(Received for publication, May 26, 1995; and in revised form, September 26, 1995)
From the
Three alternatively spliced type VIII adenylyl cyclase messages
have been identified by cDNA cloning and amplification from rat brain
cDNA. Type VIII-A was previously referred to simply as type VIII (Cali,
J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski,
J.(1994) J. Biol. Chem. 269, 12190-12195). The types
VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90
and 198 base pair exons, respectively, which encode a 30-amino acid
extracellular domain with two consensus sites for N-linked
glycosylation and a 66-amino acid cytoplasmic domain. Stable expression
of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293
(HEK-293) cells leads to the appearance of novel proteins, which are
recognized by type VIII-specific antibodies and which co-migrate with
immunoreactive species detected on immunoblots of rat brain membranes.
Types VIII-A and -C are modified by N-linked glycosylation,
while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca stimulates cAMP accumulation in HEK-293 cells stably expressing
type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase
activity of each of the variants is stimulated by
Ca
/calmodulin and the EC
for activation
of type VIII-C is one fourth of that for either type VIII-A or -B. Type
VIII-C also has a distinct K
for
substrate, which is approximately 4-12-fold higher than that for
types VIII-A or -B depending on whether Mn
or
Mg
is the counterion for ATP. The differences in the
structural and enzymatic properties of these three variants are
discussed.
A variety of hormones, neurotransmitters, and olfactants
regulate the synthesis of cAMP by adenylyl cyclases (ACs). ()Many of these agents act through three component, G
protein-coupled systems that are capable of modulating the activity of
an AC (for review, see (1) ). Alternatively, seemingly
independent signaling pathways may generate other second messengers or
activate kinases that subsequently regulate AC activity by signal
cross-talk. The importance of the latter class of regulatory mechanisms
has become apparent with the realization that the different AC isoforms
are distinguished by their ability to provide a unique integrated
response to coincident stimuli.
Eight full-length mammalian ACs have been characterized(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) , and other partial cDNA sequences have been reported(15, 16) . The existence of additional forms which are derived by alternative splicing of the messages is consistent with the sequence differences in the amino-terminal domains encoded by cDNA clones for both type V (6, 17) and type VI(7, 8, 9, 10) . A half-molecule variant of the canine type V has been described(18) , and the expression of other AC variants is suggested by the detection of multiple type V and type VIII transcripts on RNA blots under high stringency conditions(6, 9, 11, 13) . Thus far, unique functional properties have not been ascribed to any specific splice variant.
The mammalian ACs share a common topography. The
amino-terminal domains are all predicted to be cytoplasmic, but they
vary dramatically in both sequence and length. The only well conserved
sequences among the ACs are found in the so-called C and
C
regions, which are two large cytoplasmic domains of over
200 amino acids each(5, 19, 20) . The
conserved domains are homologous to each other and to the catalytic
domain of the guanylyl cyclases(2, 21) . Based on this
similarity, they are considered to be nucleotide binding domains and
have recently been shown to be sufficient to confer enzymatic
activity(22) . Each of these domains is preceded by a large
hydrophobic region of variable sequence, which includes six
transmembrane spans based on hydropathy analysis. Consensus N-linked glycosylation site(s) are always present in at least
one putative extracellular domain associated with the second set of six
transmembrane
spans(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
On the carboxyl-terminal side of the first putative nucleotide binding
domain is a highly variable region called
C
(5, 19, 20) , which is a site
for type-specific regulation(23, 24, 25) .
The corresponding region following the second nucleotide binding
domain, C
, is only present in types I, III, and VIII (2, 4, 13) . While similarities among these
isoforms may underlie their common enzymatic function, their
differences at the amino acid level presumably account for variable
responsiveness to a variety of regulatory influences.
Type VIII AC
is a Ca/calmodulin-stimulated isoform abundantly
expressed in discrete regions of rat brain(13) . The
distribution of type VIII message and its enzymatic properties are most
closely related to those of type
I(13, 19, 26) . Here we describe the
isolation of two additional type VIII cDNAs from rat brain, which are
derived by alternative mRNA splicing. The differences in the structure
and enzymatic properties of the variants are described.
The calmodulin concentration
dependence was also determined in the presence of 17 µM free Ca and 5.74 mM free
Mg
. Calmodulin-independent activity was measured in
the presence of excess EGTA and was subtracted from all values. The
concentration dependence for calmodulin activation was adequately
represented by a hyperbolic equation if the concentration of calmodulin
was considered to be the sum of the known concentration of added
calmodulin and a parameter representing the unknown concentration of
endogenous calmodulin in the membrane preparation. The activity value
determined at each concentration of calmodulin was divided by the
maximal activity computed for a given membrane preparation under these
conditions to visualize the differences in EC
values.
The mRNAs encoding
types VIII-A, -B, and -C adenylyl cyclase can be amplified from rat
brain poly(A) RNA by reverse transcription PCR using
oligonucleotide primers designed to distinguish the alternatively
spliced messages (Fig. 1A). Products of 480 and 390 bp
are amplified with primers flanking the region deleted from the type
VIII-B cDNA, while products of 636 and 438 bp are amplified with
primers flanking the region deleted from the type VIII-C cDNA. DNA
sequencing verified that the 480- and 636-bp PCR products are derived
from the type VIII-A message and the 390- and 438-bp PCR products arise
from the type VIII-B and -C messages, respectively, as predicted based
on the sequence of the cDNA clones. This confirms that the messages
encoding the three splice variants are expressed in rat brain.
Figure 1:
PCR amplification
of type VIII-A, -B, and -C mRNA and analysis of the structure of the 5`
end of the rat type VIII AC gene. A, the polymerase chain
reaction was performed as described under ``Experimental
Procedures'' using primer pairs, which flank either the region
deleted from the type VIII-B cDNA (lanes 1 and 2) or
that deleted from the type VIII-C cDNA (lanes 3 and 4). The reactions were carried out in the presence (lanes
1 and 3), or the absence (lanes 2 and 4) of cDNA reverse transcribed from 0.25 µg of rat brain
poly(A) RNA. 4% of the total reaction volume was
loaded into lanes 1 and 2 and 20% of reaction volume
into lanes 3 and 4. Products were fractionated on a
1% agarose, 0.5
TBE gel and stained with ethidium bromide. B, amplification reactions were performed using a sense strand
primer, which hybridizes to a site in the 5`-untranslated region of the
type VIII cDNA constructs and an antisense primer to a region on the 3`
side of the 5` ends of the type VIII-B and -C cDNA clones as described
under ``Experimental Procedures.'' Reactions contained 0.1
µg of rat genomic DNA template from two separate preparations (lanes 1 and 2) or no genomic DNA (lane 3).
The products from 10% of the final reaction volume were fractionated as
in A. C, 8.5 µg of rat genomic DNA was cut with PstI and SfaNI (lane 1) or PstI (lane 2) and subjected to Southern hybridization analysis as
described under ``Experimental Procedures.'' D,
schematic representation of the structure of the 5` end of the rat type
VIII AC gene. The open box represents the exon that includes
the 5` coding sequence, and the broken line represents an
intron. A, B, and C indicate positions
corresponding to the 5` ends of the type VIII-A, -B, and -C cDNAs,
respectively. Relative positions are indicated for the PstI
and SfaNI cleavage sites, and the forward (F) and
reverse (R) PCR primers used for amplification of genomic DNA
(see panels B and C, and ``Experimental
Procedures'').
Both the rat type VIII-B and -C cDNA clones extend into the 3`-untranslated region but are incomplete at their 5` ends, terminating 13 and 585 nucleotides, respectively, 3` of the ATG encoding the initiator methionine in the type VIII-A cDNA. Therefore, PCR and Southern hybridization analyses were combined to demonstrate that the three type VIII messages share the same 5` end (Fig. 1, B-D). Rat genomic DNA was used as a template for amplification with a sense strand primer corresponding to 5`-untranslated sequence that is 5` of stop codons in all reading frames in the type VIII-A cDNA, and an antisense strand primer complementary to a region 3` of the start of both the partial type VIII-B and -C cDNAs. A single 1220-bp product is detected corresponding both in size (Fig. 1B) and exact sequence to that reported for the type VIII-A cDNA. Southern hybridization was performed with a probe internal to the genomic PCR product (Fig. 1, C and D). When the rat genomic DNA is digested with PstI and SfaNI, a single fragment of the size predicted based on the cDNA sequence is observed, consistent with the PCR results (Fig. 1C, lane 1). This indicates that all three splice variants share the same initiator methionine. Digestion of the genomic DNA with PstI alone indicates that there are introns in the type VIII gene since the probe hybridized to a single band of approximately 2600 bp rather than a 1640-bp fragment that is predicted based on the cDNA sequence (Fig. 1C, lane 2).
The open reading frames within the type VIII-B and
-C messages encode proteins of 1218 and 1182 amino acids, respectively,
while the type VIII-A cDNA encodes the 1248-amino acid protein
previously described(13) . Each of the type VIII splice
variants conforms well to the topography typical of the membrane-bound,
mammalian adenylyl
cyclases(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) ,
including the two clusters of 6 potential transmembrane spans and
putative nucleotide binding domains(3, 21) . The
90-base pair region spliced out of the type VIII-B message encodes
amino acids 802-831 in type VIII-A adenylyl cyclase, which
includes two consensus N-linked glycosylation sites (Asn-814
and Asn-818). This 30-amino acid domain is flanked on the
NH-terminal and COOH-terminal sides by putative
transmembrane helices 9 and 10, respectively, and is predicted to be on
the extracellular side of the membrane (Fig. 2). The 198-bp
region spliced from the type VIII-C message encodes amino acids
635-700 in type VIII-A, which includes two consensus casein
kinase II phosphorylation sites (Ser-659 and Ser-695) and a consensus
bipartite nuclear targeting sequence (amino acids 666-682) as
determined by sequence analysis with the program, Prosite. However,
there is no evidence to indicate the sites have these functional roles in vivo. This 66-amino acid stretch is in the C
region of the first large cytoplasmic domain (Fig. 2),
which can vary considerably among the adenylyl
cyclases(5, 19, 20) . Other considerations of
topography and sequence homology discussed previously for type VIII-A
are the same for types VIII-B and C(13) .
Figure 2:
Schematic representation of predicted
structures of types VIII-A, -B and -C AC. The differences in the
topography of the three type VIII splice variants are depicted based on
analysis of their sequences and comparison to a model initially
proposed for type I AC(2) . A glycosylated 30-amino acid
extracellular domain is predicted between transmembrane spans 9 and 10
in types VIII-A and -C, but is not present in type VIII-B. The hatched lines indicate where alternative splicing of the
message has resulted in the excision of sequence encoding 66 amino
acids in the C region of type
VIII-C.
Figure 3: Immunoblot analysis of the expression of types VIII-A, -B, and -C AC in HEK-293 cells and rat brain membranes. A, whole cell lysates prepared from HEK-293 cells stably transformed with the type VIII expression constructs were subjected to SDS-PAGE and immunoblotted as described under ``Experimental Procedures.'' The amount of total cell protein loaded was 10 µg. The amount of this derived from a particular transformed cell population is indicated, and the difference was made up by the addition of vector/HEK-293 cell lysate where needed. B, membranes prepared from the stably transformed HEK-293 cells were subjected to SDS-PAGE and immunoblot analysis directly or after treatment with N-glycosidase F as described under ``Experimental Procedures.'' When the primary antibody was Ab VIII-A 1229-1248 or Ab VIII-A 666-682, 2.5 µg of vector/HEK-293 cell membranes, 0.5 µg of type VIII-A/HEK-293 cell membranes, 1.5 µg of type VIII-B/HEK-293 cell membranes, and 2.5 µg of type VIII-C/HEK-293 cell membranes were loaded. Vector/HEK-293 cell membranes were added to equalize the loads where needed. When the primary antibody was Ab VIII-A 804-813, 6 times more membrane protein was loaded. C, 25 µg of rat brain membranes were loaded in each lane (see ``Experimental Procedures''). The primary antibody was incubated with the antigenic peptide (0.1 µg/ml) for 1 h prior to incubation with the Immobilon membrane in the lane designated ``+peptide.''
Ab VIII-A 804-813 was raised against a peptide that should not be present in type VIII-B because the sequence encoding it has been removed from the message by alternative splicing. A nonspecific species of approximately 120 kDa is detected by this antiserum in all of the membranes including those prepared from the vector/HEK-293 cells (Fig. 3B). As expected, no 125-kDa immunoreactive species can be detected with this primary antibody in membranes prepared from type VIII-B/HEK-293 cells. In membranes prepared from type VIII-A/and -C/HEK-293 cells, this antiserum recognizes the deglycosylated 125-kDa species more readily than the glycosylated 165-kDa species. This result can be rationalized if the interaction of Ab VIII-A 804-813 with the 165-kDa species is sterically hindered by the presence of N-linked glycans adjacent to the epitope in types VIII-A and -C.
Ab VIII-A 666-682 was raised against a peptide that is not present in type VIII-C because the sequence encoding it has been spliced out of the message. The patterns of immunoreactivity observed with this antibody in HEK-293 cell membranes are identical to those observed with Ab VIII-A 1229-1248 except in type VIII-C/HEK-293 cells where no bands can be detected (Fig. 3B). The immunoblots confirm the expression of the type VIII-A, -B, and -C splice variants in the stably transformed HEK-293 cell populations. Neither Ab VIII-A 804-813 nor Ab VIII-A 666-682 was sufficiently sensitive or specific to unambiguously detect endogenous type VIII in a rat tissue, but the three immunoreactive species observed with Ab VIII-A 1229-1248 (Fig. 3C) are consistent with the expression of the type VIII AC variants in rat brain.
Figure 4:
cAMP accumulation and fura-2 fluorescence
in HEK-293 cell populations stably expressing type VIII-A, -B, or -C
AC. A-E, cAMP accumulation over time (mean ±
S.E., n = 4) was measured as described under
``Experimental Procedures'' with the additions indicated at
the top of each panel (final concentrations: 1 µM 4-bromo-A23187, 100 µM ATP, 100 µM Ro
20-1724). For each set of conditions, measurements were made on
Geneticin-resistant HEK-293 cell populations selected after
transfection with the pCMV5-neo vector (), type VIII-A (
),
type VIII-B (
), or type VIII-C (
) expression constructs.
The notation + or - Ca
,
indicates that the assay medium was Hepes-buffered DMEM prepared either
with or without 1.8 mM CaCl
. C, cAMP
accumulation presented in B has been scaled to the relative
expression of the type VIII variants as determined by densitometric
analysis of immunoreactivity (Fig. 3A). F,
agonist-induced changes in intracellular Ca
were
recorded over time in type VIII-B/HEK-293 cells that were loaded with
the fluorescent Ca
indicator, fura-2. Fluorescence
emission was measured at 505 nm in response to excitation at 340 nm (
F
) and 380 nm (
F
) as described under
``Experimental Procedures.'' Corrected fluorescence ratios
shown are signal averages of triplicate determinations for each set of
conditions indicated.
The P purinergic agonist, ATP, stimulates cAMP synthesis in type
VIII-A/, -B/, or -C/HEK-293 cells, although there are quantitative
differences in the responses (Fig. 4B). In the presence
of 100 µM Ro 20-1724, a cAMP phosphodiesterase inhibitor,
maximal cAMP accumulation is achieved 2-5 min after the addition
of ATP in type VIII-A/or -C/HEK-293 cells and at 1-2 min in type
VIII-B/HEK-293 cells (Fig. 4B), with no response in
control cells. The time course of the ATP-induced cAMP changes lags
behind the increase in intracellular Ca
monitored by
fura-2 fluorescence (Fig. 4F). cAMP content decreases
at times greater than 5 min, regardless of which type VIII splice
variant is expressed. Addition of a mixture of five phosphodiesterase
inhibitors attenuates, but does not eliminate the decrease in cAMP
content that occurs at longer times (data not shown), indicating that
incomplete inhibition of phosphodiesterases by Ro 20-1724 contributes
to this decay. ATP-dependent cAMP synthesis can be measured in the
absence of any phosphodiesterase inhibitor, but the decay is more rapid
and the maximal values are reduced to about half those measured in the
presence of Ro 20-1724 (Fig. 4D). Intracellular
Ca
concentration changes are also reduced in the
absence of Ro 20-1724 (Fig. 4F). This suggests that a
component of the Ca
influx observed at longer times
may be stimulated in a cAMP-dependent manner, and that the decreases in
cAMP accumulation in the absence of Ro 20-1724 may reflect both
enhanced degradation and decreased stimulation because of reduced
Ca
.
cAMP content was normalized to type VIII
immunoreactivity to obtain an estimate for the relative specific
activities of the variants in an intact cell assay (Fig. 4C). There is approximately 8-fold more type VIII
immunoreactivity detected in cells expressing type VIII-A than in those
expressing type VIII-B (Fig. 3A), and the differences
in maximal cAMP accumulation at early time points (Fig. 4B) simply reflect the relative expression levels
of these two variants (Fig. 4C). However, maximal
increases in cAMP content in HEK-293 cells expressing type VIII-C are
approximately 3-fold higher than would be expected if the ratio of cAMP
accumulation to immunoreactivity were constant for each variant (Fig. 4C). Expression of the splice variants does not
differentially affect the magnitude or time course of ATP-dependent
changes in fura-2 fluorescence (additional data not shown) indicating
that differential changes in intracellular Ca concentrations can not explain the apparent enhanced cAMP
accumulation in type VIII-C/HEK-293 cells.
Assays were performed in
medium prepared without added CaCl to determine the
contribution of extracellular Ca
to the stimulation
of cAMP synthesis (Fig. 4, E versus B). There is an
ATP-dependent release of Ca
from intracellular stores
under these assay conditions, but the maximal value of fura-2
fluorescence is decreased and the decay is accelerated (Fig. 4F). cAMP accumulation in minimal Ca
medium is essentially independent of time and is reduced to
approximately one tenth of that measured in the standard assay medium
with 1.8 mM CaCl
(Fig. 4E). This
indicates that the stimulation of the type VIII splice variants by ATP
results primarily from the influx of extracellular Ca
as previously noted for type VIII-A(13) .
Figure 5:
Kinetic characterization of type VIII AC
activity. Membranes (5 µg) prepared from HEK-293 cells stably
expressing type VIII-A, -B, or -C AC were assayed for 10 min at 30
°C in the presence of 17 µM free Ca and 5.74 mM free Mg
or 0.23 mM free Mn
as described under ``Experimental
Procedures.'' For each panel duplicate values from one of at least
three similar experiments are shown. The data were analyzed as
described under ``Experimental Procedures''. A,
MgATP dependence of type VIII AC activity stimulated by 1 µM exogenous calmodulin. B, MnATP dependence of type VIII AC
activity stimulated by 1 µM exogenous calmodulin. C, concentration of exogenous calmodulin is indicated on the abscissa, and MgATP was 0.5 mM. Activities are
expressed on a relative scale.
AC activity in membranes prepared from
type VIII-A/, -B/, or -C/HEK-293 cells increases in a
concentration-dependent manner with the addition of exogenous
Ca/calmodulin (Fig. 5C), while the
activity in control HEK-293 cells is insensitive to this treatment
(data not shown and (13) ). No calmodulin-dependent effect on
AC activity is observed in any of the membrane preparations when free
Ca
is chelated with excess EGTA, although this
treatment significantly inhibits the type VIII activity measured in the
absence of exogenous calmodulin (data not shown and (13) ). The
curves of Fig. 5C have been fitted assuming that the
total concentration of calmodulin stimulating the enzyme is the sum of
the known concentration of exogenously added calmodulin and a parameter
representing the unknown concentration of endogenous calmodulin in the
membrane preparation. The computed estimate for the latter parameter is
8.7 ± 0.5 nM for the three membrane preparations, and
the addition of this single parameter significantly improves the fit (p < 0.0002), although it increases the EC
values(13) . The calculated EC
values for
calmodulin-dependent stimulation of AC activity are 140 ± 21
nM, 116 ± 12 nM, and 30 ± 6 nM
for types VIII-A, -B, and -C, respectively, indicating that type VIII-C
is approximately 4 times more sensitive to stimulation by
Ca
/calmodulin than type VIII-A or -B. Under three
different sets of experimental conditions, the relative ratio of the
calculated EC
values for calmodulin-dependent activation
of type VIII-A or -B to that of type VIII-C was 4.5 ± 0.5 (Fig. 5C and data not shown).
Evidence for alternative splicing of the rat type VIII message was originally obtained by cDNA cloning, and expression of type VIII-A, -B, and -C messages in rat brain has been verified by reverse transcription-PCR (Fig. 1). The variant messages can arise if distinct exons are alternatively excised from the complete type VIII-A precursor transcript to generate the type VIII-B and -C messages. While the rat type VIII-B variant has been characterized here, a bovine homolog was also selected from a brain library (data not shown), and a partial human type VIII cDNA clone that apparently has the properties of the -B variant has been discussed(32) . The conservation of this variant across species indicates that splicing of the type VIII message is physiologically significant.
Three proteins detected in immunoblots of rat brain membranes appear to co-migrate with the variants as expressed in HEK-293 cells, and all of the immunoreactive species are reduced to approximately 125 kDa by treatment with N-glycosidase F (Fig. 3C). Preincubation of the antibody with the antigenic peptide prevents detection of these brain membrane proteins arguing that recognition of all three species is specific. Unfortunately neither Ab VIII-A 666-682 nor Ab VIII-A 804-813 is sufficiently specific to unambiguously identify the presence of additional type VIII sequences in these rat brain membrane proteins. The localization and identification of adenylyl cyclase isoforms in mammalian tissues has been hampered by their low abundance, and most information has therefore come from analysis of mRNA expression(1) .
The structural properties of type VIII-B are distinct from those of type VIII-A, although we have not been able to distinguish their enzymatic properties. The type VIII-B variant is insensitive to treatment with N-glycosidase F (Fig. 3), consistent with the fact that it lacks the extracellular domain between transmembrane spans 9 and 10, which includes consensus N-linked glycosylation sites in all cloned ACs(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . Additional consensus N-linked glycosylation sites are present in the sequence of several ACs in a putative extracellular domain between transmembrane spans 11 and 12(4, 6, 10, 13, 14, 33) . The single site present in this latter domain in type VIII-B is apparently not utilized when the protein is expressed in HEK-293 cells. The expression of non-glycosylated, membrane-associated forms of AC in bovine brain has been proposed based on purification studies using lectin chromatography (34, 35) . The absence of functional glycosylation sites in type VIII-B is expected to alter the post-translational processing pathway which the protein follows, and could result in its localization to a distinct membrane compartment relative to the glycosylated types VIII-A and -C. Differential processing, or localization might contribute to the fact that expression of type VIII-B is reduced relative to that of type VIII-A and -C (Fig. 3). Mutation of the glycosylation sites of type VIII-A and immunocytochemical studies with variant-specific antisera will address these possibilities.
The type VIII-C splice variant has
unique structural properties that affect its enzymatic activity when
assayed both in whole cells and membranes. The exon that is skipped to
generate the type VIII-C message encodes 66 amino acids that are found
in the C region of type VIII-A or -B (Fig. 2). The
calmodulin-binding domain of type I AC has been localized to this
region(23, 24, 25) . There is no significant
sequence homology between the calmodulin-binding domain of type I and
the corresponding sequence in type VIII-A, nor is this region in type
VIII predicted to have the amphipathic structure typical of
calmodulin-binding domains(36) . If the sequences of types I
and VIII-A are aligned, the calmodulin-binding domain of type I
terminates just 7 amino acids before the start of the 66-amino acid
region that is missing from type VIII-C. Deletion of this region
actually enhances the calmodulin-sensitivity of type VIII-C
approximately 4-fold relative to that of type VIII-A or -B as reflected
in their different EC
values for calmodulin-dependent
stimulation (Fig. 5B). This could be a conformational
effect, or the additional 66 amino acids in types VIII-A and -B may
play an inhibitory role. The enhanced calmodulin-sensitivity of type
VIII-C is reflected in whole cell assays; cAMP content in HEK-293 cells
expressing type VIII-C is approximately 3-fold higher than would be
expected if cAMP accumulation were directly proportional to the
relative expression levels of the variants (Fig. 4C).
Stimulation of the type VIII variants requires the modest increases in
intracellular Ca
that result from the influx of
extracellular Ca
( (13) and Fig. 4E). Under these conditions the concentration of
intracellular Ca
is limiting for the formation of the
stimulatory Ca
/calmodulin complex. The relative
differences in the EC
values for
Ca
/calmodulin-dependent stimulation are predicted to
result in greater relative activation of type VIII-C, as supported by
the data.
Total type VIII message is abundantly expressed throughout
the hippocampus of the rat brain, as determined with an oligonucleotide
probe that does not distinguish between the splice
variants(13) . A role for type VIII-A in long term potentiation
(LTP) in the hippocampus was suggested based on the distribution of its
message and its enzymatic properties(13) . N-methyl-D-aspartate receptor activation, which
induces LTP, also causes an influx of extracellular Ca that stimulates cAMP accumulation in the dendritic spines of
post-synaptic neurons in the CA1 field of the
hippocampus(37, 38) . Calmodulin-stimulated ACs have
also been implicated in the regulation of LTP at the mossy fiber
synapses(39) . In this case it is the depolarization-dependent
activation of Ca
channels in the presynaptic axon
terminal that leads to a Ca
influx and activation of
calmodulin-sensitive ACs. This illustrates the need to have
calmodulin-stimulated ACs capable of responding to Ca
influxes of potentially different magnitudes mediated by two
types of channels in distinct neuronal compartments. Alternative
splicing of the type VIII AC message may provide a mechanism to
generate variants with the requisite properties to contribute to the
expression of LTP in both of these pathways.