From the Department of Ophthalmology/Kresge Eye
Institute and the ¶ Department of Pharmacology, Wayne State
University School of Medicine, Detroit, Michigan 48201 and the
§ Department of Biochemistry and HHMI, University of
Washington, Seattle, Washington 98195
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ABSTRACT |
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Guanylate cyclase regulator protein (GCAP)-2 is a
Ca2+-binding protein that regulates photoreceptor
outer segment membrane guanylate cyclase (RetGC) in a
Ca2+-sensitive manner. GCAP-2 activates RetGC at free
Ca2+ concentrations below 100 nM,
characteristic of light-adapted photoreceptors, and inhibits RetGC when
free Ca2+ concentrations are above the 500 nM
level, characteristic of dark-adapted photoreceptors. We have mapped
functional domains in GCAP-2 by using deletion mutants and chimeric
proteins in which parts of GCAP-2 were substituted with corresponding
fragments of other closely related recoverin-like proteins that do not
regulate RetGC. We find that in addition to the EF-hand
Ca2+-binding centers there are three regions that contain
GCAP-2-specific sequences essential for regulation of RetGC. 1) The
region between Phe78 and Asp113 determines
whether GCAP-2 activates outer segment RetGC in low or high
Ca2+ concentrations. Substitution of this domain with the
corresponding region from neurocalcin causes a paradoxical behavior of
the chimeric proteins. They activate RetGC only at high and not at low
Ca2+ concentrations. 2) The amino acid sequence of GCAP-2
between Lys29 and Phe48 that includes the
EF-hand-related motif EF-1 is essential both for activation of RetGC at
low Ca2+ and inhibition at high Ca2+
concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of
GCAP-2 function. 3) Region Val171-Asn189,
adjacent to the C-terminal EF-4 contributes to activation of RetGC, but
it is not essential for the ability of Ca2+-loaded GCAP-2
to inhibit RetGC. Other regions of the molecule can be substituted with
the corresponding fragments from neurocalcin or recoverin, or even
partially deleted without preventing GCAP-2 from regulating RetGC.
Substitution of these three domains in GCAP-2 with corresponding
neurocalcin sequences also affects activation of individual recombinant
RetGC-1 and RetGC-2 expressed in HEK293 cells.
Guanylate cyclase activating proteins
(GCAPs)1 are
Ca2+-binding proteins that mediate regulation of cGMP
synthesis by Ca2+ in retinal rods and cones.
Ca2+ enters outer segments (OS) of vertebrate
photoreceptors through cGMP-gated Na+/Ca2+
channels in the plasma membranes. These channels are open in the dark,
but light closes them by stimulating cGMP hydrolysis. Channel closure
lowers the intracellular free Ca2+ concentrations in rods
from 700 nM to near 30 nM (1) because Ca2+ is removed from the OS by a light-independent
Na+/K+, Ca2+ exchanger (2-4). The
decrease in free Ca2+ concentration stimulates cGMP
synthesis (5). cGMP, is synthesized in photoreceptors by two membrane
guanylate cyclases, RetGC-1 and RetGC-2 (also referred to as ROSGC-1
and -2, or CG-E and GC-F, respectively) (5-11). The RetGCs are
regulated by two homologous, but distinct Ca2+-binding
proteins, GCAP-1 and GCAP-2 (6, 12-15) (Fig. 1). When free
Ca2+ concentrations decrease from 700 nM to
below 50 nM, GCAP-1 and GCAP-2 (6, 7, 9-14) activate
membrane RetGC. GCAP-1 and GCAP-2 each have four EF-hand-like domains
including three functional EF-hands that bind Ca2+ ions. At
free Ca2+ concentrations below 100 nM,
characteristic of light-adapted rods, GCAPs activate the cyclase, but
at free Ca2+ concentrations above 600 nM
(characteristic of dark-adapted photoreceptors) GCAPs inhibit it (16,
17). Inactivation of EF-hands makes GCAPs constitutive activators of
RetGC (16, 17).
Despite their functional and structural homology, GCAP-2 and GCAP-1
appear to be different in several aspects. 1) The only significant
sequence homology between GCAP-1 and GCAP-2 is within their EF-hands.
2) A naturally occurring point mutation Y99C in GCAP-1 that was linked
to human cone degeneration (18) causes a constitutive Ca2+
insensitive activation of RetGC (19, 20), while the corresponding mutation in GCAP-2 causes partial inhibition of its activity without affecting its Ca2+ sensitivity (19). 3) N-terminal fatty
acylation is reportedly essential for the regulatory properties of
GCAP-1 (21), but it is not essential for GCAP-2 (22). 4) EF-hand 3 in
GCAP-2 contributes Ca2+ sensitivity to RetGC regulation
relatively weakly compared with EF-2 and EF-4 (16), whereas EF-3 plays
a more significant role in GCAP-1 (17). 5) Krishnan et al.
(23) reported that GCAP-1 and GCAP-2 may interact with two different
sites on RetGC.
The goal of this study was to define major regulatory domain(s) of
GCAP-2 using its deletion and chimera mutants. We find that in addition
to the previously characterized EF-hand Ca2+-binding
domains, there are three regions in GCAP-2 that cannot be deleted or
substituted without loss of function. One of these regions determines
that the Ca2+-free form of GCAP-2 activates, and the
Ca2+-bound form inhibits RetGC activity in photoreceptor membranes.
Site-directed Mutagenesis (Fig. 2)--
Chimera DNAs were
constructed by using polymerase chain reaction (PCR) and "splicing by
overlap extension." DNA fragments with chimeric termini were first
amplified by PCR using Pfu polymerase (Stratagene), appropriate
cDNAs (neurocalcin- Expression of Recombinant Proteins--
Wild type and deletion
mutants of bovine GCAP-2 and GCAP-2/neurocalcin chimeric proteins were
expressed in E. coli from pET11d vector (Novagen) according
to the procedures described previously in full detail (22) except that
we used the BLR(DE3)pLysS Escherichia coli strain (Novagen).
Wild type GCAP-2 and recoverin/GCAP-2 chimeras were also expressed in
transfected HEK 293 cell culture as described previously (14). Bovine
recoverin and neurocalcin- RetGC Activity Assay--
Washed bovine outer segment membranes
depleted of endogenous activator and containing both RetGC-1 and
RetGC-2 were prepared, reconstituted with recombinant GCAPs, and
assayed as described previously (6, 22). Experiments were conducted
under infrared light using two 15 W safety lights equipped with Kodak
number 11 infrared filters at a distance of 50 cm and Excalibur dual high performance GEN II+ tube (PVS-5c) goggles. A typical reaction mixture contained 5 µM GCAP-2 or its chimera constructs
in 25 µl of 50 mM MOPS-KOH (pH 7.5), 60 mM
KCl, 8 mM NaCl, 10 mM MgCl2, 2 mM Ca/EGTA buffer, 10 µM each of dipyridamole
and zaprinast, 1 mM ATP, 1 mM GTP, 4 mM cGMP, 1 µCi of [ Recombinant Bovine RetGC-1 (ROSGC1, GC-E) and RetGC-2 (ROSGC2,
GC-F)--
These clones were expressed in HEK293 cells as described
previously for human RetGC-1 and -2 (6, 7). Bovine cDNA clones in
pcDNA3.1 vector (23) were a gift from Dr. R. Sharma. DNA constructs
were amplified in E. coli in the presence of ampicillin and
purified using a Promega plasmid DNA purification kit. Cells were
propagated in RPMI 1640 media supplemented with calf serum and
antibiotics in 100-mm diameter culture dishes until approximately 60%
confluent and then transiently transfected with 10-15 mcg of DNA/plate
using a conventional calcium phosphate precipitation. Membranes were
isolated from homogenized cells and reconstituted with GCAP-2
essentially as described (6, 7). Basal activity of recombinant RetGC-1
and RetGC-2 was less than 0.05 nmol/min/mg of protein. Nonspecific
background produced by boiled membranes (near 500 disintegrations/min)
was subtracted. Because activity of recombinant cyclases was much lower
than that of native RetGC in washed OS membranes (6, 7), time of
incubation was increased to 30 min. Approximately 1% of total GTP was
converted into cGMP after stimulation of recombinant RetGC-1 and
RetGC-2 with GCAP-2 during the reaction.
Ca/EGTA Buffers--
The buffers were prepared according to Ref.
28.
Rationale for Mutagenesis and Analyses--
GCAP-1 and GCAP-2 are
overall 40% identical (13, 14). However, most of the sequence identity
between GCAPs derives from their EF-hands and there is almost no
sequence identity in their N and C termini (Fig.
1A). GCAPs belong to a
distinct group of recoverin-like Ca2+-binding proteins (29)
that are distantly related to calmodulin. Recoverin-like proteins have
four helix-loop-helix Ca2+-binding domains, EF-hands,
usually named EF-1 through EF-4. Amino acids that form
Ca2+-binding loops are shown in boxes in Fig.
1A. Unlike calmodulin, the EF-hand loop sequence in EF-1 in
all of the currently characterized recoverin-like proteins is disrupted
and cannot function as a Ca2+-binding center. Like other
recoverin-like proteins, GCAPs also have a signal for the N-terminal
fatty acylation (13, 14). The sequence identity between neurocalcin and
GCAP-2 is almost as high as between GCAP-2 and GCAP-1. Recoverin is
nearly 28%, and neurocalcin is nearly 39% identical to bovine GCAP-2.
Yet unlike GCAPs neither neurocalcin (Fig. 1B) nor recoverin
(as indicated in the inset to Fig. 6A, and in the
accompanying article, Ref. 36) can regulate RetGC activity.
To define the regions of GCAP-2 containing specific structures that are
essential for its ability to regulate RetGC, we produced and
characterized deletion mutants of GCAP-2 as well as chimeric proteins
in which various regions of GCAP-2 were substituted by the
corresponding fragments of neurocalcin or recoverin (Fig. 2).
In order to study the effects of mutations in GCAP-2 and GCAP-1, we
used both native RetGC-containing OS membranes and recombinant RetGC-1
and RetGC-2 expressed in HEK293 cells for the following reasons. 1) OS
membranes are more likely to retain any additional unidentified
components(s) that might be potentially significant for RetGC
regulation in photoreceptors. 2) Relative contribution of RetGC-1 or
RetGC-2 to the overall cGMP synthesis in photoreceptors membranes
remains unknown. 3) It is impossible to exclude a potential existence
of any additional RetGC homologs that can be present in photoreceptors.
4) Activity of recombinant RetGCs is 10-100-fold lower than RetGC
activity in OS membranes. Therefore we consider OS membranes as a
closer in vitro approximation to the physiological conditions in order for studying RetGC regulation by GCAPs. 5) However,
even with all mentioned limitations for using recombinant cyclases for
studying GCAP-2 mutants, the recombinant RetGC-1 and RetGC-2
demonstrate their basic ability to interact with GCAPs and therefore
must reflect at least some of the regulatory properties of the native
enzymes present in photoreceptors. Hence, we also demonstrate that the
key GCAP-2 mutations affecting native RetGC activity in OS also affect
individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.
The Region between EF-2 and EF-3 Determines Whether the
Ca2+ Bound Form of GCAP-2 Activates or Inhibits
RetGC--
For other known EF-hand proteins, such as calmodulin, it is
their Ca2+-loaded form that regulates an effector protein
(29). In contrast, the apo-form of GCAP-2 and GCAP-1 activates RetGC,
and the Ca2+-loaded form inhibits RetGC (16, 17).
Apparently, exposure of regulatory domains in GCAPs is regulated by
Ca2+ binding to the EF-hand domains (16). Mutations in
EF-hands that hamper Ca2+ binding can lock GCAP-2 into its
activator conformation thus preventing the
Ca2+-dependent transition into the inhibitory
conformation (16). The greatest homology between GCAPs and neurocalcin
is within the central part of the molecule that contains all three
functional EF-hands: EF-2, -3, and -4 (Fig. 1A). We refer to
that region as the "Ca2+-binding core region." We
examined the ability of that region of neurocalcin to substitute for
the corresponding region of GCAP-2 by constructing and analyzing the
chimeric proteins shown in Fig. 2 (I-III). Surprisingly,
these chimeras do not activate RetGC at low Ca2+
concentrations. Instead, they activate RetGC only when free
Ca2+ concentrations exceed 200 nM
(Fig. 3, A and B).
Unlike wild type GCAP-2 which is Ca2+-sensitive
activator of RetGC, these chimerical proteins are
Ca2+-dependent activators of RetGC.
It should be noted that chimeras I-III are unable to activate RetGC at
low Ca2+ not because they are simply denatured, or cannot
bind Ca2+. Fig. 3 shows that they are able to activate
RetGC, but that their Ca2+ sensitivity has been reversed
(Fig. 3, A and B). Data for construct II are not
shown, but they are similar to the results with chimeras I and III.
These results imply that the Ca2+-binding core region of
GCAP-2 is not only responsible for binding Ca2+, but also
determines the direction of the switch between the "inhibitor" and
"activator" conformations of GCAP-2.
The neurocalcin structure responsible for this paradoxical effect
appears to be the region between EF-2 and EF-3. In order to verify
this, we substituted the neurocalcin sequence between EF-2 and EF-3 in
chimera III with the corresponding fragment
Phe78-Asp113 from GCAP-2 (numbers are given
according to the GCAP-2 sequence). The resulting mosaic (chimera IV)
demonstrates Ca2+ sensitivity of RetGC typical for GCAP-2
(Fig. 3B). The same GCAP-2-specific fragment,
Phe78-Asp113, when it is inserted into
constructs I and II creates mosaic chimera protein constructs V and VI
in which the length of neurocalcin-specific fragments is extended
toward the EF-4 (Fig. 2A). Unlike chimeras I-III, chimeras V
and VI demonstrate normal regulation of RetGC by Ca2+ (Fig.
3A). We therefore conclude that the direction of the
Ca2+-regulated switch between activator and inhibitor
conformations in GCAP-2 is determined by the GCAP-2-specific amino acid
sequence(s) located between Phe78 and Asp113.
Interestingly, in a related protein, recoverin, this region appears to
act as a swivel or a "hinge" between two halves of the molecule
(33). It is therefore possible that in GCAP-2 it may also form a
similar structure between regulatory domains and thus may determine the
directions of intramolecular rearrangements caused by Ca2+
binding. However, the three-dimensional structures of
Ca2+-free and Ca2+-bound GCAP-2 will be needed
to confirm this possibility.
Other regions of the GCAP-2 Ca2+-binding core structure can
be substituted with the corresponding fragments of neurocalcin (Fig. 3, chimera V and VI), or partially deleted (chimera VII,
Fig. 3C) and this does not cause complete loss of function
or reverse Ca2+ sensitivity of RetGC regulation. This shows
that except for the presence of functional EF-hands, no essential
GCAP-2-specific functions are exclusively determined by the GCAP-2
amino acid sequence located between Glu56 and
Asp77 or between Arg114 and Phe170.
The region Arg114-Phe170 includes a part of
the EF-3 and EF-4 Ca2+-binding domains. Also, the distance
between EF-3 and EF-4 in GCAP-2 is longer than in other recoverin-like
proteins, including GCAP-1 (Fig. 1A). Nevertheless, the
difference in distance between EF-3 and EF-4 does not seem to be a
critical characteristic of the GCAP-2 molecule as a RetGC regulator. An
8-amino acid fragment from that part of the molecule can be deleted
virtually without inactivation of GCAP-2 (Fig. 3C). However,
some other parts of these regions in GCAP-2, in particular, the
entering helix of EF-4 are most likely to contribute to the proper
folding and activity of GCAP-2 molecule because chimeric proteins V and
VI have 2.5-3-fold lower maximal level of RetGC activation and more
than 3-fold increase in EC50 values compared with wild type
GCAP-2 (data not shown).
The C-terminal Region Thr158-Asn189 in
GCAP-2 Contributes to Its RetGC Activation but Not to RetGC
Inhibition--
The very C terminus of GCAP-2 is not required for
activation or inhibition of RetGC. The last 4 amino acids can be
substituted with the amino acids from the recoverin C terminus (Fig.
4). A random 25-amino acid sequence
(adding an extra 10% to the length of GCAP-2, Fig. 2) can be attached
to the C terminus of GCAP-2 without affecting RetGC activation (Fig. 4,
chimera IX). Deletion of up to 9 amino acids from the C terminus does
not completely prevent GCAP-2 from activating RetGC at low
Ca2+ concentrations nor from inhibiting the cyclase in high
Ca2+ (Fig. 5). However,
longer deletions from the C terminus of GCAP-2 in mutants XI (Fig.
5A) and XII (data not shown) cause loss of its ability to
activate RetGC. Consistent with this, GCAP-2 with the EF-4
Ca2+-binding loop substituted with neurocalcin EF-4 (Fig.
3, chimera VI) still activates RetGC while substitution of the
additional C-terminal 18-amino acid sequence
Val171-Asn189 adjacent to the EF-4 loop
abolishes RetGC activation (Fig. 5B, lines XIII and
XIV).
The N-terminal fragment adjacent to the EF-4 in neurocalcin is shorter
than in GCAP-2. However, the following observations suggest that the
C-terminal GCAP-2/neurocalcin chimera protein does not lose its
activator capacity just because it is shorter than wild type GCAP-2. 1)
The length of the C-terminal fragment adjacent to the EF-4 in GCAP-2
deletion mutant X is similar to that in XIII, yet the deletion mutant X
activates RetGC while chimera XIII does not (Fig. 5A, B); 2)
The C-terminal fragment in chimera XIV is 8 amino acid residues longer
than in chimera XIII because it contains an additional fragment
(Trp193-Phe204) from GCAP-2. Chimera XIV is
therefore longer than deletion mutant X, yet it fails to activate RetGC
(Fig. 5B). Importantly, all the deletion mutant and chimeras
shown in Fig. 5 still inhibit RetGC basal activity at high
Ca2+ concentrations.
In summary, the region Val171-Asn189 appears
to contain GCAP-2-specific amino acids essential for activation of
RetGC by apo-GCAP-2. Our results also show that there are no
GCAP-2-specific sequences essential for RetGC inhibition by
Ca2+-loaded GCAP-2 in this region. Almost 30% of the
length of GCAP-2 from its C terminus does not appear to be absolutely
essential for the inhibitory effect of Ca2+-loaded GCAP-2
on the basal activity of RetGC. Even the GCAP-2 deletion mutant, XV,
truncated at Gln140 shortly after EF-3, still inhibits
RetGC basal activity at high Ca2+ concentrations (Fig.
5).
Mutations within the N-terminal Region of GCAP-2--
The N
terminus is the most variable part of GCAP-2. Its length and sequence
varies considerably across animal species. The distance between the
N-terminal glycine and EF-1 in bovine, mouse, chicken (16, 30, 31), and
zebra fish2 GCAP-2 varies
between 27 and 32 amino acid residues. Despite this variability it has
been reported that removal of the first 8 amino acids from bovine
GCAP-2 inactivates it (21). In contrast, our findings suggest that
there is no GCAP-2-specific sequence essential for RetGC regulation in
that region. That is consistent with the relatively high variability of
the N-terminal GCAP-2 sequences in different animal species. A chimera
XVIII in which the first 10 amino acids of GCAP-2 are substituted with
the corresponding region from recoverin demonstrates normal regulation
of RetGC in vitro (Fig. 6). It
is likely that the effect observed in Ref. 21 was due to misfolding
caused by excessive truncation of recombinant GCAP-2 rather than the
absence of an essential GCAP-2 specific-sequence motif.
Although we cannot conclude that the N terminus of GCAP-2 does not play
a functional role in regulation of RetGC, it appears that the total
length of the N-terminal region between Gly2 and
Trp27 (numbers correspond to bovine GCAP-2 sequence) rather
than the presence of any GCAP-2-specific sequence may be important for the proper activator conformation. We demonstrated this using two
similar neurocalcin/GCAP-2 chimeras, XIX and XX. In chimera XIX the
N-terminal sequence of GCAP-2 is substituted with the corresponding
region from neurocalcin (Fig. 2). This chimera does not activate RetGC
(Fig. 7A). However, the N
terminus of this chimera not only has a sequence different from GCAP-2,
but is also 3 amino acids longer than bovine GCAP-2 (Fig.
1A), which itself is the longest form of GCAP-2 among the
reported animal species. The N termini of chicken, mouse, and human
GCAP-2 are 3-5 amino acid residues shorter than bovine GCAP-2 (31). If we now delete 9 amino acids from the inside of the neurocalcin part of
the chimera, the N terminus (MGKQNSEESTDFTEHEIQ) is
shortened and it has very little homology with GCAP-2, but it will be
still within the range in which GCAP-2 terminus varies among different species (Figs. 1 and 2). Fig. 7B demonstrates that such
reduction in length substantially restores the ability of the chimera
protein to activate RetGC (Fig. 7B). Interestingly,
Ca2+-loaded forms of both chimeras, XIX and XX, are able to
inhibit RetGC in OS (Fig. 7, A and B). We
therefore conclude that the part of bovine GCAP-2 between its N
terminus and Leu24 does not posses major regulatory
properties that are specific to GCAP-2. Neither does the region between
Phe49 and Phe78 (which includes part of EF-2)
specifically determine the essential regulatory properties of GCAP-2.
Partial (XVI, data not shown) or complete (chimera XVII) substitution
of this region with the corresponding regions from neurocalcin does not
eliminate the ability to regulate RetGC in a Ca2+-sensitive
manner (Fig. 7C).
However, additional substitution of the region
Lys29-Phr48 in GCAP-2 (that includes the
"EF-1" motif) with the corresponding region of neurocalcin
completely inactivates GCAP-2 (Fig. 7B, chimera XXI). It
seems unlikely that the loss of function in this chimera results from
major loss of protein structure caused by the sequence exchange because
this substitution does not affect the size of the protein. Also, the
neurocalcin sequence in this region has fairly high homology with
GCAP-2 (Fig. 1A). GCAP-2 and GCAP-1 also show very little
homology to each other within their N termini except for the region
adjacent to EF-1 (which is highly homologous in all recoverin-like
proteins including neurocalcin and GCAPs) (Fig. 1A). Yet
unlike chimera XXI, chimera XXII does regulate RetGC even though the
whole N-terminal part of GCAP-2 in chimera XXII is substituted by the
corresponding amino acid sequence from GCAP-1 (Fig.
8). We conclude that the region
Lys30-Phe48 contains GCAP-specific amino acid
residues that play a key role in RetGC regulation both by apo- and
Ca2+-loaded forms of GCAP-2. The only amino acid residues
that are conserved in this region in both GCAP-1 and GCAP-2, but not in neurocalcin and other recoverin-like proteins are Lys30 and
Phe48 (numbers according to GCAP-2 sequence). It is
therefore possible that one or both of these residues participate in
the interactions between RetGC and GCAP-2. Alternatively, they may
provide for the intramolecular interactions in GCAP-2 essential for the
proper conformation of regulatory sites within the GCAP-2
structure.
There is unambiguous evidence that GCAP-1 and -2 are present in
photoreceptors and regulate RetGC (6, 13-15, 31, 32). And even though
a priori we cannot rule out that GCAPs may also be involved
in regulating some additional function(s) in photoreceptors, we would
like to emphasize that all the results presented in this paper pertain
solely to the effects of GCAP-2 mutants on stimulation or inhibition of
cGMP synthesis, not cGMP hydrolysis. The activity of cGMP PDE in assay that contains washed membranes and PDE inhibitors, zaprinast and dipyridamole, under infrared illumination typically is
insignificant. Moreover, we always directly monitor PDE activity in
every assay in order to ensure the absence of cGMP hydrolysis that
could potentially affect the recovery of cGMP synthesized by RetGC.
Fig. 7D demonstrates that regardless of free
Ca2+ concentrations used in this study, neither GCAP-2 nor
its mutants affected the recovery of [3H]cGMP added as
internal standard at the beginning of the reaction. Also, less than
10% of the total added GTP was utilized in the course of the reaction
based on TLC analysis (data not shown). Therefore the effects of GCAP-2
mutants in the conditions of our assays were directly on RetGC activity
and by the very design and conditions of the assay did not reflect the
activity of the rhodopsin Mutations in GCAP-2 That Affect Native RetGC in OS Membranes Also
Affect the Regulation of Recombinant RetGC-1 and RetGC-2--
Using
recombinant RetGC-1 and -2 for studying RetGC regulation by GCAPs may
have both limitations and advantages over using native RetGC in washed
OS membranes for a number of reasons. Interpretation of the results
using recombinant RetGCs can be potentially complicated by a possible
lack of yet unaccounted factor(s) that may contribute to the activities
of the native enzymes. It has been known, for example, that even
purification native RetGC affects its ability to react with the
Ca2+-sensor proteins (35). The inhibitory effect of WTGCAPs
and their mutants on the total RetGC activity in OS membranes can be
easily detected. However, the basal activity of each recombinant RetGC
is approximately 100-fold lower than the basal RetGC activity in OS
membranes (6, 7). That, again, might reflect the lack of some yet
unidentified factor(s) responsible for higher basal level of RetGC or
can be merely a result of lower efficiency of folding,
post-translational modifications, and/or proper transport and
incorporation of recombinant RetGCs into membranes compared with the
native RetGC in the OS membranes. For this particular reason, we find
it difficult to reliably study the inhibitory effects of GCAP-1 and
GCAP-2 using recombinant cyclases. Also, the exact contribution of
RetGC-1 and RetGC-2 to the overall RetGC activity in membranes has not
yet been determined. And last, but not the least, it is impossible to
exclude that there are additional isoforms of RetGC present in native
membranes that are significant for the net cGMP synthesis.
Nevertheless, even after taking all these limitations into account, we
think that recombinant RetGCs can be very useful tools in studying
specific mechanisms of RetGC regulation. Therefore we also verified to
what extent the effects of the key GCAP-2 mutants in the three most
important regulatory regions in GCAP-2 reflects its ability to regulate
recombinant RetGC-1 and RetGC-2. The results presented in Fig.
9 demonstrate that chimeras XIII and XXI
that fail to activate RetGC in OS membranes are also inefficient in
stimulating individual recombinant RetGC-1 and RetGC-2 (Fig. 9,
A and C). Chimeric mutant III that reverses
Ca2+ sensitivity of cGMP synthesis in OS membranes also
stimulates recombinant RetGC-1 only at high Ca2+
concentrations (Fig. 9). The second consequence of this mutation is
decreased efficiency of activation of both OS RetGC and RetGC-1 (Figs.
3 and 9). In the case of recombinant RetGC-2 its activity in the
presence of chimera III was even lower so that it made it difficult to
detect RetGC-2 activation by chimera III even at high Ca2+
(Fig. 9). However, mosaic chimera IV that activates OS RetGC in a
Ca2+-sensitive manner also efficiently activates
recombinant RetGC-2. Despite the much lower activity of recombinant
RetGCs compared with the native total RetGC in OS membranes, these data
allow us to expect that these three major regulatory regions in GCAP-2 are also engaged in regulating both RetGC-1 and RetGC-2 in
vivo. However, we cannot conclude that the interactions between
GCAP-2 and both cyclases are strictly identical. Such issue would
require identification of the exact sites of protein-protein contacts between GCAP-2 and both cyclases which is beyond the scope of our
present study.
A Map of GCAP-2 Functional Domains (Fig.
10)--
Our previous and present
observations reveal that there are six domains in the GCAP-2 primary
structure that determine its most basic characteristics as a RetGC
regulator. Three functional EF-hands (EF-2 through EF-4) are required
for GCAP-2 to be a Ca2+ sensor within the submicromolar
range of free Ca2+ concentrations. EF-2 and EF-4 contribute
to its Ca2+ sensitivity more than EF-3 (16). It is possible
that certain GCAP-2-specific amino acid residues within the EF-hands
are essential for RetGC regulation. However, the exact amino acid
sequences of the EF-hand Ca2+-binding loops can be
partially (EF-2) or even completely (EF-4) changed as long as they do
not deviate from the basic EF-hand loop motif (for the EF hand loop
structure, see Ref. 34). Chimeras IV, V, and VI each have EF-2 and EF-3
loops partially derived from neurocalcin, and the entire EF-4 loop in
chimera VI is substituted with the neurocalcin sequence, yet these
chimeras are able to regulate RetGC in a Ca2+-sensitive
manner.
The fundamental difference between GCAPs and other known EF-hand
Ca2+-binding proteins is that the apo-form of GCAPs
activates target enzyme. This means that in GCAPs Ca2+
binding is not required for RetGC activation, and only results in
inhibition of RetGC. Binding of Ca2+ to the EF-hands of
GCAP-2 drives a switch between the activator and inhibitor
conformations. The direction of the switch appears to be controlled by
the region Phe78-Asp113 located between the
EF-2 and EF-3 Ca2+-binding domains. This "switch"
domain determines that upon binding Ca2+ ions to the
EF-hands, GCAP-2 undergoes an activator-to-inhibitor transition, but
not vice versa. It should also be noted that since the corresponding
chimeras are able to stimulate RetGC in high Ca2+, we
cannot conclude from this study whether or not this domain may also
contribute to the inhibition of basal activity of RetGC in
Ca2+-loaded wild type GCAP-2.
There are two other domains in GCAP-2 that contain GCAP-2-specific
amino acid sequences essential for native RetGC regulation: the
C-terminal region, Val171-Asn189, adjacent to
EF-4 loop and the N-terminal domain,
Lys29-Phe48, that includes EF-1. The
C-terminal domain adjacent to EF-4 is important for activation of RetGC
by apo-GCAP-2, but it is not required for inhibition of the cyclase by
Ca2+-loaded GCAP-2. The N-terminal region surrounding the
EF-1 is essential for both activation and inhibition of RetGC. Even
substitution of this region with the homologous sequence of neurocalcin
disables regulation of RetGC. We speculate that this region of GCAP-2
is responsible for one of the primary interactions between GCAP-2 and
the cyclase in both apo- and Ca2+-loaded forms of GCAP-2.
Other domains may perform secondary interactions specifically required
for RetGC activation or inhibition, or for conformational changes
induced by Ca2+. In order to resolve these questions it
will be necessary to define the specific roles of particular amino acid
residues in these regulatory domains of GCAP-2 using directed
mutagenesis and analysis of the three-dimensional structure of
GCAP-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cDNA was a gift from D. Ladant) as
templates and PCR primers based on the sequences of GCAP-2,
neurocalcin, or recoverin cDNAs. The fragments were purified from
agarose gel using QIAEX II resin (QIAGEN) and spliced together by a
second round of PCR according to the splicing by overlap extension
technique (24). The resulting DNA fragments were ligated into the
NcoI/BamHI sites of the pET11d vector (Novagen). DNA sequence of the resulting constructs were verified by an automated DNA sequences using the ABI Prism system (Perkin-Elmer). Deletion mutants were constructed by a similar approach except the second round
of PCR was only used to splice fragments of DNA in order to introduce
the internal deletions.
were expressed in E. coli
according to Refs. 25 and 26 except that we used the BLR(DE3) E. coli strain (Novagen) instead of BL21(DE3). Recombinant GCAP-2 and
chimera proteins expressed in E. coli were purified using
gel filtration chromatography as described previously in detail (22).
Recombinant recoverin and neurocalcin were purified using
phenyl-Sepharose (Pharmacia) chromatography (25, 26). The recombinant
proteins were verified for the presence of N-myristoylation by reversed-phase high performance liquid chromatography and
electrospray mass spectrometry as described previously (27). All
chimeras were predominantly in myristoylated form except for the
chimeras XX and XXI.
-32P]GTP, 0.1 µCi
of [3H]cGMP, and washed bovine outer segment membranes
(3.5 µg of rhodopsin). Reaction mixtures were incubated for 12 min at
30 °C, heated for 2 min at 95 °C, chilled on ice, centrifuged for
5 min at 10,000 × g, and analyzed by TLC on
polyethylenimine cellulose plastic-backed plates with fluorescent
background (Merck). After development in 0.2 M LiCl, cGMP
spots were visualized under UV illumination, cut, eluted with 1 ml of 2 M LiCl, shaken with 10 ml of an Ecolume scintillation
mixture (ICN) and both 3H and 32P radioactivity
were counted. [3H]cGMP was used as an internal standard
to ensure the absence of cGMP hydrolysis by phosphodiesterase. In all
experiments time course of reaction was linear in time and less than
10% of GTP substrate was converted into cGMP. Basal activity of RetGC
in different preparations of washed OS membranes typically varied between 2.5 and 5 nmol of cGMP/min/mg of rhodopsin. In the absence of
protein activators the difference between RetGC basal activity measured
at 6 nM and 1 µM free Ca2+did not
exceed 20%. Data that are shown in every figure pertain to a single
experiment using one preparation of washed OS membranes, they are
representative of two or three similar independent experiments.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, sequence homology between GCAP-2 and
other recoverin-like proteins. GCAP-2 from two different species,
bovine (BGCAP2) and human (HGCAP2) (16, 30), are aligned with each
other and with other recoverin-like proteins: bovine GCAP-1
(bGCAP-1, 13), bovine neurocalcin (BovNCa,
25), and bovine recoverin (BovRv, 26). Amino acid residues
identical between HGCAP1 and HGCAP2 and other proteins of this group
are highlighted. EF-hand-related motifs, EF-1 through EF-4, are shown
in boxes. B, unlike GCAP-2, closely related to
it, the protein neurocalcin does not regulate RetGC. Washed OS
membranes depleted of endogenous GCAPs were assayed for RetGC activity
as a function of free Ca2+ concentrations in the absence
(
) or presence of either GCAP-2 (
) or neurocalcin ([/delta). See
"Experimental Procedures" for details.
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Fig. 2.
Protein constructs used in this study.
A, mutations in Ca2+-binding region (EF-2
through EF-4) of bovine GCAP-2. I, neurocalcin fragment
Ala61-Phe169 substitutes for
Ala57-Phe170 in GCAP-2; II,
neurocalcin fragment Ala61-Glu146 substitutes
for Ala57-Glu148 in GCAP-2; III,
neurocalcin fragment Ala61-Met131 substitutes
for Ala57-Leu127 in GCAP-2; IV,
neurocalcin fragments Ala61-Asn81 and
Lys118-Met131 substitute for
Ala57-Phe78 and
Asp113-Leu127 in GCAP-2, respectively;
V, neurocalcin fragments
Ala61-Asp81 and
Lys118-Glu146 substitute for
Ala57-Asp78 and
Asp113-Glu148 in GCAP-2, respectively;
VI, neurocalcin fragments
Ala61-Asp81 and
Lys118-Phe169 substitute for
Ala56-Asp77 and
Asp113-Phe170 in GCAP-2, respectively;
VII, a fragment
Val133-Gly141 in GCAP-2 is deleted.
B, mutations in the C-terminal part of GCAP-2.
VIII, recoverin fragment
Glu199-Leu202 substitutes for
Ser201-Phe204 in GCAP-2; IX, a
GCAP-2 mutant that contains mutations E80Q and D158N (16), but
unlike the (E80Q/D158N) Ca2+-insensitive mutant described
in Ref. 16, it also has a frameshift resulting in an additional
25-amino acid fragment, SEGSKDPAANKARKEAELAAATAEQ, encoded by pET11d
vector following Met203 of GCAP-2; X, GCAP-2
truncated downstream of Ser195; XI, GCAP-2
truncated downstream of Asn189; XII, GCAP-2
truncated downstream of Asn181; XIII,
neurocalcin fragment Thr158-Phe192 substitutes
for Glu159-Phe204 in GCAP-2; XIV,
neurocalcin fragment Thr158-Asp186 substitutes
for Glu159-Asn189 in GCAP-2; XV,
GCAP-2 truncated at Gln140. C, mutations within
the N-terminal part of GCAP-2. XVI, neurocalcin
fragment Ala61-Gly78 substitutes for
Ala57-Asn74 in GCAP-2; XVII,
neurocalcin fragment Gly53-Asp81 substitutes
for Phe49-Asp78 in GCAP-2; XVIII,
recoverin fragment Met1-Ser10 substitutes for
Met1-Ala10 in GCAP-2; XIX,
neurocalcin fragment Met1-Ile27 substitutes
for Met1-Leu24 in GCAP-2; XX,
fragment Leu8-Leu18 is deleted from the
neurocalcin part of the chimera protein XIX; XXI, in chimera
protein XX a neurocalcin fragment Gly32-Gly78
substitutes for Lys30-Asn74 in GCAP-2;
XXII, fragment Met1-Ala79 of GCAP-1
substitutes for Met1-Ala84 in GCAP-2. Chimera
constructs VIII and XVIII were expressed in HEK 293 cells as described
previously (14), all other constructs were expressed in E. coli as described in Ref. 22 and under "Experimental
Procedures." Fragments of GCAP-2 were substituted between the
indicated amino acid residues of GCAP-2. The diagrams representing
GCAP-2 and neurocalcin are "aligned" at the EF-1 region.
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Fig. 3.
Ca2+ sensitivity of RetGC
regulation in OS membranes is determined by the region that includes
EF-2 and EF-3. A, substitution of the
Ca2+-binding core region in GCAP-2 with that from
neurocalcin results in Ca2+-dependent
activation of RetGC. Cyclase activity is shown as a function of free
Ca2+ in the absence ( ) or presence of chimeras I (
),
V (
), or VI (
). B, the region in GCAP-2 that
determines the "sign" of Ca2+ sensitivity is located
between Phe78 and Asp113 of GCAP-2. RetGC
activity in the absence (
) or presence of wild type GCAP-2 (
),
chimera III (
), or chimera IV (
). Inset,
unlike wild type GCAP-2, chimeras I, II, and III fail to stimulate
RetGC at 12 nM Ca2+. C, RetGC
activity in the absence (
) and presence (circo]) of GCAP-2 deletion
mutant VII. Protein constructs are described in the legend to Fig. 2.
See "Experimental Procedures" for other details.
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Fig. 4.
The very C terminus of GCAP-2 is not required
for RetGC stimulation. A, RetGC activity in the absence
(a) or presence of wild type GCAP-2 (b),
GCAP-2/recoverin chimera VIII (c and d) at 6 nM (a-c), or 2 µM (d)
free Ca2+. B, RetGC activity at 6 nM
free Ca2+ in the absence (f) or presence of wild
type GCAP-2 (g), or chimera IX (h). Protein
constructs are described in the legend to Fig. 2. See "Experimental
Procedures" for other details.
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Fig. 5.
An EF-4 proximal region in GCAP-2
participates in RetGC activation at low Ca2+ but is not
required for RetGC inhibition at high Ca2+.
A, RetGC activity at 6 nM free Ca2+
in the absence (a) or presence of wild type GCAP-2
(b), GCAP-2 deletion mutants X (c) or XI
(d), GCAP-2/neurocalcin chimeras XIII (e) or XIV
(f), GCAP-2 deletion mutant XV (g).
Inset, Ca2+-loaded C-terminal deletion and
chimera mutants inhibit RetGC basal activity. B,
Ca2+ sensitivity of RetGC activation in the absence ( )
or presence of GCAP-2 truncated mutant X (
), GCAP-2/neurocalcin
chimera proteins XIII (
) or XIV (
). Protein constructs are
described in the legend to Fig. 2. See "Experimental Procedures"
for other details.
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Fig. 6.
A chimera XVIII in which first the 10 amino
acid residues of GCAP-2 are substituted with the corresponding fragment
from recoverin is capable of regulating RetGC. A,
washed OS membranes were reconstituted with soluble extracts (50 µg
of total protein) from transfected HEK293 cells as described in detail
in Ref. 14. RetGC activity was measured as a function of free
Ca2+ concentrations. Protein extract from mock-transfected
control cells ( ), cells transfected with wild type GCAP-2
cDNA (
), or chimera protein XVIII-encoding DNA (
).
Inset, unlike GCAP-2 or recoverin/GCAP-2 chimera XIII,
recoverin does not activate RetGC. RetGC activity in washed OS
membranes: in the absence (a) or presence (b and
c) of 4 µg of recombinant recoverin (b) or
GCAP-2 (c). B, a Hill plot of the data from
panel A demonstrates that recoverin/GCAP-2 chimera XVIII
regulates RetGC with the same Ca2+ cooperativity as wild
type GCAP-2. Protein constructs are described in the legend to Fig. 2.
See "Experimental Procedures" for other details.
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Fig. 7.
Regulation of RetGC by N-terminal
neurocalcin/GCAP-2 chimeras as a function of free Ca2+
concentrations. A, RetGC activity in the absence ( )
or presence (
) of chimera XIX. B, RetGC activity in the
absence (
) or presence of chimeras XX (
) or XXI (
).
C, RetGC activity in the absence (
) or presence of
chimera XVII (
). D, chimera XXI does not cause excessive
hydrolysis of cGMP. Approximately 0.1 µCi of
[3H]cGMP was added in every RetGC assay
reaction mixture (see "Experimental Procedures") prior to
incubation. After the incubation, 5-µl aliquots from each sample were
subjected to thin-layer chromatography, and [3H]cGMP was
recovered from PEI cellulose by elution in 1 ml of 2 M LiCl
and counted in 10 ml of an Ecolume scintillation mixture (ICN). 100%
level corresponds to the original amount of
[3H]cGMP standard recovered from the reaction
mixture that contained heat-inactivated OS membranes and was incubated
in parallel with other samples. No significant cGMP hydrolysis was
detected in the presence or absence of WTGCAP-2, neurocalcin, chimeras
III, XIII, or XXI or any other chimera constructs used in this
study (data not shown). Chimera constructs are described in the
legend to Fig. 2.
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Fig. 8.
Activation of RetGC by 2 µg of GCAP-1/GCAP-2 chimera protein XXII ( ) as a
function of free Ca2+ concentrations (
), no activator
added. Protein construct is as described in the legend to Fig. 2.
See "Experimental Procedures" for other details.
GTPase
PDE cascade.
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Fig. 9.
Effect of GCAP-2/neurocalcin chimeras on
Ca2+ regulation of recombinant RetGC-1 and RetGC-2.
Panels A and B, activity of recombinant RetGC-1,
panels C and D, activity of recombinant RetGC-2.
RetGC-1 and RetGC-2 were expressed in HEK293 cells and assayed as
described under "Experimental Procedures" in the absence ( ) or
presence of 5 µM wtGCAP-2 (
), 10 µM
chimera XIII (
), 10 µM chimera XXI (
), 5 µM chimera III (
), or 10 µM chimera IV
(
). Basal activities of RetGC-1 and -2 correspond to 0.04 nmol of
cGMP/min/mg of protein, maximal wtGCAP-2 stimulated activity
corresponds to 1.26 and 0.7 nmol of cGMP/min/mg of protein,
respectively.
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Fig. 10.
A map of functional domains in GCAP-2.
Explanations are as described in the text.
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ACKNOWLEDGEMENT |
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We thank Greg Niemi for mass spectrometry of GCAP-2 mutants.
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FOOTNOTES |
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* This work was supported by National Eye Institute Grants EY11522 (to A. M. D) and EY06641 (to J. B. H), and by a Career Development Award from Research to Prevent Blindness (to A. M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Ophthalmology/Kresge Eye Institute, Wayne State University School of Medicine, 4717 St. Antoine, Detroit, MI 48201. Tel.: 313-577-1573; Fax:
313-577-7635; E-mail: adizhoor{at}med.wayne.edu.
2 C. Tucker and J. B. Hurley, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GCAP, photoreceptor guanylate cyclase activating protein; EF-1-4, Ca2+-binding loops of EF-hands; OS, photoreceptor outer segment membranes; RetGC, photoreceptor membrane guanylate cyclases (other names are RosGC1 and ROSGC2 or GC-E and GC-F, respectively); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; BNC, bovine neurocalcin.
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