer (40 mmol l-1 Tris, 40 mmol
l-1 sodium acetate and 1 mmol l-1 EDTA, pH 7.2). The DNA
bands were visualized with ultraviolet light.
Subsequently, 3' and 5' RACE (rapid amplification of cDNA ends) systems (Invitrogen) were used to amplify the 3' and 5' ends of crayfish antennal gland PMCA3. For the 5' RACE, a gene-specific primer, 5'-GAGGGTGCCAGTCTTGTCA-3', and a nested primer, 5'-AGGCATCCAGGTGGCGCACCA-3', were used. For the 3' RACE, a gene-specific primer, 5'-AGGCCTCAGACATCATTCTGAC-3', and a nested primer, 5'-TGTCAAGGCTGTCATGTGGGG-3', were designed. The PCR conditions were the same as described above. The integrity of the RNA from the various tissues was checked by the presence of a fragment of 18s ribosomal RNA gene. The RNA 18s primers (sense 5'-GGCCCAGACACCGGAAGGATTGAC-3' and antisense 5'-GCCCGAGACGCGAGGGGTAGAACA-3') were designed from Procambarus clarkii.
18s ribosomal RNA gene, coding for a 518 bp fragment (accession no. AF436001)
PCR products were ligated to PCR 2.1 vector (Invitrogen) for transformation
into INVF host cells (Invitrogen). Each clone was digested with appropriate
restriction enzymes and subcloned for sequencing. Two independent clones were
sequenced from both ends. The cDNA clones were sequenced by automated
sequencing (ABI PRISM 377, 3100 and 3700 DNA sequencers; Davis Sequencing,
Foster City, CA, USA).
The complete sequence was assembled with DNASTAR (DNASTAR Inc., Madison, WI, USA). Sequence homology was revealed through a GenBank database search using the BLAST algorithm search (http://www.ncbi.nlm.nih.gov/blast).
Northern blot
Northern blot analysis was performed to determine the distribution of
PMCA3 in gill, antennal gland, cardiac muscle and axial abdominal
muscle during various stages of the molting cycle. Total RNA (25 µg) from
each tissue examined was fractionated by electrophoresis through a 0.72 mol
l-1 formaldehyde-1% agarose denaturing gel, run in MOPS buffer (5
mmol l-1 sodium acetate, 1 mmol l-1 EDTA, 20 mmol
l-1 MOPS at pH 6.6) and transferred overnight to a Nytran Plus
membrane (Schleicher & Schuell, Keene, NH, USA) by capillary elution in
10x SSC (where 1x SSC is 150 mmol 1-1 NaCl, 15 mmol
1-1 sodium citrate). RNA was fixed by ultraviolet crosslinking
using a UVC-515 ultraviolet multilinker from Ultra-Lum (Claremont, CA, USA;
120 000 µJ cm-2). An RNA molecular mass marker (a 0.24-9.5 kb
ladder) was run along with the samples, then visualized with ultraviolet light
after staining with ethidium bromide, and used for the standard curve. The
membrane was prehybridized for 4 h at 68°C in 6x SSC, 2x
Denhardt's reagent (0.4 g Ficoll type 400, 0.4 g polyvinylpyrrolidone, 0.4 g
bovine serum albumin in 1 liter water), 0.1% SDS and 100 ng ml-1
denatured salmon sperm DNA. Hybridization was performed overnight at 68°C
in the prehybridization solution with 20 ng of PMCA antennal gland
isoform cDNA probe that was randomly labeled with
[-32P]dATP. The membrane was washed four times for 15 min at
60°C in 0.1x SSC and 0.1% SDS. The membrane was exposed to X-ray
film with intensifying screens at -80°C. To normalize the hybridization
signal, 18s RNA was quantified on a corresponding formaldehyde-agarose gel.
Total RNA content was determined by OD260 and visualized by
ethidium bromide staining.
Western blot
Membrane protein from gill, antennal gland, cardiac muscle and axial
abdominal muscle was prepared from freshly isolated tissue using differential
centrifugation following published methodology
(Williams et al., 1999).
Briefly, 0.5 g tissue was dissected and ground first in liquid N2
and then in 1 ml of homogenization buffer (250 mmol l-1 sucrose, 10
mmol l-1 Tris, 10 mmol l-1 Hepes, 1 mmol l-1
EDTA; pH adjusted to 7.2 at 23°C) containing protease inhibitors for an
additional 3-5 min. Following centrifugation at 4547 g for 10
min at 4°C, the supernatant was centrifuged at 6100 g for
30 min at 4°C. The final pellet was resuspended in
100-500 µl of
homogenization buffer with protease inhibitors and stored at -80°C.
Protein concentration was determined using a Micro-BCA protein kit (Pierce,
Rockford, IL, USA).
Membrane proteins were separated by 9% SDS-polyacrylamide gel electrophoresis and transferred from unstained gels to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) in transfer buffer (192 mmol l-1 glycine, 25 mmol l-1 Tris-HCl, pH 8.3) overnight at 30 V using a Bio-Rad Trans-Blot tank apparatus. Nitrocellulose-bound protein was visualized by staining with Coomassie Brilliant Blue R-250. The nitrocellulose membrane was blocked overnight in PBS/milk (7% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) at 23°C and then incubated in PBS/milk with 1:1000 purified anti-PMCA3 polyclonal antibody for 2 h at 23°C. After three 10 min washes in PBS/milk, the nitrocellulose membrane was incubated with secondary antibody at 1:2000 dilution (horseradish peroxidase conjugated goat anti-rabbit IgG) for 1 h at 23°C in PBS/milk. After three washes in PBS, 0.1% Tween 20, bound antibody was detected using ECL western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
To generate the PMCA3 antibody, an amino acid sequence deduced from
crayfish PMCA3 cDNA sequence was used to design an antigenic
oligo-peptide (Boersma et al.,
1993). The designed oligo-peptide EGKEFNRRVRDESGC from
amino acid residues 751-775 located in the cytosolic loop before the FSBA
{
-[4-(N-2-chloroethyl-N-methylamino)] benzylamine ATP
binding} site was synthesized commercially (Genemed Biotech Inc., San
Francisco, CA, USA). To increase antigenicity, the oligo-peptide was
conjugated to cBSA (cationized BSA; Pierce). The antigenic peptide was
subsequently used for production of a polyclonal antibody in New Zealand White
rabbits in compliance with LACUC protocol AUP 245 issued to Dr Harold Stills,
WSU Veterinarian. Trained LAR staff performed all the injections and blood
collections following the euthanasia procedures. Antiserum titer was
determined by enzyme-linked immunosorbent assays (ELISAs) using synthetic
peptide as antigen (Wheatly et al.,
2001
).
Northern and western blots were repeated in triplicate and quantified through scanning the X-ray film images using KODAK 1D image analysis software (Scientific Imaging System, Eastman Kodak Company, Rochester, NY, USA).
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Results |
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Overall, the complete crayfish antennal gland PMCA3 nucleotide
sequence contains 4148 nucleotides with an open reading frame of 3546 bp
coding for 1182 amino acid residues with a molecular mass of 130 kDa
(Fig. 1A). There is a 559 bp
noncoding sequence at the 3' end before the poly(A) region and a 40 bp
noncoding sequence at the 5' end. A search of the GenBank database using
the BLAST algorithm indicated that this crayfish PMCA3 sequence is
homologous with over 55 PMCAs, including PMCA3, PMCA2, PMCA1
and PMCA4, and shares the highest homology (77.5-80.9% identity at
the DNA level) with human brain PMCA3 isoforms a and b (GenBank
accession nos U57971 and U60414; Brown et
al., 1996), rat PMCA3 (J05087;
Greeb and Shull, 1989
), mouse
retinal neuron PMCA3b (NM177236;
Krizaj et al., 2002
) and mouse
brain PMCA3 (AKO32322; Carninci
et al., 2000
). The deduced amino acid sequence also shares high
homology (85.3-86.9%) with PMCA3 from human, mouse and rat. Therefore, this
sequence was confirmed as crayfish antennal gland PMCA3. A BLAST
search indicated that the crayfish PMCA3 also shared relatively high
homology with PMCA2 sequences including mammalian PMCA2 and
PMCA2 from non-mammalian species, such as bullfrog PMCA2a
(AF337956; R. A. Dumont, U. Lins, A. G. Filoteo, J. Penniston, B. Kachar and
P. G. Gillespie, direct submission to GenBank in 2001, no accompanying
publication) and tilapia PMCA2 (AF236669; C.-H. Yang, J.-H. Leu,
C.-M. Chou, S.P. L. Hwang, C.-J. Huang and P. P. Hwang, direct submission to
GenBank in 2000, no accompanying publication).
Fig. 1B shows the alignment of
crayfish PMCA3 deduced amino acid sequence with human PMCA3a. Because PMCA3
sequences were not available from non-mammalian species, two PMCA2 sequences
(from tilapia and bullfrog) were also included in
Fig. 1.
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Comparing the crayfish PMCA3 amino acid sequence to other PMCAs, the first intracellular loop region between TM2 and TM3 is one region where major sequence differences occur (Fig. 1B). This region corresponds to the `transduction domain', thought to play an important role in the long-range transmission of conformational changes occurring during the transport cycle. The other major differences occur in the C-terminus immediately 3' to the CaM binding site. This region is believed to be the location of regulation by kinases, proteases and CaM. Amino acid sequences at this region after IQTQ of the CaM binding site vary from 40 to 107 residues in length, and the identity at this region drops to less than 67%. Furthermore, as mentioned before, crayfish PMCA3 has a 10-amino-acid insertion not found in other PMCA3 and PMCA2 sequences at the region between the FITC (fluorescein isothiocyanate) and the FSBA site.
Comparison among sequences indicates that some regions have virtually 100% identity (TM1, TM2, TM4, phosphorylation site, FITC site, FSBA site, TM5, TM6, TM8 and CaM binding site). Other regions exhibit less identity (TM7 and TM10). Interestingly, there were some regions where crayfish antennal gland PMCA3 exhibited a closer identity to mammalian PMCA3 sequences than to fish or amphibian PMCA2 sequences (TM3 and TM9).
The hydropathy profile of crayfish PMCA3 was compared with those sequences
from other species named above (Fig.
2). The predicted amino acid sequence of crayfish PMCA3 displays a
structure common to other PMCA pumps. It appears that the crayfish PMCA3
contains 10 membrane-spanning segments, as indicated in other PMCAs. Four of
these putative transmembrane domains are located near the N-terminal region
and the remaining six are located near the C-terminus, with a large
cytoplasmic loop in between. The bulk of the protein mass is facing the
cytosol and consists of three major domains: the IC loop between TM 2 and TM3,
the large unit between TM4 and TM5, and the extended `tail' following the last
transmembrane domain. The large cytosolic region (400 residues) of
crayfish antennal gland PMCA3 between membrane-spanning segments 4 and 5
contains the major catalytic domains, including the ATP binding site and the
invariate aspartate residue that forms the acylphosphate intermediate during
ATP hydrolysis.
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Crayfish PMCA3 mRNA expression during the molting cycle
The tissue distribution of this novel crayfish PMCA3 gene was
examined using a northern blot of total RNA from crayfish gill, antennal
gland, cardiac muscle and axial abdominal muscle tissues probed with the 1164
bp fragment initially cloned that corresponded to nucleotides 1488-2652. In
all four tissues examined, the probe detected a 7.5 kb mRNA
(Fig. 3). The expression of
PMCA3 mRNA in all four tissues increased during both premolt and
postmolt compared with intermolt (Fig.
3); when these changes were quantified
(Fig. 4A), the increases
observed in PMCA3 mRNA expression in the Ca2+-transporting
epithelia (gill and antennal gland) were about double (60% increase) the
increases in non-Ca2+-transporting tissues (about 25% increase in
cardiac and axial muscle). The only tissue that exhibited a significant
further increase in mRNA expression in postmolt as compared with premolt was
the antennal gland.
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|
Crayfish PMCA3 protein expression during the molting cycle
PMCA3 protein expression was confirmed through western blotting. A
polyclonal antibody against crayfish PMCA3 recognized a 130 kDa protein band
that was just detectable in all tissues in the intermolt stage
(Fig. 5). Protein expression
increased markedly during pre- and postmolt stages as compared with intermolt
in all four tissues (Fig. 5),
generally confirming the trend observed in mRNA expression. Quantification of
protein expression (Fig. 4B) in
gill and antennal gland paralleled the trends observed above in mRNA
expression (Fig. 4A), namely
that expression in antennal gland increased further in postmolt compared with
premolt while expression in gill was unchanged. A second isoform band (128
kDa) was apparent primarily in axial muscle, where it appeared to be the
prominent band in intermolt. Expression of this band did not seem to change
during the molting cycle (Fig.
5). Unexpectedly, PMCA3 protein expression in muscle increased
significantly in postmolt compared with premolt, unlike mRNA expression, which
remained unchanged.
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Discussion |
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Preliminary studies (Y.G. and M.G.W., unpublished) indicated that there are
at least two species of all other isoforms. Seemingly, there are several
splice variants in crayfish, suggesting that alternative splicing generates
significant isoform diversity as it does in mammals
(Shull and Greeb, 1988;
Strehler, 1991
;
Burk and Shull, 1992
;
Stauffer et al., 1993
;
Zacharias et al., 1995
;
Keeton and Shull, 1995
).
Alternative splicing affects two major locations in the PMCA protein that
correspond to the major regulatory domains: firstly, a region embedded between
a putative G protein binding sequence and the site of phospholipid sensitivity
in the first cytosolic loop; and secondly a region in the COOH-terminal tail
that affects regulation by CaM, phosphorylation and differential interaction
with PDZ domain-containing anchoring and signaling proteins
(De Jaegere et al., 1990
;
Strehler et al., 1990
;
Strehler, 1991
).
The present paper describes the cloning of PMCA3 from the antennal
gland of crayfish Procambarus clarkii as well as the regulation of
both mRNA and protein tissue-specific expression during the natural molting
cycle. This is the first complete sequence from an invertebrate with
the exception of several isoforms cloned as part of the Caenorhabditis
elegans genome project [Kraev et al.,
1999 initially cloned mca-1 (GenBank accession no. NM_069308) and
mca-2 (NM_067760); later, Kamath et al.
(2003
) identified mca-3
(NM_067893) with `a' (AAK685500); `b' (AAK68551) and `c' (AAM97979) splice
variants (Waterston, 1998
)]. A
partial sequence is available for the isopod Porcellio scaber
(AF455814; Ziegler et al.,
2002
). Aside from one PMCA2 sequence from a fish
(Oreochromis mossambicus, tilapia; AF23669; C.-H. Yang, J.-H. Leu,
C.-M. Chou, S.-P. L. Hwang, C.-J. Huang and P. P. Hwang, direct submission to
GenBank in 2000, no corresponding publication) and two sequences
(PMCA1bx and PMCA2av) from an amphibian (Rana
catesbiana, the bullfrog; AF337955 and AF337956, respectively; R. A.
Dumont, U. Lins, A. G. Filoteo, J. Penniston, B. Kachar and P. G. Gillespie,
direct submissions to GenBank in 2001), all other PMCA sequences in
GenBank originate in mammalian species.
The crayfish PMCA3 shares sequence homology with a range of other
published PMCAs. A phylogenetic tree showing the relationship between
the crayfish PMCA3 amino acid sequence and other PMCA
sequences available for a diversity of species is provided
(Fig. 6). When the
PMCA1-4 protein isoforms were compared, the homology of the
same isoform from different species was higher than the similarity between
isoforms within a species (Fig.
6). Thus, the crayfish PMCA3 deduced amino acid sequence shared
higher identity with mammalian PMCA3 than with PMCA2 from lower vertebrates.
Comparing sequence distances within each isoform group (PMCA1 and PMCA2), the
sequence distance was always greater between mammalian and non-mammalian
species than the distance between species among mammalian or among
non-mammalian. The same was true when crayfish PMCA3 was added to the PMCA3
isoform group (Fig. 6). This
suggests that PMCA genes diverged and their protein products
developed specialized functions early in evolution. Regions of sequence
difference included the first IC loop between TM2-3 (the `transduction'
domain) and regions in the C-terminus after the CaM binding site that are
regulatory sites for various kinases and proteases. The major differences
between the deduced crayfish PMCA3 amino acid sequence and other PMCA3
sequences occur in regions close to the sites where alternative splicing
originates. Regions that are predictably highly conserved are those that are
critical to the catalytic and transport functions of the pump. Several of
these regions are located in the large catalytic domain between TM4 and TM5
(phosphorylation site, FITC site, FSBA site, CaM binding site and TM1, 2, 4,
5, 6 and 8). Certain regions where crayfish antennal gland PMCA3 bore a closer
resemblance to mammalian PMCA3 than to lower vertebrate PMCA2 (such as TM3 and
TM9) presumably differentiate the two isoforms. The regions of high diversity
between isoforms are likely to reflect isoform-specific regulatory and
functional specializations that allow each pump to fulfill a unique role in
the specific cell or tissue in which it is expressed. Regions where there is
high diversity among the same isoforms from different species (TM7 and TM10)
are presumably less critical to the function of either isoform. Hydropathy
analysis of the crayfish PMCA3 suggested common structural membrane topography
to PMCAs described in other species
(Strehler, 1991;
Monteith and Roufogalis,
1995
).
|
A probe to the novel PMCA3 crayfish gene hybridized with a single
band of 7.5 kb in all tissues tested, suggesting that it is ubiquitously
expressed in intermolt at approximately comparable levels. The mRNA species
detected in crayfish is the same size as reported in human erythrocyte
(Strehler et al., 1990) and
rat brain and skeletal muscle (Greeb and
Shull, 1989
). It is considerably longer than the cDNA sequence
represented in Fig. 1A,
indicating the presence of an extended untranslated sequence.
The ubiquitous distribution of PMCA3 in crayfish tissue appears to
be in contrast to the tissue distribution in mammals, where it has been
restricted primarily to excitable tissues (brain and skeletal muscle
Greeb and Shull, 1989;
Burk and Shull, 1992
;
Carafoli and Stauffer, 1994
;
Stauffer et al., 1995
; hair
cells Furuta et al.,
1998
). It has also been found at lower levels in regions of the
mammalian digestive system and in testis. In mammals, the predominant
PMCA isoform in transporting epithelia (uterus, liver, kidney and
lactating mammary glands) appears to be PMCA2
(Stauffer et al., 1997
;
Furuta et al., 1998
;
Street et al., 1998
;
Reinhardt and Horst, 1999
).
PMCA3 mRNA expression increased in both premolt and postmolt stages
compared with intermolt (Fig.
3) in all crayfish tissues examined irrespective of their
involvement in net vectorial Ca2+ influx (gill, antennal gland) or
not (control tissues, muscle); however, increases were numerically greater in
Ca2+-transporting epithelia than in
non-Ca2+-transporting tissues, partially supporting the hypothesis
on which this study was based. These data support transcriptional regulation
of PMCA during pre- and postmolt compared with intermolt. The
upregulation of PMCA3 mRNA in premolt antennal gland was greater than
observed in gill and increased significantly in postmolt, confirming an
earlier prediction from organismal studies
(Wheatly et al., 1999
) that
postmolt Ca2+ influx at the antennal gland would necessitate
proliferation of Ca2+ pumps while gills were engineered with an
overcapacity to pump Ca2+ in postmolt. Our findings in crayfish
confirm an earlier study using semi-quantitative RT-PCR that showed increased
expression of PMCA from the non-Ca2+-transporting stages (early
premolt) to the stages of CaCO3 deposition/degradation (late
premolt/intramolt) in the sternal epithelia of the isopod Porcellio
scaber (Ziegler et al.,
2002
). Collectively, both studies would suggest that PMCA plays a
role in the vectorial epithelial Ca2+ transport.
Antibody raised against crayfish PMCA3 recognized a protein of 128-130 kDa
in crayfish tissues, which was the same size as reported in mammalian species
(Strehler et al., 1990;
Carafoli et al., 1996
).
Importantly, the protein expression patterns (Figs
4B,
5) generally confirmed the mRNA
expression patterns described above (Fig.
3), namely that the PMCA3 protein was expressed in all tissues and
that expression increased in pre- and postmolt in all tissues examined
compared with intermolt. For gill and antennal gland, protein expression
patterns paralleled those observed for mRNA, indicating that PMCA3
was transcriptionally regulated. In muscle, however, protein expression
increased in postmolt compared with premolt even though mRNA expression had
been unchanged. The best available explanation would be that rate of mRNA
translation was increased or that the appearance of a second protein band
confounded the image analysis.
The expression pattern observed for PMCA3 during the molting cycle
directly opposes the pattern previously reported for SERCA, the
Ca2+ pump located on endomembranes. In both epithelial
(hepatopancreas) and non-epithelial tissues (muscle), SERCA expression was
highest in intermolt and decreased in both pre- and postmolt to expression
levels that were roughly comparable (Zhang
et al., 2000; Chen et al.,
2002
). Thus, in crayfish non-mineralizing tissues, PMCA and SERCA
expression patterns seem to be inversely regulated during molting stages.
Overexpression of PMCA in rat aortic endothelial cells similarly
resulted in downregulation of SERCA (Liu
et al., 1996
). It has been suggested that the genes for all the
major Ca2+-transporting pathways are linked for regulatory
purposes.
Interestingly, in an arthropod mineralizing tissue, the anterior sternal
epithelium of the isopod Porcellio scaber
(Hagedorn et al., 2003), an
increase in SERCA expression was observed from the non-transporting early
premolt stage to the Ca2+-transporting late premolt and intramolt
stage. These changes were not seen in nervous tissue. This would suggest a
role for SERCA in transcellular Ca2+ transport in mineralization
processes. Related studies revealed that IC Ca2+ hotspots represent
SER cisternae (Ziegler, 2002
)
and that SERCA activity increased by fivefold from early premolt to the
Ca2+-transporting late premolt and intramolt
(Hagedorn and Zeigler, 2002
).
Similarly, SERCA activity has been shown to increase in rat dental ameloblasts
during calcification. Mineralizing tissues display Ca2+ flux rates
that are much higher than other Ca2+-transporting epithelia (such
as kidney) and it seems plausible that they may have evolved routes for
Ca2+ transit that exceed typical transepithelial rates. Seemingly,
there are differences in expression of Ca2+ import/export proteins
between mineralizing and non-mineralizing tissues that warrant further
investigation.
The Ca2+ secretory model of pregnant and lactating rats revealed
that five different Ca2+ pumps in mammary tissue (PMCA1b, 2b and 4b
and SERCA2 and 3) were all upregulated by the 14th day of lactation
(Reinhardt and Horst, 1999).
In this model, a large amount of Ca2+ moves across the cell from
the blood to the milk. In this case, PMCA and SERCA trends were the same,
suggesting that the lactating mammary gland model has more in common with the
isopod mineralizing model.
Since PMCA3 has such a restrictive tissue-specific distribution in mammals
(primarily neuronal/excitable) there are relatively few studies that have
examined regulation of expression, particularly in response to Ca2+
flux. However, evidence is accumulating that regulation of
Ca2+-associated genes is centrally regulated by changes in IC
Ca2+ itself (Zacharias and
Strehler, 1996; Kuo et al.,
1997
; Carafoli et al.,
1999
; Guerini et al.,
1999
). For example, rat cerebellar granule cells kept under
depolarizing conditions for several days (leading to increased Ca2+
influx) showed a marked upregulation of PMCA3 at both the mRNA and protein
levels (also of PMCA1a and PMCA2; Guerini
et al., 1999
). Functionally, the PMCAs appear to play an important
role in cellular Ca2+ dynamics. A number of different regulatory
mechanisms may alter their functionality
(Carafoli, 1991
;
Monteith and Roufogalis, 1995
;
Penniston and Enyedi, 1998
).
Primarily, PMCAs are activated by Ca2+-calmodulin, acidic
phospholipids and serine/threonine phosphorylation
(James et al., 1988
;
Enyedi et al., 1989
; Falchetto
et al., 1991
,
1992
). In addition to
mediating regulation by Ca2+-calmodulin, the COOH-terminal region
of the calcium pump has also been shown to be the target of phosphorylation by
protein kinases A and C (Wuytack et al.,
1992
; Monteith and Roufogalis,
1995
; Penniston and Enyedi,
1998
) and to be affected by proteases such as calpain
(Carafoli, 1994
;
Wang et al., 1994
).
The present study has cloned and sequenced the entire PMCA3 from the crayfish Procambarus clarkii. Sequence data will inform our general understanding of the molecular evolution of this important PMCA isoform. Expression of PMCA3 mRNA and protein increased in all tissues examined in the pre- and postmolt stage, a period during which Ca2+ influx at transporting epithelia contributes to overall Ca2+ conservation as the organism seeks to remineralize its cuticle. As we seek to more fully understand the regulation of PMCA and other Ca2+-associated membrane proteins (SERCAs, Ca2+ channels and NCX), the crustacean molting cycle will continue to offer an interesting non-mammalian model.
The PMCA3 sequence from the crayfish Procambarus clarkii antennal gland has been accepted by GenBank (accession no. AY455931).
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