(Received for publication, August 21, 1995; and in revised form, November 27, 1995)
From the
Alternate splicing of human plasma membrane calcium pump isoform
4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a
and hPMCA4b, which have different downstream regulatory regions. Of the
two, hPMCA4a has a lower affinity for calmodulin and a lower effective
affinity for Ca (Enyedi, A., Verma, A. K., Heim, R.,
Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J.
T.(1994) J. Biol. Chem. 269, 41-43). Additional
consequences of the alternate splice were studied by analyzing the
characteristics of constructs (expressed in COS-1 cells) containing
different portions of the carboxyl terminus of hPMCA4a. Our results
show striking differences in the structure of the calmodulin-binding
and autoinhibitory domains of the two variants. The calmodulin-binding
region of hPMCA4b is a region of about 28 residues, whereas that of
hPMCA4a is about 49 residues long and is probably interrupted by a
region not involved in the binding. The autoinhibitory region of
hPMCA4b (a part of the downstream region that keeps the molecule
inactive in the absence of Ca
-calmodulin) is divided
between the 28-residue calmodulin-binding region and a downstream
region, whereas in hPMCA4a, all of it is contained within the
49-residue calmodulin-binding region.
The plasma membrane Ca pump (PMCA) (
)is a calmodulin-regulated P-type ATPase that is an
essential element in controlling intracellular Ca
concentration. The calmodulin regulation of the pump has been
extensively studied. Most studies have been performed on red cell
membrane preparations, which contain hPMCA4b plus a small amount of
hPMCA1b. Ca
-calmodulin binds tightly to the pump and
activates it by increasing its V
and apparent
affinity for Ca
. A similar activation of the pump can
be achieved by removing about 15 kDa from its carboxyl terminus by
proteolysis. After the calmodulin-binding domain had been located at
the carboxyl terminus of the enzyme (James et al., 1988; Verma et al., 1988), a synthetic peptide containing the 28 residues
believed to be responsible for calmodulin binding was shown to bind
calmodulin even more tightly than the intact enzyme (Enyedi et
al., 1989). This peptide also inhibited the pump that was
activated by proteolytic removal of the calmodulin-binding domain and
the whole carboxyl terminus. This was the first report that
demonstrated that the calmodulin-binding domain itself may also serve
as an internal inhibitor of the enzyme.
More direct evidence for the dual role of the calmodulin-binding domain was provided later utilizing an overexpression system in COS-1 cells. After the successful expression of the wild type hPMCA4b (Adamo et al., 1992), a mutant of hPMCA4b called ct120 was made. This mutant, which lacked the calmodulin-binding domain and all residues downstream of it, was fully active and did not bind or respond to calmodulin (Enyedi et al., 1993). Adding back all 28 residues of the calmodulin-binding domain, which made a construct called ct92 (Verma et al., 1994; see also Fig. 1), induced a substantial inhibition of the pump in the absence of calmodulin. This inhibition, however, was only about of the inhibition that was observed in the full-length pump, suggesting that other segments of the downstream region are also involved in self-inhibition of isoform 4b. ct92, on the other hand, had an apparent affinity for calmodulin just as high as that of the full-length enzyme, indicating that the portion of hPMCA4b downstream of the 28-residue calmodulin-binding domain played no role in calmodulin binding.
Figure 1: Carboxyl-terminal sequences of hPMCA4a, hPMCA4b, and some of their truncated versions. The truncated mutants are named according to the number of residues cut off at the carboxyl terminus (i.e. 4a(ct56) is hPMCA4a lacking the last 56 residues). 4b(ct120) lacks the entire region shown here, so that it ends just before the sequence LRRG at position 1085 (Enyedi et al., 1993). Because the alternate splice only changes the structure of the molecule downstream of residue 1104, all constructs are identical upstream of this residue. Basic amino acid residues are underlined, and acidic ones are boxed.
The upstream 19 residues of the
calmodulin-binding domain are conserved in all the PMCA isoforms, but
an alternate RNA splice affecting the middle of the sequence coding for
this region changes the structure of the rest of the calmodulin-binding
domain and the entire carboxyl terminus. The isoforms generated from
transcripts containing an additional spliced-in exon are called
``a'' (or CII) and those produced from mRNA lacking this exon
are called ``b'' (or CI). Because the change to the a forms
involves the entire carboxyl-terminal regulatory region and produces a
less basic calmodulin-binding domain, these forms are expected to have
different regulatory properties. Indeed, studies using synthetic
peptides corresponding to the calmodulin-binding domain showed that the
peptide representing an a form had ten times lower affinity for
calmodulin than the peptide representing a b form (Enyedi et
al., 1991). Subsequently, the full-length a and b forms of hPMCA4
were overexpressed in COS-1 cells, and their calmodulin-response curves
were compared (Enyedi et al., 1994). As expected, hPMCA4a
showed lower apparent affinity for calmodulin than hPMCA4b. This change
in the calmodulin affinity resulted in a reduced sensitivity of the
pump isoform 4a to Ca, which is probably the most
important physiological consequence of the alternate splice.
In this
report, additional consequences of the alternate splice were analyzed
by studying the characteristics of constructs containing different
portions of the carboxyl terminus of hPMCA4a. 4a(ct56), a construct
similar to 4b(ct92) containing the 28 residues believed to constitute
the calmodulin-binding domain, had been used to show that the
differences in the Ca sensitivity between the
isoforms originated from the differences in their respective
calmodulin-binding domains. Closer inspection of 4a(ct56), however,
showed that unlike 4b(ct92), it has lower affinity for calmodulin than
its full-length parent and that like 4b(ct92) its activity is higher in
the absence of calmodulin than the activity of its parent. To further
study this region, more residues of the carboxyl terminus were added to
4a(ct56) and two additional constructs, called 4a(ct44) and 4a(ct35)
were produced. The characteristics of 4a(ct35) appeared to be identical
to those of full-length hPMCA4a, indicating that in hPMCA4a both
calmodulin-binding and inhibitory characteristics reside in a region of
about 49 residues at the carboxyl terminus.
To analyze the calmodulin-binding regulatory region of hPMCA4a, constructs containing different portions of the carboxyl terminus have been synthesized. The differences in the carboxyl-terminal sequences of the mutants are shown in Fig. 1. 4a(ct56) contains 28 residues of the putative calmodulin-binding domain but lacks everything (56 residues) downstream of it. 4a(ct44) and 4a(ct35) are 12 and 21 residues longer than 4a(ct56) (lacking only 44 and 35 residues at the carboxyl terminus), respectively. As judged by SDS gel electrophoresis followed by immunoblotting (Fig. 2A), each construct was expressed at a high level similar to that of the full-length enzyme and migrated with the expected size, which was in between full-length hPMCA4a and ct120. ct120 is only 28 residues shorter than 4a(ct56), but it has been called ct120, indicating that it lacks 120 residues from the carboxyl terminus of full-length hPMCA4b, which is 36 residues longer than hPMCA4a (Enyedi et al., 1993).
Figure 2:
Demonstration of expression of
functionally active hPMCA4a and its truncated versions 4a(ct56),
4a(ct44), and 4a(ct35) in COS-1 cells. A, immunoblots of
microsomes from cells transfected with cDNA encoding hPMCA4a and the
mutants. 2 µg of membrane protein was applied on each lane of a
7.5% SDS-polyacrylamide gel according to Laemmli. After the proteins
were separated on the gel they were immunoblotted. The slower migrating
faint band seen on each lane corresponds to the endogenous calcium pump
of COS-1 cells. B, formation of the phosphoenzyme intermediate
of hPMCA4a constructs. 10 µg of each of the membrane preparations
shown in A was subjected to phosphorylation from
[-
P]ATP on ice in the presence of 50
µM Ca
and 50 µM La
as described under ``Materials and
Methods.'' After trichloroacetic acid precipitation, 2 µg of
each phosphorylated sample was applied on a 7.5% acidic
SDS-polyacrylamide gel according to Sarkadi et al.(1986). An
autoradiography of the phosphorylated proteins is shown after overnight
exposure of the dried gel.
As shown in the autoradiogram of Fig. 2B, each construct had the capability to form the
phosphorylated intermediate (EP) of the enzyme from
[-
P]ATP in the presence of
Ca
. The phosphorylation of the mutants was greatly
enhanced by La
(the Ca
+
La
-dependent EP formation is shown in the figure) as
is characteristic of this pump. That the strong phosphorylated bands
correspond to the mutants can be seen from their characteristic
migration pattern (compare with Fig. 2A). The slower
migrating faint radioactive bands also seen on the immunoblots of Fig. 2A are due to the endogenous Ca
pump of COS-1 cells, whereas the faster migrating faint
radioactive bands are most probably due to the incomplete inhibition of
the EP formation of the endoplasmic reticulum Ca
pump
by thapsigargin. Each construct showed high ATP-powered,
phosphate-enhanced Ca
uptake into microsomes from the
transfected COS-1 cells when measured in the presence of calmodulin.
The Ca
transport activity varied within the range of
6-12 nmol/mg membrane protein/min depending on the level of
expression. In order to compare the maximum activities of the mutants
and the wild type, each activity was corrected for expression level, as
determined by a scanned Western blot. When this was done, no
significant differences were found between the activities. Both the
phosphorylation and Ca
transport experiments prove
that each mutant was functionally active. This was expected because the
mutations affected only the regulatory region, and no mutations were
done at the active sites of the molecule.
To study the
characteristics of the mutants, their Ca transport
activities were tested as a function of calmodulin concentration at a
relatively high, fixed Ca
concentration. In Fig. 3, the calmodulin response curves of the constructs are
compared with that of the full-length hPMCA4a. 4a(ct56), containing all
28 residues of the putative calmodulin-binding domain, showed lower
sensitivity to calmodulin than full-length hPMCA4a; it required about 3
times higher calmodulin concentration for half-maximal activation. This
result was rather surprising because ct92, a similar construct of
hPMCA4b, had been shown to have the same sensitivity to calmodulin as
full-length isoform 4b (Verma et al., 1994). Thus in hPMCA4b
no residues beyond the 28 residue region are involved in calmodulin
binding. In contrast, hPMCA4a required an additional 21 residues
downstream of the 28-residue region for full calmodulin affinity; the
calmodulin response curve of 4a(ct35) was indistinguishable from that
of the full-length enzyme, whereas 4a(ct44) still had slightly lower
calmodulin affinity. To put this finding in context, it is important to
remember that full-length hPMCA4a has about a 5-fold lower apparent
affinity for calmodulin than full-length hPMCA4b (Enyedi et
al., 1994).
Figure 3:
4a(ct56) is less sensitive to calmodulin
stimulation than is full-length hPMCA4a. Calmodulin concentration
dependence of Ca uptake by microsomal vesicles
isolated from COS-1 cells transfected with hPMCA4a (circles)
and the truncated mutants 4a(ct56) (diamonds), 4a(ct44) (filled triangles), and 4a(ct35) (open triangles) is
shown. The membrane vesicles were incubated at 37 °C in the
presence of the appropriate concentration of calmodulin at a free
Ca
concentration of 1.2 µM for 3 min,
after which Ca
uptake was initiated by the addition
of ATP. After subtracting the values of control membrane vesicles,
Ca
uptake was expressed as f = (v - V
)/(V
- V
), where V
is the activity
at 1.2 µM free Ca
in the absence of
calmodulin, v is the activity in the presence of the
appropriate calmodulin concentration, and V
is the
maximum Ca
uptake at 1.2 µM Ca
, which was determined by fitting the data of
activity versus calmodulin concentration to the
Michaelis-Menten equation. In all cases, the highest activity measured
was at least 90% of the V
calculated. V
varied from one transfection to the next,
because of variation in the transfection level ranging from 5 to 10
nmol, mg membrane protein
, min
.
The expression level of each membrane preparation was judged by digital
scanning of the immunoblots. Data points are averages of two to three
independent determinations on two to three different preparations. The K
values ± standard deviation were:
hPMCA4a, 0.102 ± 0.004 µM; 4a(ct35), 0.115 ±
0.012 µM; 4a(ct44), 0.180 ± 0.032 µM;
and 4a(ct56), 0.303 ± 0.098
µM.
In Fig. 4, the Ca transport activities of the mutants without calmodulin are
compared with that of full-length hPMCA4a. The procedure used for
preparation of the membranes removed calmodulin and avoided the
activation of the pump protein by proteolysis as much as possible so
that the activities measured in this experiment would represent the
activity of the intact mutant without calmodulin. The maximum activity
of each mutant was also determined in the presence of saturating
concentrations of Ca
and calmodulin, and the
activities measured in the absence of calmodulin were expressed as a
percentage of this maximum. Thus, the possibility that the observed
differences in the activities were due to a variation in the expression
level of the different mutants in the COS cell membranes could be
avoided. When measured in the absence of calmodulin, 4a(ct56) showed
higher activity than full-length enzyme. These data were consistent
with results obtained with a similar construct of hPMCA4b (Verma et
al., 1994). Maximal self-inhibition was achieved by adding 12 more
residues to 4a(ct56); in the absence of calmodulin, 4a(ct44) had the
same low activity as full-length enzyme. It is worth mentioning that
the activity of hPMCA4a in the absence of calmodulin was measured at
several Ca
concentrations and was always higher than
the activity of hPMCA4b (data not shown). The greater self-inhibition
observed in hPMCA4b is under investigation.
Figure 4:
The activity of 4a(ct56) is higher than
that of full-length hPMCA4a when measured in the absence of calmodulin.
Ca uptake by microsomal vesicles isolated from COS-1
cells transfected with hPMCA4a, 4a(ct35), 4a(ct44), and 4a(ct56) was
measured in the absence of calmodulin at 7 µM free
Ca
concentration. Maximum Ca
uptake
was also determined for each construct at 7 µM free
Ca
and at a saturating calmodulin concentration, and
the activities shown were expressed as a percentage of this maximum.
Ca
uptake by control vesicles has been subtracted
from all data points. The data points represent the average of three
independent determinations on three different preparations ±
standard deviation.
Finally, the
characteristics of each mutant were analyzed by measuring the
dependence of Ca uptake on free Ca
in the presence and the absence of calmodulin (Fig. 5). In
the absence of calmodulin, at all Ca
concentrations
tested, 4a(ct56) was more active than full-length hPMCA4a, whereas
4a(ct44) had a low activity similar to that of its parent enzyme. When
measured in the absence of calmodulin, the 12 residues added to
4a(ct56) affected only the maximum velocity without having any effect
on the apparent affinity for Ca
(for all three
mutants, K
for Ca
was in the
range 0.5-0.6 µM). In the presence of a high
calmodulin concentration (4.7 µM), the picture was quite
different. The maximum velocity of the constructs was the same (it
varied only somewhat with the expression level of the mutants), whereas
the apparent Ca
affinity was different. When measured
in the presence of high calmodulin, 4a(ct56) was less responsive to
Ca
stimulation, with a K
of
0.55 ± 0.05 µM, than the full-length enzyme (K
of 0.35 ± 0.04 µM), and a
slight difference in the Ca
response curves was
observed even between 4a(ct44) and full-length hPMCA4a. This difference
in the apparent Ca
affinities came from the
difference observed in the apparent calmodulin affinity of the
constructs, similar to the difference previously observed between
full-length hPMCA4b and hPMCA4a (Enyedi et al., 1994). The
Ca
curve of 4a(ct35) was indistinguishable from the
parent enzyme, as expected (not shown). Because 4a(ct35) had the same
characteristics as the full-length hPMCA4a, we conclude that the
calmodulin-binding and inhibitory domains in hPMCA4a reside in the same
region of about 49 residues (Fig. 6), with the inhibitory domain
being slightly shorter than the calmodulin-binding domain.
Figure 5:
Characteristics of hPMCA4a constructs as a
function of free Ca concentration. Ca
uptake by microsomal vesicles from hPMCA4a (circles),
4a(ct44) (triangles), and 4a(ct56) (diamonds) was
measured in the absence (open symbols) and in the presence (filled symbols) of 4.7 µM calmodulin. Maximum
activities of the constructs were determined at a saturating free
Ca
and calmodulin concentration, and Ca
uptake activities were expressed as a percentage of this maximum
activity. The control values were subtracted from each data point. The
data points represent the average of three independent determinations
on three different preparations. The lines represent the best
fit to the data given by the Hill equation. In the absence of
calmodulin, V
for 4a(ct56) was 63 ± 3%,
whereas V
for 4a(ct44) was 34 ± 4% and
for hPMCA4a was 42 ± 1.5% of the corresponding V
in the presence of calmodulin. The
characteristics of 4a(ct35) were indistinguishable from those of the
full-length hPMCA4a, but this is not shown in the
figure.
Figure 6: Structural differences between regulatory regions of hPMCA4a and hPMCA4b. In hPMCA4a both calmodulin binding and self-inhibition reside within a 49-residue region. In hPMCA4b the calmodulin-binding domain is only 28 residues long, but additional residues contributing to self-inhibition reside in a region further toward the carboxyl terminus. The location of this second inhibitory region has not been determined yet. Basic and acidic residues are marked as in Fig. 1.
All of the results concerning the calmodulin binding and
inhibitory properties of the calmodulin-binding domain of PMCA isoform
4a must be considered in the context of its overall lower calmodulin
affinity and less effective autoinhibition when compared with isoform
4b. The lower affinity of the full-length hPMCA4a for calmodulin has
already been described (Enyedi et al., 1994), and its less
effective autoinhibition is currently under study and has been
described in an abstract (Enyedi et al., 1995). The unexpected
characteristics of the calmodulin-binding domain of hPMCA4a described
here show the presence of a region that is less efficient in both of
its functions, a property that must be desirable in certain kinds of
cells. Studies utilizing polymerase chain reaction techniques have
shown that transcripts for hPMCA4a are present in substantial amounts
in brain, in tissues containing smooth muscle, (uterus, stomach, small
intestine, and colon), and in the pancreas. PMCA4a mRNA is also present
in skeletal muscle, heart, and lung (Keeton et al., 1993;
Stauffer et al., 1993). Because the lower calmodulin
sensitivity of 4a leads to a lower Ca sensitivity
(Enyedi et al., 1994), it is probable that pump isoform 4a
occurs in cells that have more intense Ca
spikes and
higher sustained levels of intracellular Ca
.
Because the calmodulin-binding site of isoform 4b is restricted to the 28 residues originally envisioned for this role (Verma et al., 1994), the novel properties of the 4a calmodulin-binding site are interesting. Inspection of the sequences of these two forms (Fig. 1) discloses the probable structural basis for the differences observed here. In isoform 4b, the 20 residues downstream of the 28-residue calmodulin-binding domain constitute a region with a net charge of +l that contains few hydrophobic residues. Such a region would not be expected to make a significant contribution to calmodulin binding. The calmodulin-binding domain of isoform 4a shows quite a different structure, because it is interrupted at its 20th residue by a negatively charged aspartic acid. The nine residues from this aspartic acid to the next glycine downstream have no positively charged amino acids so that this region has a net charge of -1. This contrasts with the net charge of +2 for the corresponding residues in 4b, which are combined with an appropriate number of hydrophobic residues.
The next region immediately downstream in 4a appears to be much more favorable for binding of calmodulin. The next 16 residues(1114-1129) have three positively charged side chains and a substantial number of hydrophobic residues. Indeed, motifs that appear at the beginning of the calmodulin-binding domain are repeated here. The first seven residues of the calmodulin-binding domain are LRRGQIL; the LRR is repeated in residues 1120-1122, and the GQIL is echoed by GQHL in residues 1126-1129. Its positive charge and content of hydrophobic residues render this region a probable contributor to calmodulin binding, in contrast with the relatively hydrophilic nature and low positive charge present in the corresponding region of isoform 4b. In the similar isoforms 1a, 1c, and 1d, the presence of an analogous second putative calmodulin-binding region was noted by Kessler et al.(1992), who suggested that it might contribute to calmodulin binding.
This analysis of the structure suggests that the calmodulin-binding domain of hPMCA4a consists of two shorter calmodulin-binding regions interrupted by the region containing the aspartic acid at position 1105. It is interesting to speculate that the region surrounding this aspartic acid may not be in direct contact with calmodulin, but rather may be folded out. If so, the calmodulin-enzyme complex would consist of two helical regions interacting directly with calmodulin separated by a hairpin that is folded out of the interaction surface. Thus, it appears that isoform 4a has a two-part calmodulin-binding domain, which is less effective than the domain in isoform 4b but still binds calmodulin tightly, so that calmodulin remains a physiologically important regulator for this pump.
The differences between the structures of the calmodulin-binding
domains and inhibitory domains of isoforms 4a and 4b are summarized in Fig. 6. In isoform 4b, a substantial part of the inhibition
contributed by the carboxyl terminus is due to a region in an as yet
undefined position beyond the end of the 28-residue calmodulin-binding
domain. In isoform 4a, the calmodulin-binding domain is much longer,
and we have shown here that the inhibitory domain is included within
the calmodulin-binding domain. Although the inhibitory region is
slightly shorter, most of the determinants of calmodulin binding also
contribute to inhibition, and there is no inhibitory region beyond the
end of the calmodulin-binding domain in isoform 4a. These interesting
structural differences provide a framework that in large part accounts
for the differences in calmodulin affinity and basal activity between
isoforms 4a and 4b, a pattern that is probably followed by the a and b
isoforms of the other three gene products of the plasma membrane
Ca pump.
Other calmodulin-regulated enzymes also
have calmodulin-binding and autoinhibitory regions. In most cases these
regions overlap only partially, if at all; in smooth muscle myosin
light chain kinase (Krueger et al., 1995) and in
calmodulin-dependent protein kinases I (Yokokura et al., 1995)
and II (Brickey et al., 1994), the autoinhibitory region is
upstream of the calmodulin-binding region. In calmodulin-dependent
protein kinase I, there is little or no overlap between the regions.
Thus isoform 4a of the plasma membrane Ca pump is
unusual because all of its inhibitory determinants are contained within
the calmodulin-binding domain.