(Received for publication, August 30, 1995)
From the
In order to characterize the structure and regulation of members
of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4
family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel
member of this family, which we call RNPDE4A8. Sequencing of the
pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but
to differ from two other PDE4A transcripts, RD1 (pRPDE8;
RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at
its 5` end, consistent with alternative mRNA splicing. The pRPDE39 cDNA
encodes a predicted protein of 763 amino acids, of which all but 21,
located at the extreme amino terminus, are found in the pRPDE6 protein.
Expression of pRPDE39 in COS cells produced a protein of 98 ±
1.4 kDa, as determined by immunoblotting with an antiserum specific to
the carboxyl-terminal regions of all PDE4A proteins, compared to a
predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39
mRNA only in testis. Immunoblotting of testis extracts demonstrated two
bands of 97 ± 2 and 87 ± 3 kDa, the larger of which
co-migrated with the band seen in COS cells expressing pRPDE39. COS
cell expressed pRPDE39 partitioned between a high speed pellet
(particulate) fraction (15% of protein; 8% of activity) and a cytosolic
fraction. The particulate fraction had a K for cAMP of 3.3 ± 0.6 µM, and the
cytosolic fraction a K
of 5.4 ±
2.8 µM. The V
values for the
pRPDE39 protein, relative to the RD1 protein, were 0.16 ± 0.06
and 0.29 ± 0.05 for the particulate and cytosolic forms,
respectively. The pRPDE39-encoded PDE activity could not be removed
from the particulate fraction by high salt concentrations, or by
nonionic detergents. The pRPDE39-encoded enzyme was inhibited by
rolipram at an IC
of 0.5 ± 0.2 µM for
the particulate form and 1.0 ± 0.2 µM for the
cytosolic form, which are values typical of PDE4 family members. The
highly tissue-specific distribution of the pRPDE39 mRNA suggests that
the pRPDE39 protein functions to modulate a cAMP signaling pathway that
is present largely, if not exclusively, in the testis.
The cAMP-specific phosphodiesterases (PDEs) ()are a
large family of enzymes that are differentiated from other cyclic
nucleotide PDEs by their high specificity and affinity for cAMP and by
their inhibition by a specific class of compounds, including the
antidepressant drug rolipram(1, 2) . They are also the
closest mammalian homologs of the dunce gene of Drosophila
melanogaster, which was first isolated as a mutation affecting
learning and memory(3) . The cAMP-specific PDE proteins are
encoded by four genes in mammals (PDE4A, PDE4B, PDE4C, and PDE4D), and at least three of these genes encode multiple PDE
isoforms, generated from different alternatively spliced mRNA
transcripts (reviewed in Refs. 2 and 4). The physiologic significance
of this diversity of cAMP-specific PDE isoforms is not known. However,
the rat PDE4 genes have different patterns of expression in
tissues, suggesting, but not proving, that they have different
functions (5, 6, 7, 8, 9, 10) .
The various alternatively spliced mRNAs from each of at least three
different PDE4 genes also have different regional patterns of
expression in the brain(9) . The proteins encoded by various
alternatively spliced mRNAs from some of the mammalian PDE4 genes have, in some cases, been shown to have functional
differences: for example, the different proteins encoded by various rat PDE4D mRNAs differ in their regulation by
phosphorylation(10) , and those encoded by different rat PDE4A transcripts differ in cellular localization (see below).
We and others have isolated cDNA clones derived from different mRNA transcripts of the rat PDE4A gene, studied the tissue distribution of these mRNAs, and analyzed the biochemical and pharmacologic properties and tissue localization of their encoded proteins(5, 6, 9, 11, 12, 13, 14, 15) . The first rat PDE4A transcript to be described, RNPDE4A1, represented by the RD1(5) , and pRPDE8 (9) cDNA clones, encodes an enzyme of 610 amino acids that is expressed primarily in the brain, particularly the cerebellum(9, 12, 14) . The RD1 protein is present exclusively in the high speed pellet, or particulate, fraction of cellular extracts, consistent with its associating directly or indirectly with membranes. A second rat PDE4A transcript, RNPDE4A5, represented by the pRPDE6 cDNA, is also expressed in brain and encodes a protein of 844 amino acids that is present in both particulate and cytosolic forms(9, 15) . We now report a novel alternatively spliced mRNA transcript from the rat PDE4A locus, which we call RNPDE4A8 (using the accepted nomenclature(1) ), and demonstrate that it is expressed almost exclusively in the testis. RNPDE4A8 encodes a rolipram-inhibited, cAMP-specific PDE of 763 amino acids that is present in both particulate and cytosolic forms. The highly specific tissue-specific distribution of the RNPDE4A8 protein suggests that it functions in a signal transduction pathway that is present largely, or exclusively, in the testis.
Figure 2: Alignment of the amino acid sequences of the amino-terminal regions of the proteins encoded by pRPDE6 (RNPDE4A5), pRPDE39 (RNPDE4A8), and pRPDE8 (RNPDE4A1; identical to the RD1 protein(9) ). Underlined regions indicate upstream conserved region 1 (UCR1), upstream conserved region 2 (UCR2), and the approximate extent of the catalytic region (Catalytic) of the PDEs. A represents the location of alternative splicing in mRNAs from the dunce gene of D. melanogaster(3) and in transcripts from the human PDE4A and PDE4D genes(18) . B represents the methionine used as the initiation codon in the met26RD1 construct. Only the first 284 amino acids encoded by pRPDE39 are shown in the figure, as the remaining sequence is identical with that of the corresponding regions of the pRPDE6 and pRPDE8 proteins, as described previously(5, 9) , and also to the corresponding region of the putative amino acid sequence encoded by the ratPDE2 partial cDNA(6) .
Figure 1: Structure of mRNA transcripts from the rat PDE4A gene. The numbers indicate cDNAs derived from each transcript, as follows: 1, pRPDE6 (RNPDE4A5); 2, pRPDE39 (RNPDE4A8); 3, pRPDE8 (RNPDE4A1; also represented by the RD1 cDNA); 4, met26RD1, an engineered construct containing a deletion of all of the upstream regions of transcripts 1, 2, and 3, and using the methionine at amino acid position 26 of pRPDE8 as the initiation codon(12) . The heavy line indicates sequences common to D. melanogaster and mammalian cAMP-specific PDEs, which contain the consensus regions upstream conserved region 1 (UCR1), upstream conserved region 2 (UCR2), and the catalytic region (Catalytic), as indicated in the bottom of the figure. Thinner lines indicate sequence regions unique to each cDNA. The lines merge where the sequences of the cDNAs join that of the consensus sequence. Small dark boxes indicate initiation codons, and the star indicates the common termination codon.
Figure 3: Expression of the alternatively spliced mRNAs from the PDE4A gene in tissues. RNase protection assays with probes corresponding to the 3` terminus of pRPDE8 (A) and the alternatively spliced regions of pRPDE8 (B), pRPDE6 (C), and pRPDE39 (D) was performed using total RNA (10 µg) from various rat tissues. Arrowheads indicate the position of the RNA fragment protected by each probe. Size markers (in nucleotides) are indicated in the outside lanes, or as short horizontal lines on the sides of the panels, representing MspI-digested pBR322. Lanes are as follows: 1, probe without RNase; 2, kidney; 3, testis; 4, heart; 5, lung; 6, spleen; 7, liver; 8, skeletal muscle; 9, olfactory bulb; 10, brainstem; 11, cerebellum; 12, occipital cortex; 13, parietal cortex; 14, temporal cortex; 15, frontal cortex; 16, tRNA (10 µg).
Figure 4: Expression of pRPDE39 protein in various tissues and in COS cells, as measured by immunoblotting. Immunoblots were performed with an antiserum specific for the carboxyl-terminal region of PDE4A proteins. A, extracts from testis tissue and COS cells expressing various plasmids. Lane 1, 150 µg of testis protein; lane 2, 50 µg of testis protein; lanes 3, 4, and 5, extracts from COS cells expressing the indicated plasmids (10 µg of protein). The positions of proteins used as size markers are indicated. All size determinations were done on a minimum of six independent determinations, using unstained molecular weight markers, as described previously(15) . B, extracts from COS cells containing vector only (C) or vector expressing pRPDE39 (T). The immunoblots were done in the presence of various concentrations (µg/ml) of the peptide used to generate the antiserum. The single immunoreactive band found in the transfected cells and indicative of the pRPDE39 protein is marked with an arrow.
To determine whether a naturally occurring species of a molecular weight similar to the pRPDE39 protein is present in tissues, extracts from testis were analyzed by immunoblotting. Testis was chosen because our RNase protection experiments had demonstrated that pRPDE39 mRNA is present in testis. Immunoblotting of PDE4A species in testis, using our antibody to the carboxyl terminus, demonstrated the presence of a 97 ± 2-kDa and an 85 ± 3-kDa species (Fig. 4). The larger of these species co-migrated with COS cell expressed pRPDE39 (Fig. 4). By comparison, bands co-migrating with COS cell expressed RD1 and pRPDE6 proteins were seen in immunoblots of brain tissue(12, 15) , where RD1 and pRPDE6 mRNAs are expressed(9) . We did not see bands co-migrating with the RD1 or pRPDE6 proteins in testis immunoblots, consistent with our RNase protection data (Fig. 3). The presence of an 85-kDa band in testis immunoblots was unexpected. This could reflect partial proteolysis of the 97-kDa band or, alternatively, a novel PDE4A protein, which could be encoded by a separate alternatively spliced mRNA.
To determine the relative distribution of the pRPDE39 protein between the particulate fraction and cytosol, extracts of COS cells expressing pRPDE39 were fractionated into a low speed pellet (P1), a high speed pellet (P2; i.e. the particulate fraction, which includes membranes), and a high speed supernatant (cytosol), and the fractions were analyzed by immunoblotting (Fig. 5). A single band of 97 ± 2 kDa was seen in homogenates and also in the high speed supernatant and high speed pellet (P2) fractions, with a trace amount in the low speed (P1) pellet. These data demonstrate that the pRPDE39 protein, when expressed in COS cells, partitions between both particulate and cytosolic fractions. Approximately 14.5 ± 2.5% of the PDE protein, as determined by immunoblotting, was present in the particulate fraction. In these respects, the pRPDE39 protein is similar to the pRPDE6 protein, but with one difference, that the pRPDE6 protein was found to be equally distributed between the high speed (P2) and the low speed pellet (P1) fractions(15) .
Figure 5: Partitioning of pRPDE39 protein between particulate and cytosolic fractions. Homogenates from COS cells expressing pRPDE39 were fractionated into a low speed pellet (P1), a high speed pellet (P2), and cytosol, as described under ``Experimental Procedures.'' The resulting fractions were then immunoblotted.
We have determined previously that the RD1 protein can be released from the particulate fraction by nonionic detergents, whereas the pRPDE6 protein cannot be released from this fraction by either detergents or by high salt concentrations(15) . Therefore, we tested whether the pRPDE39 protein could be released from the particulate fraction by these treatments. COS cells expressing pRPDE39 were fractionated into high speed pellet and supernatant fractions. The high speed pellet fraction was then treated with various concentrations of either Triton X-100 or NaCl, and the release of PDE protein from the pellet fraction was analyzed by immunoblotting (Fig. 6). Unlike RD1, but like pRPDE6, the pRPDE39 protein could not be released from the high speed pellet fraction by either of these treatments.
Figure 6: Association of pRPDE39 enzyme with particulate fractions is unaffected by detergents or high salt concentrations. The high speed pellet (P2) fraction was isolated from COS cells expressing pRPDE39 and treated with various concentrations of Triton X-100 or NaCl. The preparations were then centrifuged as described under ``Experimental Procedures,'' and the resulting high speed pellet (P2) fractions were analyzed by immunoblotting. The treatments were as follows: lane 1, 10% Triton X-100; lane 2, 1% Triton X-100; lane 3, 0.1% Triton X-100; lane 4, 5% Triton X-100, 2.5 M NaCl; lane 5, 5 M NaCl; lane 6, 0.5 M NaCl; lane 7, 0.05 M NaCl; lane 8, KHEM buffer as negative control.
Figure 7: Lineweaver-Burk plots for cAMP as substrate utilization by the pRPDE39 enzyme. Extracts were prepared from cytosolic (A) or particulate (B) fractions isolated from COS cells expressing pRPDE39, as described under ``Experimental Procedures.'' Activity is given in nanomoles of cAMP hydrolyzed/min/mg of protein. In each case, a plot typical of 4 independent experiments is shown.
We
also compared the partitioning of pRPDE39 enzyme activity between the
particulate and cytosolic fractions by enzymatic assays, to confirm the
data obtained by immunoblotting. On the basis of PDE activity, 8.8
± 0.6% (± S.D.; n = 4) of pRPDE39 enzyme
activity was associated with the particulate fraction. These data are
consistent with the relative V of the
particulate enzyme being about one-half of the cytosolic form. If the
enzymatic activity of the particulate form is corrected for the
differences in V
, then approximately 16% of the
protein would be predicted, on the basis of enzyme activity, to be in
the particulate fraction. This value agrees well with that obtained by
immunoblotting (see above).
Figure 8: Dose-dependent inhibition of pRPDE39 enzyme activity by rolipram, as determined in cytosolic (A) or particulate (B) fractions isolated from COS cells expressing pRPDE39. Assays were performed with 1 µM cAMP as substrate. Data are shown as means ± S.D. for data obtained from 4 separate experiments, with PDE activity in the absence of inhibitor considered as 100%.
Many components of signal transduction pathways exist as
multiple, closely related isoforms. In some cases, the different
members of each of these families are encoded by separate genes.
Additional diversity can be generated by other mechanisms, including
alternative mRNA splicing, differential protein cleavage, and RNA
editing. The functional consequences of this diversity are often
unknown. In this study, we describe pRPDE39 (RNPDE4A8), which encodes a
new cAMP-specific PDE isoform that differs from other proteins encoded
by the rat PDE4A gene. When compared to the structure of two
other PDE4A mRNAs, pRPDE8 (RD1; RNPDE4A1) and pRPDE6
(RNPDE4A5), the pRPDE39 mRNA has a structure consistent with
alternative splicing at its extreme 5` end. Compared with other PDE4A transcripts, the pRPDE39 mRNA has a unique pattern of
expression, as it is found largely, if not exclusively, in testis. The
pRPDE39 mRNA encodes a protein of 763 amino acids, which migrates as a
98 ± 1.4-kDa species when expressed in COS cells. A species of
very similar size (97 ± 2 kDa) is seen on immunoblotting of
testis extracts, suggesting that the 97-kDa testis band is encoded by
the pRPDE39 mRNA. Like the pRPDE6 protein, but unlike the pRPDE8 (RD1)
protein, the pRPDE39 protein is present in both cytosolic and
particulate fractions. Neither high salt concentrations nor the
nonionic detergent Triton X-100 could release the pRPDE39 protein from
the particulate fraction. The K for cAMP and
IC
for rolipram of the pRPDE39 protein are of the same
order of magnitude as those of the proteins encoded by other rat PDE4A mRNAs(7, 8, 12, 13, 14, 15, 26, 27) and
also for other mammalian cAMP-specific PDEs ( (9) and (18) and references therein), demonstrating that the pRPDE39
protein is a typical member of the cAMP-specific PDE4 family.
Five different cDNAs encoded by the rat PDE4A gene have been reported to date(5, 6, 9) : RNPDE4A1, represented by the RD1 (5) and pRPDE8 (9) cDNAs; RNPDE4A2, represented by the RD2 cDNA (5) ; RNPDE4A3, represented by the RD3 cDNA(5) ; RNPDE4A5, represented by the pRPDE6 cDNA(9) ; and pRPDE39 (this article), which we tentatively call RNPDE4A8. The partial ratPDE2 cDNA clone (6) could belong to any of these groups, except RD2. We have discussed elsewhere why we believe that RD2 and RD3 most likely represent artifacts of cloning(2, 9) . Therefore, the three cDNAs described in Fig. 1represent all the known PDE4A mRNAs. However, other PDE4A cDNAs may be isolated in the future, as additional tissues are examined.
Our data
demonstrate that there are multiple mechanisms for PDE4A regulation in
the cell. First, there is regulation at the level of transcription or
mRNA splicing, as the various alternatively spliced transcripts encoded
by this locus are present in different levels in various tissues. We
have previously shown this to be the case in the brain(9) .
Secondly, each of the proteins encoded by the various alternatively
spliced transcripts may be differentially regulated in the cell. In
this paper, we show that the cytosolic forms of both the pRPDE6 and
pRPDE39 proteins have a roughly 2-fold difference in the V values, as compared with that of their
particulate forms. These differences, although small in magnitude, are
reproducible and suggest that interaction with a membrane-associated
component may be important in the regulation of PDE activity. Because
the interaction of the pRPDE6 and pRPDE39 proteins with the particulate
fraction could not be disrupted by either high salt, nor by nonionic
detergents, it is likely that the proteins interact with a non-lipid
moiety in this fraction, possibly a protein associated with
detergent-insoluble cytoskeletal components. One potential problem with
our data is that we have analyzed the biochemical properties of the
pRPDE39 protein only by expressing it in COS cells. It is possible that
the recombinant pRPDE39 protein may be processed differently in COS
cells, as opposed to testis, or that overexpression of the protein in
COS cells may affect its intracellular distribution. However, we
consider that data obtained by expressing PDE cDNAs in COS cells is
likely to provide a good representation of the native enzyme. This is
based on our previous studies which yielded similar biochemical data
for pRPDE6 and RD1 expressed in COS cells, compared to the
corresponding native proteins in the brain(15) .
Previously,
we proposed that the different amino-terminal regions of the PDE4
proteins, like the amino-terminal regions of other PDEs, may regulate
their biochemical properties(15) . Our isolation of different
members of the rat PDE4A family, all with identical catalytic regions,
but differing in their amino-terminal regions, allows us to test this
hypothesis. Within these amino-terminal regions are two novel and
highly conserved regions of amino acid sequence, which we call upstream
conserved regions 1 and 2 (UCR1 and UCR2; (18) ). UCR1 and UCR2
are conserved in organisms as evolutionarily distant as D.
melanogaster and humans and appear to be distinct, as they have no
homology to each other, and are separated by a region of very low
homology. We have now shown that the pRPDE6 and pRPDE39 proteins, both
of which have extensive amino-terminal regions which include UCR1 and
UCR2 (Fig. 1), have a V that is 3- to
5-fold lower than that of the RD1 protein, which lacks UCR1 and the
amino-terminal half of UCR2. This suggests that the amino-terminal
regions of these proteins affect the conformation of the protein in a
highly specific way, by attenuating the V
, but
not the K
. These conformational and activity
changes are a mechanism for regulating the PDE4A proteins.
One
interesting property of the pRPDE39 mRNA is that it appears to be
expressed largely or exclusively in testis. Partial cDNA clones derived
from transcripts from all four PDE4 genes have been isolated
from rat testis cDNA
libraries(6, 7, 8, 10) , and
complete cDNAs encoded by two of these genes, PDE4B and PDE4D, have been isolated from these
libraries(7, 8) . A 67-kDa protein which reacts with
antisera specific to PDE4D proteins has been purified from rat testis
and is probably encoded by the RNPDE4D1 mRNA(28) . However, all
of the rat PDE4B and PDE4D cDNAs that have been
isolated from testis have also been detected in tissues outside the
testis, including the brain (9) . ()A complete cDNA
from the rat PDE4C gene has not been reported, but PDE4C transcripts are expressed in a number of
tissues(6, 9) . pRPDE39 is the first complete PDE4A transcript to be isolated from testis. Our RNase
protection analysis and immunoblotting of extracts from testis are
consistent with pRPDE39 being a major PDE4A-encoded protein in
testis.
The physiologic role of the pRPDE39 protein is not known. However, it may play a role in a cAMP signaling pathway that is largely, or exclusively, present in the testis. In males, the gonadotrophic peptide hormones follicle-stimulating hormone and luteinizing hormone act almost exclusively on the testis, binding to serpentine seven-transmembrane G-protein-coupled receptors. Activation of these receptors increases adenylyl cyclase activity(29, 30, 31) . The pRPDE39 protein, as well as other cyclic nucleotide PDEs in the testis, may modulate the actions of these hormones, as suggested previously by other groups(7, 8, 27, 32, 33) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L36467[GenBank].