The GAFa Domains of Rod cGMP-phosphodiesterase 6 Determine the Selectivity of the Enzyme Dimerization*
Khakim G.
Muradov
,
Kimberly K.
Boyd
,
Sergio E.
Martinez§,
Joseph A.
Beavo§, and
Nikolai O.
Artemyev
¶
From the
Department of Physiology and Biophysics,
University of Iowa College of Medicine, Iowa City, Iowa 52242 and
the § Department of Pharmacology, University of Washington,
Seattle, Washington 98195
Received for publication, August 19, 2002, and in revised form, January 9, 2003
 |
ABSTRACT |
Retinal rod cGMP phosphodiesterase (PDE6 family)
is the effector enzyme in the vertebrate visual transduction cascade.
Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of
and
subunits. A
system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6
and PDE5, which contain the N-terminal GAFa/GAFb domains, or
portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or
FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed
using gel filtration and sucrose gradient centrifugation. The
composition of formed dimeric enzymes was analyzed with Western
blotting and immunoprecipitation. Consistent with the selectivity of
PDE6 dimerization in vivo, efficient heterodimerization was
observed between the GAF regions of PDE6
and PDE6
with no
significant homodimerization. In addition, PDE6
was able to form
dimers with the cone PDE6
' subunit. Furthermore, our analysis
indicated that the PDE6 GAFa domains contain major structural
determinants for the affinity and selectivity of dimerization of PDE6
catalytic subunits. The key dimerization selectivity module of PDE6 has
been localized to a small segment within the GAFa domains,
PDE6
-59-74/PDE6
-57-72. This study provides tools for the
generation of the homodimeric 
and 
enzymes that will allow
us to address the question of functional significance of the unique
heterodimerization of rod PDE6.
 |
INTRODUCTION |
Photoreceptor rod and cone cGMP phosphodiesterases
(PDE6 1 family) are the
effector enzymes in the vertebrate visual transduction cascade. The
cascade is initiated by photoexcitation of the visual receptor
rhodopsin and leads to hydrolysis of intracellular cGMP by
transducin-activated PDE6 (1, 2). PDE6 enzymes belong to a large
superfamily of phosphodiesterases of cyclic nucleotides that are
critical modulators of cellular levels of cAMP and cGMP. Currently,
eleven PDE families have been identified in mammalian tissues based on
primary sequence, substrate selectivity, and regulation (3, 4). Rod
PDE6 is composed of two homologous catalytic
- and
-subunits of
similar size and two copies of an inhibitory
-subunit (1, 5-8).
Cone PDE6 catalytic dimer is made up of two identical PDE
' subunits
(9). A cone-specific inhibitory P
-subunit is highly homologous to
the rod P
(10). The
-subunit associates with soluble rod and cone
PDEs. It interacts with the methylated prenylated C termini of PDE6
catalytic subunits and regulates the enzyme attachment to the membrane
(11). The role of the PDE
-subunit in phototransduction is not
well-defined, although it may modify the activity of the cascade by
uncoupling transducin and PDE (12).
PDE6 enzymes have catalytic domains of about 280 aa residues in the
C-terminal part of the molecule, which is highly conserved among all
known cyclic nucleotide phosphodiesterases (3, 4). The catalytic region
of photoreceptor PDEs closely resembles the catalytic site of cGMP
binding, cGMP-specific PDE (PDE5) (45-48% sequence identity) (13).
Furthermore, PDE6 and PDE5 share a strong substrate preference for cGMP
and have similar patterns of inhibition by competitive inhibitors,
including zaprinast, dipyridamole, and sildenafil (13-15). In addition
to the C-terminal catalytic domain, PDE6 contains two N-terminal GAF
domains (GAFa and GAFb). GAF domains have been recognized as a large
family of domain homologues and named for their presence in
cGMP-regulated PDEs, adenylyl cyclases, and the
E. coli protein Fh1A (16). Besides PDE6, several
other PDE families possess GAF domains, including cGMP-stimulated PDE
(PDE2), PDE5 (13, 17, 18), PDE10 (19), and PDE11 (20). At least one of
the two GAF domains in the PDE2, PDE5, and PDE6 catalytic subunits
serves as a site for noncatalytic binding of cGMP. Noncatalytic cGMP
binding to GAF domains affects the catalytic properties of PDE2 and
PDE5 (21-24). In PDE6, noncatalytically bound cGMP appears to enhance the affinity of the inhibitory interaction between P
and the catalytic core (25, 26). The second major function of the GAF domains
is their role in dimerization of the PDE catalytic subunits. Earlier
biochemical studies of PDE2 and PDE5 indicated that their dimerization
occurs within the N-terminal parts of the molecules (18, 27). Ultimate
evidence on the intersubunit interface of PDE2 has been recently
provided by a solution of the crystal structure of PDE2A GAFa-GAFb
domains (28). The crystal structure revealed that the PDE2A regulatory
region forms a dimer with the interface formed by the two GAFa domains
(28). The role of PDE6 GAF domains in dimerization is supported by
recent electron microscopy imaging of rod PDE6
(29). The imaging showed that the main intersubunit interaction occurs between the very
N-terminal domains, presumably involving GAFa modules, and indicated
that the GAFb domains may also contribute to the interface.
Almost all PDEs known to date are dimeric. However, the role of
dimerization in enzyme function is not understood. Dimerization is not
required for catalytic activity, because the isolated monomeric catalytic domain of PDE5 (30) and the monomeric short splice variant
PDE4D2 (31) have been shown to be catalytically active. Furthermore, a
majority of PDEs, with a notable exception of rod PDE6
, form
catalytic homodimers (3, 4). Although the possibility of minor
homodimeric species, 
and 
, has not been ruled out, the
dominant catalytic species of rod PDE6 is clearly a heterodimer 
(32, 33). The functional significance of heterodimerization of rod PDE
remains unclear. It is as yet unknown if the catalytic characteristics
of PDE6
and PDE6
in the dimer are equivalent. To circumvent the
problem of a lack of efficient expression of PDE6 in various cell types
(34, 35), we have previously developed a robust system for expression
of PDE6
'/PDE5 chimeras in insect cells (36). In this study, we
extended this approach to chimeras between PDE6
and PDE5 to
investigate the structural basis for specific dimerization of the rod
PDE6 catalytic subunits. A series of chimeric proteins between PDE6 and
PDE5 have been constructed that contain the GAFa and/or GAFb domains of
PDE6 or PDE5. These chimeras were expressed in Sf9 cells in
various combinations as His-, myc-, or FLAG-tagged proteins. The
dimerization of chimeric PDE6 subunits was assessed using gel
filtration, sucrose gradient centrifugation, Western blotting, and
immunoprecipitation with anti-FLAG- and anti-myc-specific antibodies.
The patterns of dimerization of chimeric PDE6/PDE5 subunits are
consistent with the selectivity of PDE6 dimerization in rod
photoreceptor cells. Our results indicate that the PDE6 GAFa domains
contain major structural determinants for the affinity and selectivity
of dimerization of PDE6 catalytic subunits.
 |
EXPERIMENTAL PROCEDURES |
Materials--
cGMP was obtained from Roche Molecular
Biochemicals. [3H]cGMP was a product of Amersham
Biosciences. All restriction enzymes were purchased from New England
BioLabs. AmpliTaq® DNA polymerase was a product of PerkinElmer Life
Sciences, and Pfu DNA polymerase was a product of
Stratagene. All other reagents were purchased from Sigma. Bovine
holo-PDE6 was extracted from bleached rod outer segment membranes and
purified as described previously (33).
Cloning, Expression, and Purification of PDE6/PDE5
Chimeras--
To construct PDE6
/PDE5 chimera FLAG-
-
-5 (see
Fig. 1), the FLAG tag DNA sequence was first inserted into the
pFastBacHTb vector (Invitrogen) replacing the His6 sequence. A DNA
fragment was PCR-amplified using pFastBacHTb as a template, a 5'-primer containing an RsrII site and the FLAG tag sequence,
and a 3'-primer containing a BamHI site. This fragment was
then ligated with the large RsrII/BamHI fragment
of pFastBacHTb to produce pFastBacFLAG. A bovine retinal cDNA
library kindly provided by Dr. W. Baehr (University of Utah) was used
as a template for PCR amplifications of PDE6
sequences. DNA
coding for PDE6
-1-443 was PCR-amplified using primers carrying
BamHI and HindIII sites. DNA coding for PDE5-506-865 amino acids was PCR-amplified using the pFastBacHTb-PDE5 template (36) and primers with the HindIII and
XhoI sites. The two PCR products were ligated into the
pFastBacFLAG vector using the BamHI and XhoI
sites. The myc tag DNA sequence was inserted into the pFastBacHTb
vector using a PCR-directed cloning procedure similar to the insertion
of the FLAG tag sequence. The construct for chimera myc-
-
-5 (see
Fig. 1) was generated by ligation of the PCR-amplified PDE6
-1-441
and PDE5-506-865 DNAs into the BamHI and XhoI
sites of pFastBacmyc.
The His-tagged PDE6
/PDE5 chimeras were constructed by amplifying
appropriate regions of the PDE6
subunits with primers containing
unique restriction sites. When unique restriction sites were not
available at the desired location, first-round PCR products coding
chimeric junctions were extended to the nearest unique sites in a
second-round PCR amplification. To improve the recognition of the
His6 tag by commercial antibodies, the His6
flanking sequence in the original pFastBacHTb vector was replaced by
the His6 flanking sequence from pET-15b (Novagen). The DNA
sequences of all constructs were confirmed by automated DNA sequencing
at the University of Iowa DNA core facility.
Generation of the recombinant bacmids, transfection of Sf9
cells, and viral amplifications were carried out according to the manufacturer's recommendations (Invitrogen). For protein expression, Sf9 cell cultures (10 ml, 2 × 106 cells/ml)
were infected with one or two different viruses at a multiplicity of
infection of 3-10. Sf9 cells were harvested at 48 h after
infection by centrifugation and stored at
80 °C until use.
Sf9 cells were resuspended in 3 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgSO4 and
CompleteTM Mini protease inhibitor mixture (one-third
tablet) (Roche Molecular Biochemicals) and sonicated with two 10-s
pulses using a microtip attached to a 550 Sonic Dismembrator (Fisher
Scientific). Sf9 cell lysates were cleared by centrifugation
(100,000 × g, 90 min, 2 °C), dialyzed against 30 mM Tris-HCl buffer (pH 8.0) containing 130 mM
NaCl, 2 mM MgSO4, and 50% glycerin, and then
centrifuged again at 100,000 × g for 1 h at
2 °C. Dialysis against 50% glycerin allowed a concentration of PDE
samples by ~4-fold and subsequent storage at
20 °C without
freezing. The presence of glycerin did not affect the behavior of PDEs
in gel filtration.
Gel Filtration and Fraction Analysis--
Aliquots of dialyzed
PDE samples (50-200 µl) were injected into a Superose® 12 10/30
column (Amersham Biosciences) equilibrated at 25 °C with 30 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 2 mM MgSO4. Proteins were eluted at
0.4 ml/min, and 0.4-ml fractions were collected starting at 17 min
post-injection. Each fraction was assayed for PDE activity, protein
concentration, and the presence of chimeric PDEs by Western blotting.
PDE activity was measured using 10- to 20-µl aliquots from fractions
and 5 µM [3H]cGMP as described previously
(37, 38). Protein concentrations were determined by the method of
Bradford using IgG as a standard (39). The column was calibrated with
the following protein standards: bovine thyroglobulin (670 kDa, 85 Å),
horse ferritin (440 kDa, 61 Å), sweet potato
-amylase (200 kDa),
rabbit aldolase (158 kDa, 48.1 Å), bovine serum albumin (67 kDa, 35.5 Å), and chicken ovalbumin (45 kDa, 30.5 Å). The Stokes radii for PDEs
were estimated using the correlation of elution volume with the Stokes
radius proposed by Porath (40). Gel filtration analyses were performed two or more times with similar results for each PDE chimera combination from at least two different preparations of Sf9 cell extracts. Results of a typical analysis are shown.
Western blot analysis of the gel filtration fractions (20-µl
aliquots) was performed following SDS-PAGE in 10% gels (41). Monoclonal anti-polyhistidine, M2 monoclonal anti-FLAG, and monoclonal anti-c-myc (clone 9E10) antibodies (Sigma) with the respective dilutions of 1:1500, 1:5000, and 1:5000 were utilized. The
antibody·antigen complexes were detected using anti-mouse
antibodies conjugated to horseradish peroxidase (Sigma) and ECL reagent
(Amersham Biosciences). The compositions of PDE complexes in fractions
corresponding to dimeric enzymes were examined by immunoprecipitation
(IP). Aliquots of the gel filtration fractions (80 µl) were incubated
with or without anti-FLAG or myc-antibodies (1 µl) for 30 min at
25 °C followed by the addition of 5 µl of protein G-Agarose
(Sigma) and incubation for 40 min at 25 °C. The agarose beads were
washed four times with 300 µl of phosphate-buffered saline (pH 7.1), and the bound proteins were eluted with an SDS-PAGE sample buffer. PDE
complexes were separated on 10% gels and analyzed by Western blotting
using appropriate antibodies.
Sucrose Gradient Centrifugation--
Sucrose density gradients
(5-35%) were prepared in 30 mM Tris-HCl (pH 8.0) buffer
containing 100 mM NaCl, 2 mM MgSO4,
and 4 mM 2-mercaptoethanol using a GradiFrac gradient
former (Amersham Biosciences). Protein standards or aliquots of 200 µl from peak PDE gel filtration fractions were loaded onto the
gradients in 14- × 89-mm centrifugation tubes and centrifuged for
24 h at 40,000 rpm in a Beckman SW41 rotor at 4 °C. Fractions
of 300 µl were collected starting from the bottom of the tubes.
Fractions from the tubes with protein standards were analyzed for
protein concentration, whereas fractions from the tubes containing PDE
samples were analyzed for PDE activity. Sucrose density centrifugations
were performed two times with similar results for each PDE preparation.
Results of a typical analysis are shown. The protein standards were:
bovine liver catalase (250 kDa, 11.3 S), rabbit aldolase (158 kDa, 7.3 S), bovine serum albumin (67 kDa, 4.6 S), and chicken
ovalbumin (45 kDa, 3.5 S). Sedimentation coefficients
(s20,w) for chimeric PDEs were
estimated using a linear plot of distances traveled by standards from
meniscus versus the s20,w values of standards (42). The molecular weights of PDEs were calculated
using the estimated sedimentation coefficients, the Stokes radii
obtained from the gel filtration data, and the following equation
(43),
|
(Eq. 1)
|
where s is the sedimentation coefficient;
NA is Avogardo's number;
is the viscosity
of the medium (0.01 g·cm
1·s
1),
r is the Stokes radius; v is the partial specific
volume of a protein (0.73 cm3·g
1), and
is the density of the medium (1 g·cm
3).
 |
RESULTS |
Selectivity of Dimerization of the GAFa-GAFb Domains of Rod
PDE6--
Dimerization of PDE6 catalytic subunits is very tight, and,
apparently, there is no exchange of subunits between dimers once they
are formed following the synthesis and folding of the polypeptide chains. The dimer formation between chimeric PDE6/PDE5 subunits was
therefore assessed following co-expression of these chimeras in
Sf9 cells. Chimeric PDEs (Fig. 1)
from soluble fractions of Sf9 cells as well as native PDE6 and
recombinant wild-type PDE5 were examined by FPLC gel filtration on a
calibrated Superose 12 HR 10/30 column and by sucrose gradient
centrifugation. The elution profiles of native PDE6 and recombinant
wild-type PDE5 on the gel filtration column were very similar and
corresponded to an apparent molecular mass of ~210 kDa
(Fig. 2A), which is consistent
with dimerization of the catalytic subunits. Molecular masses of rod
holoPDE6 (PDE

2) and the PDE5 dimer calculated from
the sequences were ~217 and 197 kDa, respectively. Only relatively small fractions of the enzymes (~15% PDE5 and ~10% PDE6) migrated as aggregates with high molecular mass. The previously developed chimeric PDE, Chi4 (termed hereafter His-
'-
'-5), containing the
cone PDE6
' GAFa-GAFb region and the catalytic domain of PDE5 (36),
is predicted to form homodimers. The chromatographic behavior of
His-
'-
'-5 on a Superose 12 HR 10/30 column indicated an apparent molecular mass of ~200 kDa (Fig. 2A), which is in good
agreement with the theoretical molecular mass of 183 kDa for the
'-
'-5 homodimer. The fraction of aggregates in the chimeric PDE
(~10%) was similar to those in PDE5 and PDE6 preparations.

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Fig. 1.
Schematic representation of PDE6/PDE5
chimeras. Constructs are designated as follows: name of tag,
composition of GAFa domain, composition of GAFb domain, catalytic
domain. Subscripts a and indicate replacements
PDE6 -59-74 and PDE6 -57-72 within the PDE6 and PDE6 GAF
regions, respectively.
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Fig. 2.
Gel filtration profiles of PDE5, PDE6, and
chimeric PDEs
'- '-5,
- -5, and
- -5. A,
rod holoPDE6 (50 µg) (smaller dashed line) mixed with the
dialyzed cell extract of noninfected Sf9 cells (100 µl) or
dialyzed cell extracts of Sf9 cells (100,000 × g, 90 min) infected with viruses for expression of PDE5 (50 µl) (solid line) and '- '-5 (100 µl) (larger
dashed line) were subjected to FPLC gel filtration on a
Superose® 12 10/30 column (Amersham Biosciences). Fractions of 0.4 ml
were collected and analyzed for PDE activity. B and
C, chimeras FLAG- - -5 (B) and myc- - -5
(C) were expressed individually in Sf9 cells and the
dialyzed soluble cell extracts (100,000 × g, 90 min)
were subjected to FPLC gel filtration. Fractions of 0.4 ml were
collected and analyzed for protein concentration (dashed
line), PDE activity (solid line), and by Western
blotting with anti-FLAG or anti-myc antibodies.
|
|
We first constructed PDE6/PDE5 chimeras containing both the GAFa and
GAFb domains from either rod PDE6
(
-
-5) or PDE6
(
-
-5) and the catalytic domain from PDE5 (Fig. 1). As generally defined by
the PDE sequence alignment and the crystal structure (17, 28), the
boundaries of the GAFa domains are PDE6
-54-220 and PDE6
-52-218,
and the boundaries of the GAFb domains are PDE6
-255-443 and
PDE6
-253-441. The nonconserved N termini, PDE6
-1-53 in
-
-5 and PDE6
-1-51 in
-
-5, were from the respective
catalytic subunits. Chimera
-
-5 was constructed for expression as
a FLAG-tagged protein, whereas
-
-5 was generated as a myc-tagged
polypeptide. Gel filtration fractions were analyzed by Western blotting
for the presence of chimeric PDE proteins and for PDE activity with cGMP as the substrate. Individual expression of FLAG-
-
-5 or myc-
-
-5 resulted in the formation of only high molecular weight aggregates that migrated with the exclusion volume of the Superose 12 column (Fig. 2, B and C). Furthermore, the
aggregates also migrated with the exclusion volume on a Superose 6 HR
10/30 column (not shown), which is capable of separating proteins
weighing up to 1 × 106 kDa. Notably, these aggregates
were capable of hydrolyzing cGMP. When FLAG-
-
-5 and myc-
-
-5
were co-expressed in Sf9 cells, a peak of PDE activity appeared
in the fractions corresponding to dimeric PDE species with an apparent
molecular mass of ~200 kDa (Fig.
3A). Approximately 50% of the
total soluble FLAG/myc-tagged protein formed the dimeric enzyme. In the
peak activity fractions 11 and 12, the enzyme hydrolyzed cGMP with a
Km value of 3.8 µM and a
Vmax of 21 nmol/min/mg of protein. The Western blot analysis of these fractions using anti-FLAG and anti-myc antibodies confirmed the presence of both FLAG-
-
-5 and
myc-
-
-5 (Fig. 3A). A sizable peak of PDE activity was
also present in fractions corresponding to aggregates of FLAG-
-
-5
and myc-
-
-5 (Fig. 3A). The aggregation appears to be
irreversible, because re-chromatography of the fractions with
aggregates did not generate dimeric enzymes (not shown). Considering
the intensity of immunostaining, the catalytic activity of these
aggregated PDE species is 1.5- to 2-fold lower than that of the dimeric
PDE species.

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Fig. 3.
Analysis of dimerization of PDE chimeras
- -5 and
- -5. A,
chimeras FLAG- - -5 and myc- - -5 were co-expressed in
Sf9 cells and examined by gel filtration on the Superose® 12 column. Fractions of 0.4 ml were collected and analyzed for protein
concentration (dashed line), PDE activity (solid
line), and by Western blotting with anti-FLAG or anti-myc
antibodies. IP, an aliquot (80 µl) of the dimeric PDE peak
fraction 12 was incubated with (+) or without ( ) anti-FLAG
antibodies. Protein·antibody complexes were isolated using protein
G-agarose and analyzed by Western blotting with anti-myc as described
under "Experimental Procedures." B, combined PDE peak
front fractions 9-11 (solid line) and tail fractions 13-16
(dashed line) were concentrated by dialysis against 30 mM Tris-HCl buffer (pH 8.0) containing 130 mM
NaCl, 2 mM MgSO4, and 50% glycerin and then
reapplied onto the column. C, combined peak PDE gel
filtration fractions 10-13 were concentrated to a volume of 200 µl
using the YM-10 Microcon® devices (Millipore Corp., Bedford, MA) and
loaded onto the 5-35% sucrose density gradients. Following
centrifugation for 24 h at 40,000 rpm in a Beckman SW41 rotor,
fractions of 300 µl were collected starting from the bottom of the
tubes and analyzed for PDE activity. Arrows indicate
sedimentation of protein standards. PDE5, holoPDE6, and '- '-5
migrated at the same position of the gradient indicated as
PDE*.
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|
To verify the nature of the dimeric species, chimeric PDE was
immunoprecipitated with anti-FLAG antibodies and then probed with
anti-myc antibodies using Western blotting. The results of the IP
experiments proved the formation of a heterodimer between FLAG-
-
-5 or myc-
-
-5 (Fig. 3A). The peak of
dimeric PDE activity on the gel filtration column was relatively broad.
To determine if this PDE might be heterogeneous, the combined
concentrated front fractions 9-11 and tail fractions 13-16 were
reapplied onto the column. The resulting PDE activity profiles were
nearly identical, suggesting the presence of a single dimeric form of
PDE and trace amounts of aggregates (Fig. 3B).
The molecular shape of a protein significantly influences its migration
on a gel filtration column. Sucrose density centrifugation was utilized
as an additional independent approach to estimate the molecular weight
of chimeric PDEs (42). The sedimentation rates of native rod holoPDE6,
recombinant PDE5, His-
'-
'-5, and the
FLAG-
-
-5·myc-
-
-5 complex were similar (Fig.
3C). An estimated s20,w value
of 8.1 for the FLAG-
-
-5·myc-
-
-5 complex corresponds well
to the dimeric structure. Using this sedimentation coefficient and the
Stokes radius of 52 Å derived from the gel filtration data (Fig.
3A) (40), Equation 1 (43) yields a molecular mass of 176 kDa, which is comparable to the theoretical molecular mass (184 kDa)
for the FLAG-
-
-5·myc-
-
-5 dimer.
Next, we examined the possibility of dimerization of PDE6
and
PDE6
with cone PDE6
' and PDE5. FLAG-
-
-5 and myc-
-
-5
each were co-expressed with His-
'-
'-5 or His-PDE5. Because
PDE6
' and PDE5 form catalytic homodimers, a peak of PDE activity in gel filtration fractions corresponding to dimeric PDE species cannot be
used as evidence for heterodimerization. Instead, we relied on Western
blot analysis of the fractions for the presence of FLAG-
-
-5 and
myc-
-
-5. The Western blot analysis of the gel filtration
fractions of PDE species formed upon co-expression of FLAG-
-
-5
and His-
'-
'-5 indicated heterodimerization between the two
subunits (Fig. 4A). The
FLAG-
-
-5 signal appeared in fractions 10-14 corresponding to
dimeric PDE. The immunoprecipitates of these fractions with anti-FLAG
antibodies contained His-
'-
'-5, demonstrating the heterodimeric
composition of the dimers (Fig. 4A). Co-expression of
myc-
-
-5 and His-
'-
'-5 has not led to any significant dimer
formation between the two chimeric PDEs (Fig. 4B).
Similarly, co-expression of FLAG-
-
-5 or myc-
-
-5 with PDE5
and examination of formed PDE species revealed no detectable dimerization between the GAF regions of rod PDE6 and PDE5 (not shown).
The patterns of dimerization
-
-5 or
-
-5 were consistent with the selectivity of PDE6 dimerization in rod photoreceptor cells,
allowing us to use them as templates to further probe the role of the
PDE6 GAFa and GAFb domains.

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Fig. 4.
Analysis of dimerization of PDE chimeras
- -5 and
- -5 with
'- '-5.
His- '- '-5 was co-expressed with FLAG- - -5 (A) or
myc- - -5 (B) in Sf9 cells. Soluble Sf9
cell extracts (100,000 × g, 90 min) were applied onto
a FPLC Superose® 12 10/30 gel filtration column. Aliquots (20 µl)
from 0.4-ml gel filtration fractions were analyzed by Western blotting
with anti-His, anti-FLAG, or anti-myc antibodies. IP, an
aliquot (80 µl) of the dimeric FLAG- - -5·His- '- '-5 peak
fraction 12 was incubated with (+) or without ( ) anti-FLAG
antibodies. Protein·antibody complexes were isolated using protein
G-agarose and analyzed by Western blotting with anti-His as described
under "Experimental Procedures."
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Roles of the GAFa and GAFb Domains in Rod PDE6
Dimerization--
To determine the contributions of the rod PDE6 GAFa
and GAFb domains to the enzyme dimerization, two new PDE6/PDE5 chimeras have been constructed. Chimera His-
-
-5 contained the GAFa and GAFb domains from PDE6
and PDE6
, respectively (Fig. 1). The GAF
domains were swapped in the second chimera, His-
-
-5. If the rod
PDE6 catalytic subunits associate in a symmetrical "head-to-tail" fashion, His-
-
-5 and His-
-
-5 might have been capable of
self-dimerization. However, the gel filtration and Western blot
analyses of His-
-
-5 and His-
-
-5 expressed individually in
Sf9 cells revealed no formation of dimeric PDEs (not shown). In
contrast, catalytically active dimeric PDE species were observed
following co-expression of His-
-
-5 and His-
-
-5 (not shown),
suggesting a "head-to-head" dimerization of the PDE6
and PDE6
subunits. The lack of self-dimerization of FLAG-
-
-5,
myc-
-
-5, His-
-
-5, and His-
-
-5 allowed us to probe the
role of the GAFa and GAFb domains by co-expressing His-
-
-5 and
His-
-
-5 with either FLAG-
-
-5 or myc-
-
-5. Two of the
four combinations, FLAG-
-
-5·His-
-
-5 and
myc-
-
-5·His-
-
-5, yielded no dimeric PDE species (not
shown). The other two combinations, FLAG-
-
-5·His-
-
-5 and
myc-
-
-5·His-
-
-5, produced functional dimeric enzymes as
evidenced from the gel filtration and sucrose density centrifugation
data (Fig. 5). The migrations of
FLAG-
-
-5·His-
-
-5 and myc-
-
-5·His-
-
-5 in gel
filtration and in the sucrose density gradient were similar to those of
the FLAG-
-
-5·myc-
-
-5 complex. The catalytic
characteristics of FLAG-
-
-5·His-
-
-5
(Km = 2.7 µM,
Vmax = 20 nmol/min/mg) and
myc-
-
-5·His-
-
-5 (Km = 4.9 µM, Vmax = 17 nmol/min/mg) assayed
in the peak fractions #11 (Fig. 5, A and B) were
comparable to those of FLAG-
-
-5·myc-
-
-5. The results of
these experiments suggest that the main selectivity determinants of
dimerization of PDE6
and PDE6
reside within the GAFa domains.

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Fig. 5.
Dimerization of
- -5 with
- -5 and
- -5 with
- -5. A and
B, combinations of chimeras, FLAG- - -5·His- - -5
(A) and myc- - -5·His- - -5 (B), were
co-expressed in Sf9 cells and analyzed by FPLC gel filtration on
a Superose® 12 10/30 column (dashed line, protein
concentration; solid line, PDE activity) and Western
blotting using anti-His, anti-FLAG, or anti-myc antibodies as described
under "Experimental Procedures." C, combined peak
FLAG- - -5·His- - -5 (dashed line) and
myc- - -5·His- - -5 (solid line) gel filtration
fractions 10-13 were concentrated to a volume of 200 µl using the
YM-10 Microcon® devices (Millipore Corp., Bedford, MA) and loaded
onto the 5-35% sucrose density gradients. Following centrifugation
for 24 h at 40,000 rpm in a Beckman SW41 rotor, fractions of 300 µl were collected starting from the bottom of the tubes and analyzed
for PDE activity.
|
|
The GAFb domains of PDE6
and PDE6
are more homologous than the
GAFa domains and may contribute to the affinity of dimerization without
influencing its selectivity. Chimera His-
-5-5 containing the GAFa
domain of PDE6
and the GAFb domain of PDE5 was generated to test
this possibility. His-
-5-5 did not exhibit any propensity for
self-dimerization (not shown) but was able to efficiently form
heterodimers with FLAG-
-
-5 when the two proteins were
co-expressed in Sf9 cells (Fig.
6A). Sucrose density
centrifugation of FLAG-
-
-5·His-
-5-5 produced a
s20,w value of 8.1 confirming the
dimeric nature of the complex (not shown). Therefore, the GAFb domains of PDE6 do not significantly contribute to the dimeric interface.

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|
Fig. 6.
Dimerization of
- -5 with
-5-5,
 - -5, or
 - -5. Chimera
FLAG- - -5 was co-expressed with His- -5-5 (A),
His- - -5 (B), or His- - -5
(D) in Sf9 cells. Chimera
His- - -5 alone was expressed in Sf9 cells to
test its capacity for homodimerization (C). Dimer formation
was examined by the Western blot analysis of the fractions following
separation of the chimeric PDEs on a Superose® 12 10/30 column.
|
|
Mapping Dimerization Selectivity Determinants within the GAFa
Domains of PDE6--
The dimerization properties of His-
-
-5 and
His-
-
-5 indicated that the major selectivity determinants are
localized within PDE6
-1-235 (PDE6
-1-233). Approximately
45-residue-long N-terminal sequences of the PDE6
,
, and
'
subunits are very dissimilar and are followed by modestly conserved
N-terminal portions of the GAFa domains (~aa 45-90). The degree of
conservation between the GAFa domains is higher in the remaining GAFa
segments (~aa 90-230). This was taken into consideration in
designing chimeras His-
-
-5 and His-
-
-5 (Fig. 1).
His-
-
-5 and His-
-
-5 included the PDE6
sequences
1-93 and 94-235, respectively, substituting corresponding sequences
of PDE6
. After His-
-
-5 and His-
-
-5 showed no
capacity for self-dimerization, these chimeras were co-expressed with
FLAG-
-
-5 or myc-
-
-5. The combinations
FLAG-
-
-5·His-
-
-5, myc-
-
-5·His-
-
-5, or
myc-
-
-5·His-
-
-5 produced no dimeric PDE species as
judged by the gel filtration and Western blot analysis (not shown). On
the contrary, His-
-
-5 and FLAG-
-
-5 were capable of
assembly of catalytically active dimeric PDE (Fig. 6B).
Sedimentation of FLAG-
-
-5·His-
-
-5 in the sucrose
gradient (s20,w 8.1) and the
Km value for cGMP hydrolysis (4.5 µM)
were equivalent to the properties of other chimeric PDEs. In addition, His-
-
-5 formed dimers with His-
-
-5 (not shown). These
data indicate that at least some PDE6
dimerization selectivity
determinants are localized to within ~90 N-terminal residues of the
PDE6
and
subunits.
The major role of the GAFa domains in the dimerization of both PDE2
(28) and PDE6 suggests similar topographies of the intersubunit interfaces. The structure of the PDE2 (GAFa-GAFb)2 dimer
shows two main sites in each monomer that participate in the
intersubunit interface, one comprising a portion of the
-helix 1 and
the
1/
2 loop, and the second including the helix connecting GAFa
and GAFb (28). On the basis of a homology model of the
PDE6
GAFa-PDE6
GAFa dimer (Fig. 7,
A and B), the former site corresponds to ~16
residue segments in PDE6
(aa 59-74) and PDE6
(aa 57-72), which
are situated within the N-terminal 90-residue dimerization selectivity
regions. Chimeras His-
-
-5 and
His-
-
-5 (Fig. 1) have been constructed to test the
possibility that these PDE6
and
segments are responsible for
the selectivity of PDE6 dimerization. Gel filtration tests (Fig.
6C) and sucrose density centrifugation (not shown) revealed
that, unlike other constructed rod PDE6 chimeras (Table
I), His-
-
-5
was able to form homodimers. His-
-
-5 and
His-
-
-5 were then tested for the ability to form
heterodimers with FLAG-
-
-5 or myc-
-
-5. Consistent with the
role of PDE6
-59-74/PDE6
-57-72 as determinants for PDE6
dimerization, His-
-
-5 failed to dimerize with
myc-
-
-5, whereas His-
-
-5 did not produce dimers with FLAG-
-
-5 (not shown). Furthermore, although
His-
-
-5 was unable to dimerize with myc-
-
-5
(not shown), His-
-
-5 displayed a gain of
dimerization with FLAG-
-
-5 (Fig. 6D). The formation of
FLAG-
-
-5·His-
-
-5 dimers was also confirmed using immunoprecipitation with anti-FLAG antibodies followed by Western
blotting with anti-His antibodies (not shown).

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|
Fig. 7.
A model of the
PDE6 GAFa-PDE6 GAFa
dimer. A, a homology model of the
PDE6 GAFa-PDE6 GAFa dimer was generated with Swiss-PdbViewer
(version 3.7b2) and SWISS-MODEL (44) using the coordinates of the PDE2A
GAFa-GAFb dimer as a template (28). The image was produced using Sybyl
(version 6.6, Tripos). The PDE6 GAFa and PDE6 GAFa domains are
shown in green and red, respectively. Putative
intersubunit contact regions PDE6 -59-74 (white),
PDE6 -216-224 (cyan), PDE6 -57-72 (yellow),
and PDE6 -214-222 (blue) are indicated by
arrows. B, sequence alignments (45) of the PDE6
, , ' and PDE2 segments corresponding to PDE6 -59-74 and
PDE6 -216-224. PDE2 intersubunit contact residues within
PDE2-220-235 are underlined. C, from the model,
PDE6 -59-74 (white) interacts with PDE6 -57-72
(yellow) and PDE6 -214-222 (blue), and
PDE6 -57-72 interacts with PDE6 -59-74 and PDE6 -216-224
(cyan). D, schematic depiction of probable
interaction defects excluding homodimerization of PDE6 and
PDE6 .
|
|
 |
DISCUSSION |
The structural basis and functional role of PDE dimerization are
poorly understood because dimerization is not required for catalytic
function. The first molecular insights into PDE dimerization have been
revealed by the structure of the regulatory domains of PDE2A (28). This
crystal structure demonstrates that the GAFa domain is responsible for
the dimerization of PDE2A. One apparent implication from the PDE2A
structure is that other GAF domain-containing PDEs, such as PDE5, PDE6,
PDE10, and PDE11 utilize GAF modules for dimerization. Yet, it remains
unclear how well the dimerization interfaces of PDE5 and PDE6 parallel
that of PDE2. Although at a relatively low resolution, electron
microscopy imaging of PDE5 and PDE6 showed molecular shapes that are
somewhat different from the PDE2 structure (29). Furthermore, the
electron microscopy study indicates that, in addition to GAFa, GAFb
domains may contribute to the intersubunit interaction in PDE5 and
PDE6. Analysis of the dimerization interface of rod PDE6 catalytic
subunits permitted us to address two unresolved questions. What are the key structural determinants for dimerization of PDE6, and are they
similar to those identified in the PDE2A structure? What are the
selectivity determinants of rod PDE6 heterodimerization? The
heterodimerization of rod PDE6
and
subunits is unique among
known PDEs. It may be critical in the forming of rod-specific photoresponses. However, a potential significance of PDE6
heterodimers and the properties of individual subunits cannot be
assessed in the absence of 
and 
homodimers. Identification
of the selectivity determinants would be a first major step toward
generation of the homodimeric species.
Our analysis of dimerization of chimeric PDE6
/PDE5 proteins using
gel filtration, immunoprecipitation, and sucrose gradient centrifugation demonstrated selective heterodimerization between the
GAF regions of PDE6
- and
-subunits with no significant homodimerization. This observation is in accordance with the
established heterodimeric nature of the rod enzyme. Interestingly, the
GAFa-GAFb region of PDE6
, but not PDE6
, was capable of forming a
dimer with the GAFa-GAFb region of PDE6
'. Although dimerization of PDE6
and PDE6
' does not have physiological implications, as the
subunits are expressed in different types of photoreceptor cells, it
may provide additional clues to understanding PDE6 intersubunit interfaces. The patterns of dimerization (or lack thereof) (Table I)
show that the GAFa domains are the major contributors to the dimer
assembly of PDE. The finding that the PDE6 GAFa domains, similar to the
PDE2 GAFa domains, are responsible for its dimerization suggests a
common structural organization of the intersubunit interfaces of the
GAF domain-containing PDEs.
The subsequent identification of the PDE6
dimerization
selectivity determinants was carried out using chimeras carrying complementing portions of the PDE6
and
GAFa domains. The
ability of His-
-
-5 to dimerize with FLAG-
-
-5 has
implicated the N-terminal segment of the GAFa domains in the exclusive
PDE6
association. Further evidence was provided by the analysis
of the chimeric PDEs, His-
-
-5 and
His-
-
-5, containing short replacements PDE6
-57-72 and PDE6
-59-74 within the PDE6
and PDE6
GAF
regions, respectively. PDE6
-57-72 and PDE6
-59-74 correspond to
a region of PDE2GAFa,
1 helix-
1/
2 loop, that is involved in
the PDE2 dimer interface (28). Not only did the replacements prevent heterodimerization between His-
-
-5 and
myc-
-
-5, and between His-
-
-5 and
FLAG-
-
-5, but His-
-
-5 gained the ability for
self-dimerization and heterodimerization with FLAG-
-
-5. Dimerization of His-
-
-5 with FLAG-
-
-5
suggests that the lack of homodimerization of PDE6
is caused by a
defect in the interaction between the two PDE6
-59-74 segments (Fig.
7D). However, homodimerization of
His-
-
-5 coupled with the absence of association of
His-
-
-5 with myc-
-
-5 indicates that the lack
of homodimerization of PDE6
cannot be accounted for by the defective
interaction between the two PDE6
-57-74 segments. Homology modeling
of the PDE6
GAFa dimer using the structure of PDE2GAFa dimer as a
template indicates that PDE6
-57-72 may participate in two sets of
interactions involving PDE6
-59-74 and PDE6
-216-224 (Fig. 7,
A-C). PDE6
-216-224 resides at the start of the helix connecting GAFa and GAFb. Table I shows that no dimers had been formed
that would entail the interaction between PDE6
-57-72 and the
beginning of the connecting helix from PDE6
. Therefore, this structural constraint appears to disallow homodimerization of PDE6
(Fig. 7D). Additional negative dimerization determinants within the GAFa domain of PDE6
cannot be ruled out. A significant number of residues within PDE6
-59-74/PDE6
-57-72 and
PDE6
-216-224/PDE6
-214-222 are not conserved (Fig.
7B) and may play a role in the selective assembly of
PDE6
.
The finding that the selectivity determinants of PDE6 dimerization
are confined to relatively short segments in the GAFa domains supports the feasibility of generating mutant PDE6
and
subunits capable of homodimerization. Mutant homodimeric rod PDE6
expressed in transgenic animals would allow us to study the
individual catalytic subunits and elucidate the functional
significance of rod PDE6 heterodimerization.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health (NIH) Grant EY-10843. The services provided by the Diabetes and
Endocrinology Research Center of the University of Iowa were supported
by NIH Grant DK-25295.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.
¶
Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 319-335-7864; Fax:
319-335-7330; E-mail: nikolai-artemyev@uiowa.edu.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M208456200
 |
ABBREVIATIONS |
The abbreviations used are:
PDE, cGMP
phosphodiesterase;
P
,
subunit of PDE6;
PDE6
',
' subunit of
cone PDE6;
PDE5, cGMP-binding, cGMP-specific PDE (PDE5 family);
aa, amino acid(s);
IP, immunoprecipitation;
FPLC, fast-protein liquid
chromatography.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.