(Received for publication, January 16, 1997, and in revised form, February 28, 1997)
From the § Howard Hughes Medical Institute and the
Department of Pharmacology, University of Texas
Southwestern Medical Center, Dallas, Texas 75235-9050
We recently described two eye guanylyl cyclases (GC-E and GC-F) that contain an apparent extracellular domain potentially capable of binding ligands (Yang, R.-B., Foster, D. C., Garbers, D. L., and Fülle, H.-J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 602-606). Here, Northern and Western analyses showed that both cyclases are expressed in the retina and enriched in photoreceptor outer segments. By the use of specific GC-E and GC-F antibodies coupled to different sized gold particles both cyclases were colocalized within the same photoreceptor cells raising the possibility of homomeric and/or heteromeric interactions. A point mutant of GC-E (D878A) was constructed and expressed; it contained no detectable cyclase activity but acted in a dominant negative fashion to abolish the activity of native GC-E and GC-F in coexpression studies. These results suggested that GC-E and GC-F could form either homomers or heteromers, at least when overexpressed in COS-7 cells. Immunoprecipitation with GC-E and GC-F antibody followed by Western analysis confirmed that both homomers and heteromers could be formed. However, similar experiments using retina or outer segments revealed that a vast majority of GC-E and GC-F were precipitated as homomers in the eye. Therefore, like other members of the membrane guanylyl cyclase subfamily, GC-E and GC-F appear to preferentially form homomers.
Rod and cone cells convert the energy of an absorbed photon to an electrophysiological signal in a process called phototransduction. The photoexcitation beginning with rhodopsin leads to an enzymatic cascade resulting in the increased hydrolysis of cyclic GMP (cGMP) and closure of the cGMP-gated cation channel.
Photoreceptor guanylyl cyclases have been suggested to be regulated by Ca2+-sensitive guanylyl cyclase-activating proteins (1, 2); thus a decrease in cytoplasmic Ca2+ in response to light is suggested to relieve inhibition of the cyclases, for subsequent restoration of cGMP concentrations, and for a return to the dark state (3, 4). Two membrane forms of guanylyl cyclase have been found specifically expressed in the eye of the rat (5), human (6, 7), and bovine (8). In the rat, they are termed GC-E1 and GC-F (5), whereas their human homologs are designated RetGC-1 and RetGC-2, respectively (7). These cyclases contain an apparent extracellular domain that retains a number of conserved cysteine residues found in guanylyl cyclases with known ligands (5, 9). The eye cyclases also contain an intracellular protein kinase homology domain and a cyclase catalytic domain homologous to all other membrane guanylyl cyclases.
Although other membrane guanylyl cyclases are known to form homomers (10-12), the existence of two cyclases in the eye raises the important question of whether both are located within the same cell, and if so, whether these membrane cyclases could form heteromers, a characteristic of the soluble forms of guanylyl cyclase (13, 14).
Based on Northern and Western analyses, we showed that GC-E and GC-F are expressed in a retinal-specific manner with enriched partition to the outer segments. GC-E and GC-F not only form homo-oligomers but also associate in a heteromeric state in co-transfected COS cells. In addition, a point mutant of GC-E (D878A) exerts a dominant negative effect when coexpressed with wild-type GC-E and GC-F. Therefore, in cultured cells overproducing GC-E or GC-F, either hetero- or homo-oligomers can be formed. Using immunogold labeling, colocalization of both cyclases was evident within the same photoreceptor cells raising the possibility that hetero- and homo-oligomers also exist normally. However, solubilization of the cyclases from the retina or outer segment membranes followed by immunoprecipitation and subsequent Western analysis demonstrated that GC-E and GC-F exist principally as homomers.
The full-length cDNAs for GC-E
and GC-F were cloned into the mammalian expression vector pCMV5 and
termed pCMV5-GC-E and pCMV5-GC-F, respectively (5). The FLAG (DYKDDDDK)
and HA (YPYDVPDYA) epitopes were positioned immediately after the
cleavage site of the signal peptide by PCR-mediated mutagenesis (15).
Briefly, the resulting PCR fragments containing the desired epitope
insertions were used to replace 5 wild-type
EcoRI/XhoI or BglII/NsiI
fragments in pCMV5-GC-E or pCMV5-GC-F constructs, respectively. A
similar approach was used to generate a GC-E D878A point mutation. The
PCR product with the D878A mutation was digested with SalI
and NdeI and then ligated with additional wild-type 5
EcoRI/SalI and 3
NdeI/SalI fragments into the pCMV5 vector. Modified sequences in all constructs were confirmed by sequencing of both strands.
The following primers were used in
the constructions (modified sequences are underlined): FLAG-E1S,
5-GAC TAC AAG GAC GAT GAC GAT AAG GCT GTG TTC AAA GTG GGG
GTG-3
; FLAG-E1A, 5
-CTT ATC GTC ATC GTC CTT GTA GTC GGA
GAA GGC AGA CGG AGA TGG-3
; HA-E1S, 5
-TAT CCT TAC GAC GTA CCT
GAT TAC GCT GTG TTC AAA GTG GGG GTG-3
; HA-E1A, 5
-AGC GTA
ATC AGG TAC GTC GTA AGG ATA GGA GAA GGC AGA CGG AGA TGG-3
;
M13-20, 5
-TAA AAC GAC GGC CAG TGA GCG -3
; E09A, 5
-TCC TGG TCT AAT
AGC TCG-3
; FLAG-F1S, 5
-GAC TAC AAG GAC GAT GAC AAG CTC
CCC TAC AAG ATA GGG GTC-3
; FLAG-F1A, 5
-CTT ATC GTC ATC GTC CTT
GTA GTC TGC TTG CCC CCA TAT GAG-3
; HA-F1S, 5
-TAT CCT TAC
GAC GTA CCT GAT TAC GCT CTC CCC TAC AAG ATA GGG GTC-3
; HA-F1A,
5
-AGC GTA ATC AGG TAC GTC GTA AGG ATA TGC TTG CCC CCA TAT
GAG-3
; F-PCR2S, 5
-AGA TCT CCT CCA GTA TCT CAT CAC-3
;
F-5A, 5
-AGT GTG TCT GAG TCT CTC-3
; E878DA-1S, 5
-GTG GAA ACA ATT GGA
GCT GCA TAC ATG GTG GC-3
; E878DA-1A, 5
-GCC ACC ATG TAT
GCA GCT CCA ATT GTT TCC AC-3
; E878-2S, 5
-GAA GAG GTG ACA
CTC TAT TTC-3
; E878-2A, 5
-GCA GTA CCG AGG CAT GGT GAG-3
. FLAG-E1S/E1A or HA-E1S/E1A primer pairs combined with M13-20 and E09A
primers were used to construct the pFLAG-GC-E or pHA-GC-E vector.
FLAG-F1S/F1A, HA-F1S/F1A, F-PCR2S, and F-5A primers were used to create
PFLAG-GC-F and PHA-GC-F constructs. Other primers were designed for the
point mutation pE-D878A plasmid.
Whole eyes were collected from
Sprague-Dawley rats after decapitation. The retinas were dissected from
the posterior chamber of the eye cup, quickly frozen in liquid
nitrogen, and stored at 80 °C until use. Total RNAs were isolated
from the whole eye, retina, and retina-depleted eye by homogenization
in guanidinium thiocyanate followed by phenol/chloroform extraction and
precipitation with isopropyl alcohol (16). 30 µg of RNA was separated
on 2.2 M formaldehyde, 1% agarose gels and transferred to
nylon membranes in 20 × SSC. Blots were hybridized overnight at
42 °C in a solution containing 50% formamide, 5 × SSC, 5 × Denhardt's, 1% SDS, and 100 µg/ml denatured salmon sperm DNA
with a 1.6-kilobase pair GC-E or a 1.0-kilobase pair GC-F cDNA
probe (5). Blots were washed twice at room temperature and twice at
65 °C in 0.5 × SSC, 0.1% SDS (30 min per wash).
Autoradiography was performed at
80 °C for 3 days.
Two peptides, NH2-IPPERRKKLEKARPGQFTGK-COOH and NH2-AEIAAFQRRKAERQLVRNKP-COOH, corresponding to the COOH-terminal amino acid sequence of GC-E and GC-F were synthesized (5). Peptides were conjugated to tuberculin (Serumstaatensinstitut, Copenhagen, Denmark). Each conjugated complex mixed with complete Freund's adjuvant was injected subcutaneously in female New Zealand White rabbits. Booster injections were given every 3 weeks. Anti-FLAG (M2) and HA (12CA5) monoclonal antibodies were purchased from Eastman Kodak Co. and Boehringer Mannheim, respectively.
Cell Culture and TransfectionCOS-7 cells were maintained
in Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in an
atmosphere of 95% air and 5% CO2 at 37 °C.
Transfection was by the DEAE-dextran method on 100-mm plates (17). Two
days following transfection, cells were washed twice with cold PBS (pH
7.4) and scraped into centrifuge tubes. The cell pellet was frozen
quickly in liquid nitrogen and stored at 80 °C until use.
To determine guanylyl cyclase activity, membrane proteins
from transfected cells or chromatographic fractions were added to a
total reaction volume of 100 µl containing 25 mM Hepes
(pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 0.2 mM 3-isobutyl-1-methylxanthine, 4 mM
MnCl2, 0.1 mM GTP, and 1 µCi of
[-32P]GTP. Incubations were for 10 min at 37 °C and
terminated by the addition of 0.5 ml of 110 mM zinc acetate
and 0.5 ml of 110 mM Na2CO3.
[32P]cGMP was purified by Dowex chromatography and
quantitated by liquid scintillation counting as described (18). To
determine cellular cGMP, transfected cells in one 100-mm dish were
split into a 6-well plate. The next day transfected cells were washed with serum-free medium and then incubated in serum-free medium containing 0.25 mM 3-isobutyl-1-methylxanthine for 10 min
at 37 °C. 0.5 N perchloric acid was used to terminate
the reaction. Cyclic GMP in the acidified cell extracts was purified by
column chromatography and quantitated by radioimmunoassay (18).
Bovine eyes were freshly collected from the local
slaughterhouse. All procedures were performed in ambient light and at
4 °C. Ten retinas were dissected and washed twice with cold PBS and homogenized in 25 ml of cold sample buffer containing 25 mM
Hepes (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 20% glycerol. The
homogenates were centrifugated for 10 min at 1,000 × g. The resulting supernatant fluids were centrifugated for
30 min at 20,000 × g to obtain the crude retinal membranes, which were then washed once by the sample buffer. Washed retinal membranes were solubilized by 5 ml of the same buffer containing 1% Triton X-100. After 30 min on ice, lysate was clarified by centrifugation for 20 min at 350,000 × g to remove
insoluble material, and the supernatant fraction was stored at
20 °C until use. The outer segments were prepared by a
modification of the discontinuous sucrose density gradient technique as
described (19). Purity of the outer segment membranes was confirmed by the enriched presence of rhodopsin (greater than 90%).
Proteins were separated by SDS-PAGE. After electrophoretic transfer, the nitrocellulose membranes were blocked with PBS (pH 7.5) containing 5% nonfat dry milk and 0.1% Tween 20 overnight at 4 °C. The membranes were incubated with anti-GC-E or GC-F antisera at 1:10,000 or 1:5,000 dilution for 1 h at room temperature. For the control, each antiserum was incubated with 1 mg/ml peptide antigen in PBS (pH 7.5) overnight at 4 °C. Following two washes (15 min with PBS containing 0.1% Tween 20), the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (Tago Inc.) at a 1:10,000 dilution for 1 h. After washing the membranes, the enhanced chemiluminescence system (Amersham Corp.) was used for detection. For immunoprecipitation, transfected cells were solubilized in 0.5 ml of cold lysis buffer (25 mM Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 20% glycerol) for 40 min on ice. Lysates were clarified by centrifugation at 4 °C for 20 min at 350,000 × g. 2.5 µl of polyclonal antiserum were added to the clarified lysates. The immunocomplexes were precipitated by adsorption to protein A-agarose (Pierce), washed four times with lysis buffer, and subjected to Western blot analysis.
Immunoelectron MicroscopeLight-adapted female Sprague-Dawley rats maintained on a 12-h light/dark cycle were sacrificed. The posterior portion of each eye was dissected and immediately placed into a fixative containing 4% formaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at 4 °C. The specimens were then rinsed three times (1 h each) in 0.1 M phosphate buffer (pH 7.4) containing 0.9% NaCl and incubated in the same rinse solution overnight at 4 °C. The next day the samples were transferred into the same rinse buffer containing 0.1 M glycine. The ultrathin sections were prepared from LR White-embedded tissue and used to perform colloidal gold labeling as described (20).
Northern blot analysis
using RNA (30 µg) isolated from the whole eye, retina, and
retina-depleted eye demonstrates that GC-E and GC-F mRNA is
enriched in the retina but not detectable in samples from the
retina-depleted eye (Fig. 1). Thus, both eye guanylyl
cyclase mRNAs are expressed in a retinal-specific manner.
Generation and Characterization of Polyclonal Antibodies
Polyclonal antibodies were generated against the
COOH-terminal segment of rat GC-E or GC-F (see "Experimental
Procedures"). These peptide antigens share sequence similarity among
their potential homologs in mouse, human, or bovine (6-8, 21) and yet
are unique compared with other guanylyl cyclases. To examine the
specificity of antibodies, membrane proteins (10 µg) extracted from
the GC-E- or GC-F-transfected COS cells were analyzed for antibody
cross-reactivity by Western blot. As shown in Fig. 2,
the anti-GC-E polyclonal antibody reacts with a single 120-kDa
polypeptide in COS cells expressing GC-E. COS cells expressing GC-F or
vector alone show no reactivity. Likewise, the anti-GC-F antiserum
specifically recognizes a polypeptide with slightly greater mobility
(~116 kDa) in COS cells transfected with the GC-F construct. Again, vector alone or GC-E-transfected cells show no reaction to this antibody. Moreover, these two antisera do not cross-react with other
membrane guanylyl cyclases, GC-A, GC-B, GC-C, or GC-D (not shown).
With respect to tissue distribution, the corresponding GC-E or GC-F
immunoreactive bands were observed in rat retina (Fig. 3), but not in other tissues such as brain, heart,
kidney, liver, lung, skeletal muscle, small intestine, spleen, or
testes (data not shown). This is consistent with the retinal expression
of GC-E and GC-F determined by Northern analyses (Fig. 1 and Ref. 5).
The immunolabeled bands were not detected when the primary antibodies
were preincubated with the individual peptide antigen, which confirms
the specificity of these antisera (Fig. 3). There is apparent
cross-species reactivity in mouse and bovine retinal membranes,
presumably owing to the existence of orthologous GC-E and GC-F proteins
in these species (not shown).
Localization of GC-E and GC-F in Photoreceptor Outer Segments
To examine the partitioning of GC-E and GC-F receptors
in the retina, we determined their distribution by Western blot
analysis with membranes solubilized from the retina, photoreceptor
outer segment, or outer segment-depleted preparations. The
immunolabeled bands corresponding to GC-E and GC-F are enhanced in the
outer segments where phototransduction occurs (Fig. 4).
These data clearly verify that both GC-E and GC-F are predominantly
expressed in the photoreceptors but do not exclude much lower
expression in areas outside the photoreceptors (Fig. 4).
Immunolocalization of Photoreceptor Guanylyl Cyclases
Enrichment of both GC-E and GC-F in the outer segments leads to an important question of whether they are colocalized in the same photoreceptors or not. To determine their subcellular localization at the ultrastructural level, the rat retinas were used for subsequent immunoelectron microscopy studies with specific anti-GC-E and GC-F antisera.
A double immunolabeling technique was used in which sections were
immunolabeled on one side with 5-nm gold-labeled anti-GC-E antiserum
and the other with antiserum against GC-F coupled to 10-nm gold
particles (Fig. 5B). The data clearly showed
that both cyclases are located within the same rod cells (Fig.
5B). In addition, the gold particles of either size are
primarily distributed over the marginal region of both disk and plasma
membranes in the outer segments. Therefore, if GC-E and GC-F are
receptors, ligands apparently are present either outside the
photoreceptors or in the disk itself.
No labeling was seen in the connecting cilium, inner segment, and photoreceptor cell body (not shown). In addition, the immunogold label of GC-E over the outer segments appears to be consistent with a proposed role of RetGC in phototransduction in several species (22-25). Due to the rod-rich rat retina that contains very few cones, we were not able to compare the expression differences between rods and cones. In our experiments, peptide antigens do not completely abolish the immunogold label, which may be due to a higher affinity of the antibody for the true antigens rather than peptide alone. Regardless, the gold labels are considered significant since the anti-GC-E and GC-F antibodies invariably give rise to a stronger immunogold signal compared with a negligible background by the preimmune serum (Fig. 5A).
Homo- and Hetero-oligomerization in COS CellsPrevious studies have documented that other membrane guanylyl cyclases can assemble into homodimeric or higher ordered homomeric complexes in the absence of ligand (10, 11). However, soluble cyclases are known to form heterodimers and therefore the existence of GC-E and GC-F within the same photoreceptors raised the important question of whether or not heteromeric forms of the retinal cyclases exist. We constructed cDNAs encoding HA or FLAG epitope-tagged GC-E and GC-F proteins and examined their association by co-immunoprecipitation assays from both singly and co-transfected COS cells.
We first examined whether GC-E or GC-F formed homomeric complexes. COS
cells were transfected with cDNAs for FLAG-GC-E and/or GC-E tagged
with a HA epitope (HA-GC-E). Lysates of these cells were
immunoprecipitated with the anti-FLAG M2 monoclonal antibody, then the
precipitates were analyzed by immunoblotting with M2 or the anti-HA
monoclonal antibody 12CA5, respectively. A 120-kDa immunoreactive band
recognized by the anti-HA antibody was observed in the M2
immunoprecipitates from cells coexpressing FLAG-GC-E and HA-GC-E
proteins, but not from cells transfected with individual tagged
constructs alone (Fig. 6A). Similarly, the M2
monoclonal antibody immunoprecipitates both FLAG-GC-F and HA-GC-F in
cells co-transfected with both epitope-tagged GC-F constructs (Fig. 6B). These results demonstrated that photoreceptor guanylyl
cyclases are capable of forming homomeric complexes.
Whether heteromeric complexes could form was ascertained by co-transfection of FLAG-GC-E and untagged GC-F constructs in COS cells. Cells were then subjected to assay for complex formation by immunoblotting of anti-FLAG M2 immunoprecipitates with GC-F or GC-E antisera. If a stable interaction is maintained by coexpressing COS cells, it is predicted to co-immunoprecipitate the FLAG-GC-E·GC-F complexes. The anti-FLAG M2 immunoprecipitates from coexpressing cells resulted in a coprecipitation of GC-F, but not from cells expressing either FLAG-GC-E or GC-F alone (Fig. 6C). Likewise, the reciprocal immunoprecipitation of GC-F results in the coprecipitation of the overexpressed GC-E (not shown).
Furthermore, association between the expressed receptors appears to require coexpression and is not an artifact formed only after cell lysis, since a mixture of lysates containing separately expressed proteins is not sufficient for complex formation in heterologous expression systems (not shown).
Dominant Negative Effects of the GC-E D878A MutantThe apparent ability to form heteromers was confirmed by introduction of an alanine (D878A) at the same relative position as Asp-893 of GC-A. The D893A mutation results in an inactive cyclase that forms dimers with wild-type GC-A, thus acting as a dominant negative protein (26).
No cGMP production could be detected in cells expressing the GC-E D878A construct (Table I). However, coexpression of GC-E D878A and wild-type FLAG-GC-E constructs resulted in a substantial decrease (75%) in guanylyl cyclase activity as well as a 92% decrease in overall cGMP production (Fig. 7A and Table I). Likewise, coexpression of this mutant with wild-type GC-F also resulted in inhibition of cyclase activity and intact cell cGMP concentrations to 30 and 4% of wild-type controls, respectively (Fig. 7B and Table I). These results are in agreement with the observation that photoreceptor cyclases are capable of forming oligomers in the overexpressing COS cells (Fig. 6). Importantly, the expression of wild-type GC-E and GC-F, as monitored by immunoblots, remained unchanged in the presence of either vector plasmid or the GC-E D878A mutant construct (Fig. 7, A and B).
|
Immunoprecipitation of GC-E and GC-F from the Retina
That
GC-E and GC-F could form heteromers and homomers in overexpressing
cells leads to the question of which forms are normally present in the
retina. GC-E or GC-F was immunoprecipitated from the Triton-solubilized
retinal membranes with either anti-GC-E or GC-F-specific antibodies.
The immunoprecipitates were then analyzed for the presence of GC-E and
GC-F by Western blot analyses. As shown in Fig.
8A, GC-E antibody predominantly
immunoprecipitated GC-E, but a signal was only barely detectable with
GC-F. Likewise, anti-GC-F antiserum also quantitatively
immunoprecipitated GC-F and a limited amount of GC-E.
The same patterns of precipitation were also observed in the outer segment membranes (Fig. 8B). Examination of the unbound supernatant fluids from the immunoprecipitation assay indicates that all photoreceptor guanylyl cyclases were precipitated (Fig. 8B). These results demonstrate that individual photoreceptor guanylyl cyclases preferentially interact to form a homomeric complex in the outer segments; only a small portion of cyclases appear to assemble as heteromers.
Molecular Size of Receptor ComplexesThe molecular size of receptor oligomers was further determined by gel filtration chromatography. The freshly detergent-solubilized retinal cyclase activity was eluted in a relatively broad peak within a range of estimated molecular mass of 240-480 kDa (not shown); a similar elution profile was also reported in other studies (27, 28). This apparent size is consistent with the oligomeric state of GC-E and GC-F shown by immunoprecipitation assay, suggesting an enzymatic complex of a dimer or higher ordered structure (Fig. 6). Therefore, like other membrane receptor guanylyl cyclases (10-12), photoreceptor GC-E and GC-F appear to preexist in an oligomeric state.
In summary, we have demonstrated that rat GC-E and GC-F are photoreceptor guanylyl cyclases that can be expressed within the same cells. These two photoreceptor guanylyl cyclases, therefore, appear unique compared with various other components of the phototransduction cascade that are expressed in rod- or cone-specific manners (29-35). Using immunoprecipitation followed by immunoblotting, GC-E and GC-F expressed in COS cells could assemble in homomeric or heteromeric complexes. This is further sustained by a point mutation of D878A GC-E, which exerts a dominant negative effect to block the activity of wild-type GC-E and GC-F in coexpression studies. However, similar experiments with retina and outer segments showed that both cyclases appear to preferably form homomers.
We thank Deborah E. Miller and Lynda Doolittle for sequencing and oligonucleotides synthesis. We also thank Dr. Ted Chrisman for advice in the gel filtration chromatography, Dr. Michaela Kuhn for carefully reading the manuscript, and Dr. John J. Bozzola of Southern Illinois University for performing the immunoelectron microscopical studies.