From the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114
Received for publication, October 13, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Retinitis pigmentosa (RP) is a blinding retinal
disease in which the photoreceptor cells degenerate. Mutations in the
gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is
thought to be a putative guanine nucleotide exchange factor for an
unknown G protein. Ablation of the RPGR gene in mice
suggested a role in maintaining the polarized distribution of opsin
across the cilia. To investigate its function, we used a protein
interaction screen to identify candidate proteins that may interact
physiologically with RPGR. One such protein, designated
RPGR-interacting protein (RPGRIP), is expressed specifically in rod and
cone photoreceptors. It consists of an N-terminal region predicted to
form coiled coil structures linked to a C-terminal tail that binds
RPGR. In vivo, both proteins co-localize in the
photoreceptor connecting cilia. RPGRIP is stably associated with
the ciliary axoneme independent of RPGR and is resistant to extraction
under conditions that partially solubilized other cytoskeletal
components. When over-expressed in heterologous cell lines, RPGRIP
appears in insoluble punctate and filamentous structures. These data
suggest that RPGRIP is a structural component of the ciliary axoneme,
and one of its functions is to anchor RPGR within the cilium. RPGRIP is
the only protein known to localize specifically in the photoreceptor
connecting cilium. As such, it is a candidate gene for human
photoreceptor disease. The tissue-specific expression of RPGRIP
explains why mutations in the ubiquitously expressed RPGR confer a
photoreceptor-specific phenotype.
Genetic defects underlying
RP1 are heterogeneous.
The X-linked RPGR gene (1, 2) is clinically important
because of the greater severity of disease and the higher percentage of
patients associated with mutations in this gene (3). Neither the
disease mechanism nor the physiological function of RPGR is fully
understood. Analyses of the RPGR protein sequence, however, provide
some clues. The N-terminal domain of RPGR shares sequence similarity
with the regulator of chromatin condensation (RCC1), a nuclear protein that catalyzes guanine nucleotide exchange for the small GTPase Ran. RCC1 has an essential role in regulating nuclear import and export (4) by catalyzing the GTP exchange reaction for Ran. The
presence of an RCC1 homology domain thus raises the possibility that
RPGR may also regulate intracellular transport in photoreceptors via an
unknown G protein.
RPGR is ubiquitously expressed. In photoreceptor cells, however, it is
concentrated in the connecting cilium (5). We have previously studied
the retinal phenotype of RPGR knockout mice (5).
Photoreceptor defects are detected at an early age in mice lacking
RPGR. Cone photoreceptors exhibit ectopic localization of cone opsin
(the photopigment in cones similar to rhodopsin in rods) in the cell
body and synapse. Although ectopic distribution of rhodopsin is not
apparent in rods by immunofluorescence, there is nevertheless a reduced
level of rhodopsin in the outer segments. Subsequently, both cone and
rod photoreceptors degenerate. These data are consistent with the
proposal that RPGR plays a role in maintaining the polarized protein
distribution across the connecting cilium by facilitating directional
transport or restricting redistribution (5).
To investigate the in vivo function of RPGR and the disease
mechanism associated with RPGR mutations, we used a yeast two-hybrid screen to identify potential interacting partners of RPGR. This effort
led to the discovery of a novel protein that interacts with the RCC1
domain of RPGR. After substantial completion of this work, two reports
(6, 7) were published describing the identification of a bovine and
human RPGR interacting protein. Comparison of the sequences shows that
the protein we have identified is the murine ortholog of the bovine and
human RPGR-interacting protein. Adopting their designation, we refer to
this protein in the present report as RPGRIP (for RPGR interacting
protein). We further demonstrate that RPGRIP is a structural component
of the ciliary axoneme. We propose that RPGRIP serves as a scaffold to
anchor regulatory complexes including RPGR within the connecting cilium.
Yeast Two-hybrid Screening--
A GAL4-based two-hybrid system
was employed. Cloning vectors, yeast host cells, and reagents were
purchased from CLONTECH Laboratory (Palo Alto, CA).
A retinal cDNA library was constructed using poly(A)+
RNA from C57BL/6 mouse retinas. The cDNAs were inserted into the
pACT2 plasmid vector downstream from the GAL4 activation domain. The
resulting cDNA library contained ~1 × 106
independent colonies. The bait plasmid was constructed by inserting a
cDNA encoding the bait protein into the pGBKT7 plasmid vector downstream from the GAL4-DNA binding domain. The bait protein consisted
of the RCC1 homology domain of murine RPGR (residues 39-460).
Reference to the numbering of the RPGR sequence in this report is based
on a previously published sequence (GenBankTM accession
number AAC40190) (8). A sequential transformation protocol was used to
introduce bait and library plasmids into yeast. Yeast AH109 cells were
first transformed with the bait plasmid, and the bait protein
expression was verified by immunoblotting. Competent cells were then
prepared from a yeast clone harboring the bait plasmid and transformed
with the library plasmids. Positive colonies were isolated based on
their ability to express nutritional markers HIS3 and ADE2 and the
lacZ reporter, driven by different Gal4-responsive promoters
to minimize false positives due to fortuitous activation of a
particular promoter. To rule out bait-independent activation, the
candidate plasmids isolated from the initial screen were co-transformed
with an irrelevant control bait and shown not to activate the
nutritional markers.
Cloning of Full-length RPGRIP cDNA--
To isolate cDNAs
encompassing the entire coding region of RPGRIP, one of the cDNA
clones isolated from the initial screen was used as a probe to screen a
second mouse retinal cDNA library constructed in a Northern Blotting--
Total RNA isolated from a variety of
mouse tissues was separated on denaturing agarose gels, blotted to
nylon membranes, and hybridized to radiolabeled probes under high
stringency conditions, with the final wash carried out in 0.1× SSC,
0.1% SDS at 70 °C. Four different probes were used in separate
experiments to determine the origins of the bands seen in different
tissues. P1 was a double-stranded DNA probe spanning ~1000 base pairs
of the coding region near the C terminus (beginning at amino acid codon
1025) plus 750 base pairs of the 3' untranslated region. P2 was also a
double-stranded DNA probe located upstream from P1 (corresponding to
codons 77-773). The P3 and P4 probes spanned the same region as P1 but
were synthesized in vitro using T7 or T3 RNA polymerases as
single-stranded RNA probes in either the sense (P3) or the antisense
(P4) orientation. The locations of the probes are shown at the bottom
of Fig. 1.
Antibodies, Immunoblotting, and Immunofluorescence
Microscopy--
A polyclonal RPGRIP antibody was generated by
immunizing a rabbit with a truncated recombinant RPGRIP. A cDNA
fragment corresponding to amino acid codons 991-1331(inclusive of the
RPGR binding domain) of RPGRIP was inserted into the pGEX4T-2
expression vector (Amersham Pharmacia Biotech) and expressed in
Escherichia coli as a GST fusion protein. The GST moiety was
removed from the purified fusion protein by thrombin digestion and the
purified RPGRIP fragment used as the immunogen. Affinity purification
of the specific antibodies was carried out with the same immunogen
immobilized in an AminoLink column (Pierce). A polyclonal antibody
raised against the C-terminal 253 amino acids of mouse RPGR (RPGR-253)
was described previously (5). The C-terminal portion of RPGR is
variable because of alternative splicing (3). To follow all the major
variants, the RPGR-253 antibody was further fractionated by affinity
chromatography using as ligands recombinant proteins representing
smaller fragments within the original immunogen. One such fraction,
RPGR-S1, would be specific for residues 494-563 common in all
variants. RPGR-S1 was used for immunofluorescence detection of RPGR in
this study. Antibodies against actin, Immunogold Labeling and Transmission Electron
Microscopy--
Mouse eyes were fixed in 2% formaldehyde, 0.1%
glutaraldehyde in 0.1 M phosphate buffer for 1 h.
Retinas were dissected out, soaked in 30% sucrose in PBS overnight,
and frozen in liquid nitrogen. Thin (70 nm) sections were cut on a
Leica cryo-ultramicrotome and collected on Formvar-coated nickel grids.
Grids were incubated sequentially with 0.15 M glycine/PBS,
TTBS (Tween/Tris-buffered saline) supplemented with 100 mM
2-mercaptoethanol, and blocked in 5% goat serum, 1% fish gelatin in
TTBS. Incubation with the primary antibody diluted in the blocking
solution proceeded overnight at room temperature. After washing in
TTBS, grids were incubated with goat anti-rabbit secondary antibody
conjugated to 5 nm gold and washed again. To perform silver
enhancement, a goat anti-rabbit secondary antibody conjugated to 1 nm
gold particles was used instead. Sections were then treated with the
Aurion R-Gent silver enhancement reagents (Electron Microscopy
Sciences) for 8 min. Sections were post-stained with 4% uranyl
acetate, washed through drops of methyl cellulose, and air dried.
Sections were viewed and photographed on a JEOL 100CX electron microscope.
In Vitro GST Pull-downs and Co-immunoprecipitation--
Purified
GST-RPGRIP (residues 991-1331) fusion protein and the native RPGR
present in mouse brain lysate were used in the pull-down assays. Mouse
brain was mechanically homogenized in a lysis buffer (20 mM
Tris, pH 7.4, 1% Nonidet P-40, 5 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride,
and a mixture of protease inhibitors (Roche Molecular
Biochemicals)). The homogenate was cleared by centrifugation at
20,000 × g for 30 min. GST-RPGRIP or GST (negative
control) coupled to glutathione-Sepharose was incubated with the
indicated amounts of brain lysates adjusted to the same final volume in
lysis buffer at 4 °C overnight. The glutathione-Sepharose beads were
washed three times with washing buffer (20 mM Tris, pH 7.4, 0.5% Nonidet P-40, 5 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors). Bound proteins were eluted by boiling in SDS-PAGE sample buffer and
analyzed by SDS-PAGE and immunoblotting.
Expression and Analyses of RPGRIP and RPGR in Cultured
Cells--
COS-7 cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum at
37 °C in 5% CO2. Transfection was carried out using the
Geneshuttle 40 reagent (Quantum Biotechnologies) according to the
manufacturer's instructions. To express RPGRIP, three expression
plasmids were constructed. The full-length coding region of RPGRIP was
inserted into the pcDNA3 (Invitrogen) to generate the pRPGRIP
plasmid for expression of untagged RPGRIP. To generate recombinant
RPGRIP that were tagged at the N terminus with either a Myc or
an EGFP epitope, coding sequences for myc or EGFP were
introduced into pRPGRIP upstream and in frame with the RPGRIP coding
sequence, generating plasmids pMyc-RPGRIP and pEGFP-RPGRIP,
respectively. For co-immunoprecipitation of recombinant RPGRIPs, COS
cells were co-transfected with pmyc-RPGRIP and pEGFP-RPGRIP. After
48 h, cells were washed with PBS and lysed in an
immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5mM EDTA, 1% Nonidet P-40, and a
protease inhibitor mixture). The lysate was centrifuged at 16,000 × g for 15 min. Immunoblot analyses indicated that about
1-5% of the total RPGRIP expressed in COS cells was recovered in this
supernatant. Immunoprecipitation was allowed to proceed overnight at
4 °C by incubating the cleared lysate with either an anti-Myc
monoclonal antibody (clone 9E10) and anti-mouse IgG-agarose or with an
anti-GFP polyclonal antibody (Santa Cruz) and protein A-agarose. After
washing, bound proteins were eluted by boiling in SDS-PAGE sample
buffer, separated by SDS-PAGE, and detected by immunoblotting with the
anti-RPGRIP antibody. For chemical cross-linking, cells transiently
transfected with pRPGRIP were washed with PBS and incubated with the
indicated amount (Fig. 6B) of dimethyl pimelimidate (DMP) in
100 mM HEPES buffer (pH 8.2) for 1 h at 4 °C. The
reaction was terminated with a quenching buffer (100 mM
Tris, pH 7.4, in PBS). After removal of the quenching buffer, the cells
were lysed in SDS-PAGE sample buffer.
Cellular Fractionation and Extractions--
The procedure
consisted of collecting and extracting the inner and outer segment
enriched fraction produced by shaking freshly dissected retinas in a
homogenization buffer (11). Under Nomarski optics, these preparations
were seen to contain outer segments linked to the connecting cilia with
variable portions of inner segment membranes attached (10). The
remainder of the retinas usually remained as intact sheets, thus
providing an enrichment of photoreceptor-derived material in the
suspension. Unless otherwise noted, tissues were kept on ice or at
4 °C during the procedure. In a typical experiment, 16 freshly
dissected mouse retinas were vortexed for 1 min in 0.6 ml of
homogenization buffer (34% sucrose, 25 mM Tris, pH 7.4, 5 mM EDTA, 150 mM NaCl, 10 mM
iodoacetamide, and a mixture of protease inhibitors). The suspension
was centrifuged at 500 × g for 2 min to remove large
debris. The supernatant was diluted 1:3 with a dilution buffer
(homogenization buffer without sucrose) and centrifuged again at
170,000 × g for 15 min. The supernatant was removed to
fresh tubes and was designated the cytosol fraction. The pellet was
resuspended in the dilution buffer containing 1% Nonidet P-40.
Aliquots of 100 µl each were spun at 170,000 × g for
20 min. The supernatant was pooled and designated as the
detergent-soluble fraction and the pellets the axoneme-enriched fraction. Although the scarcity of mouse retinal tissues precluded the
use of sucrose gradient centrifugation, which would have yielded a
cleaner axonemal preparation (12), the enrichment of Identification of RPGRIP--
To identify interacting proteins, we
performed a yeast two-hybrid screening of a mouse retinal cDNA
library using the RCC1 homology domain of RPGR as bait. From among
106 co-transformants, six independent clones were isolated.
Sequence analysis revealed that one clone coded for the
To confirm that the coding sequence of RPGRIP was full-length at the 5'
end, the 5' RACE procedure was performed. Sequences of the RACE
products provided an additional 30 base pairs at the 5' end but
did not further extend the open reading frame. Subsequent immunoblotting analyses showed that the native RPGRIP from mouse retinas and a recombinant protein from transfected COS cells expressing the 1331 open reading frame had the same apparent molecular weight on
SDS-PAGE (Fig. 2A), lending
further support to the claim that this open reading frame is
full-length. The apparent molecular mass of RPGRIP is 210 kDa,
larger than the predicted 150 kDa. The reduced motility of the
polypeptide on SDS-PAGE may be accounted for by the high glutamic acid
content (14%) in this protein.
Confirmation of a direct interaction between RPGR and RPGRIP was
provided by an in vitro GST pull-down assay. A GST-RPGRIP fusion protein containing the RPGR binding domain was shown to co-sediment with RPGR from tissue lysate in the presence of
glutathione-Sepharose (Fig. 2B). Therefore, the recombinant
RPGRIP interacts with native RPGR in vitro. A demonstration
of the interaction between two native proteins by
co-immunoprecipitation was not technically feasible because RPGRIP
could not be solubilized under native conditions (see below).
Tissue-specific Expression of RPGRIP--
The tissue expression
pattern of RPGRIP was examined by Northern blotting (Fig.
3A) and immunoblotting (Fig.
3B). RNAs from a number of mouse tissues were examined. Initially a
double-stranded DNA probe P1 (see bottom of Fig. 1) revealed
a doublet band around 10 kb in the retina only and a 4.4-kb band that
is more widely distributed. To determine which band(s) represented the
authentic RPGRIP transcript, three additional probes were synthesized
and used to probe Northern blots. Probe P2 was located upstream from P1
and entirely within the coding region. Probe P2 detected the 10-kb
doublet but not the 4.4-kb band. Furthermore, the 10-kb doublet was
present only in the wild type retina but not in the adult rd
mouse retina in which the photoreceptors had degenerated, indicating a
photoreceptor-specific origin of these transcripts. Thus, if the 4.4-kb
band represented an RPGRIP transcript, it would have only the capacity
to encode a protein comprising essentially the RPGR binding domain
alone. Existence of such a variant would have functional implications.
Alternatively, this band might be derived from an overlapping but
otherwise unrelated gene. A data base search indicated that the gene
for a chromatin-specific transcription elongation factor (16) overlaps
with RPGRIP at the 3' end and is transcribed in the opposite
orientation. To explore the latter possibility, probes P3 and P4,
covering the same region as P1 but synthesized as single-stranded RNA
probes in the antisense (P3) or sense (P4) orientation, were used to
probe Northern blots. P3 detected only the 10-kb doublet, whereas P4
detected only the 4.4-kb band. This finding confirmed that the
4.40-kb band was derived from an overlapping gene transcribed in the
reverse orientation. Therefore the 10-kb doublet represents the
authentic RPGRIP transcripts, and both should encode the full-length
RPGRIP protein. The size difference is likely to reside in the 3'
untranslated region because of alternative use of transcription
termination signals. Finally, probe P2 detected a much smaller
transcript (3 kb) found only in the testis. The functional significance
of this variant, if any, is not clear. It should be noted that the
testis is thought to express irrelevant transcripts which, in somatic
tissues, may be tightly regulated (17).
Immunoblotting of RPGRIP protein indicates a similar
photoreceptor-specific expression (Fig. 3B). A polyclonal
antibody raised against the RPGR binding domain of RPGRIP detects the
210-kDa polypeptide in the retina but not in a variety of other
tissues. The RPGRIP band is also present in the retinas of RPGR
knockout mice.
RPGRIP Is Localized in the Photoreceptor Connecting
Cilium--
Photoreceptors are highly polarized sensory neurons with
their outer segments, inner segments, nuclei, and synapses organized into distinct layers on retinal sections, which makes it relatively simple to determine the subcellular localization of a protein on
retinal sections. By immunofluorescence microscopy, we previously determined that RPGR is localized in the connecting cilia of rod and
cone photoreceptors (5). To determine the subcellular location of
RPGRIP in photoreceptor cells, we performed immunostaining of retinal
sections using the polyclonal RPGRIP antibody. The data show RPGRIP is
located at the junction between the inner and outer segments (Fig.
4A), suggesting a localization
in the connecting cilia. This staining pattern is identical to that of RPGR (Fig. 4A). The RPGRIP localization remains the same in
the absence of RPGR (Fig. 4A). This would suggest that
RPGRIP is independently targeted to the connecting cilium, whereas RPGR
is anchored to this location via its interaction with RPGRIP.
Immunostaining of frozen sections of the lungs indicated that RPGRIP is
not present in the motile cilia of airway epithelia (not shown).
Photoreceptor connecting cilium originates from a pair of basal bodies,
located at the apical inner segment and related structurally to
centrioles. The axonemal microtubules grow out of one of the basal
bodies and continue into the outer segment (18). To determine whether
RPGRIP is confined to the connecting cilium, or whether it extends
proximally to the basal body and distally into the outer segment, we
performed double labeling for RPGRIP and
Because mouse photoreceptors are overwhelmingly rods, the data
described above provided no evidence concerning the localization of
RPGRIP in cones or whether it is present in cones at all. To address
this question, we performed immunostaining for RPGRIP on the
cone-dominant retinas of 13-lined ground squirrels. In contrast to
mice, ground squirrel retinas are ~95% cones (19). On retinal
sections, immunostaining with the RPGRIP antibody prominently labeled
the connecting cilia in this cone-dominant retina (Fig. 4D).
The RPGRIP antibody, although generated with a murine antigen, recognizes a region in RPGRIP that is highly conserved among the mammalian species, suggesting that immunostaining in the ground squirrel was likely to be valid. These data demonstrate that RPGRIP is
localized in the connecting cilia of both rod and cone photoreceptors.
Immunogold labeling and electron microscopy corroborated the findings
by immunofluorescence. An examination of multiple sections showed that
all the connecting cilium profiles are labeled with little background
staining in the inner and outer segments. As shown in Fig.
5, labeling was restricted to the
connecting cilium but was not found in the basal body. In the
transverse view of connecting cilia, the 5-nm gold particles, on
average, do not overlap with the profiles of the microtubule doublets.
They appear on the external side of the ring of microtubule doublets.
In this regard, the ultrastructural labeling of RPGRIP is somewhat
similar to that of myosin VIIa (20). An examination of longitudinal sections revealed a similar finding (not shown).
Expression of RPGRIP in Heterologous Cell Lines--
The presence
of the coiled coil domain in RPGRIP suggests that it may normally exist
as dimers or higher order polymers. To examine the state of existence
of RPGRIP, recombinant RPGRIP, either tagged or untagged, was expressed
in COS cells. By immunofluorescence or by following the green
fluorescence protein tag, RPGRIP in transfected cells was seen in
punctate and filamentous patterns (Fig.
6A). Double labeling
experiments showed that RPGRIP did not co-localize with microtubules or
several intermediate filament proteins including vimentin and
cytokeratins. Furthermore, the filamentous staining pattern of
RPGRIP was preserved in cells in which the microtubules had been
disrupted by treatment with nocodazole (data not shown). These data
suggest that RPGRIP may exist in large polymers in the cultured cells.
Consistent with this notion, cellular fractionation and SDS-PAGE
analyses showed less than 5% of RPGRIP could be recovered in the
supernatant; the remainder was present in the detergent-insoluble
pellet. Limited cross-linking produced higher molecular weight species
consistent with dimer and higher order oligomers (Fig. 6B).
As a second test for oligomer formation, COS cells were co-transfected
with plasmids carrying c-Myc- or EGFP-tagged RPGRIP. The cell
lysate was then immunoprecipitated with either a c-myc or an
EGFP antibody. As shown in Fig. 6C, either antibody was able
to co-precipitate both recombinant proteins, providing further evidence
for homodimer or higher oligomer formation by RPGRIP in the
transfected cells. Taken together, these data suggest that RPGRIP
polymerizes into a higher molecular weight complex in transfected
cells.
RPGRIP Is a Structural Component of the Ciliary Axoneme--
To
examine the nature of its association with cilium in greater detail, a
series of cellular fractionation and extraction experiments were
carried out. In these experiments, photoreceptor inner and outer
segments were shaken off, collected and extracted with detergent or
high salt. As shown in Fig.
7A, RPGRIP was not detectable
in the cytosol, nor could it be solubilized by treatment with the
detergent Nonidet P-40. It was found exclusively in the detergent-insoluble fraction enriched for ciliary axoneme. Treatment with saponin, or with ATP and its nonhydrolyzable analog AMP-PNP, produced identical results (not shown), indicating that RPGRIP was not
associated with detergent-insoluble lipids (21) or indirectly tethered
to the axoneme via an association with kinesins. Thus, RPGRIP is likely
to be directly associated with, or is a part of, the axonemal
cytoskeleton. This is in contrast to RPGR, which exhibited a sizable
cytosolic pool in addition to the fraction associated with the cilium.
Considering the cilium is such a minute structure relative to the inner
and outer segments, the data is fully consistent with our observation
that RPGR is concentrated in the connecting cilium.
The detergent-insoluble fraction from above was further extracted with
high salt or denaturants. The results are shown in Fig. 7B.
RPGRIP was completely resistant to extraction by high salt and was only
solubilized by the strong denaturants, SDS and urea. In contrast, a
substantial fraction of RPGR could be solubilized by high salt
extraction, which is consistent with the suggestion that RPGRIP is
specifically targeted to the cilium while RPGR is recruited to this
cellular compartment as a secondary event. We then compared this
extraction profile with that of ciliary cytoskeletons and tektin
filament. Ciliary microtubules are of the stable, acetylated form (22),
and microtubules in the basal bodies contain Although the importance of RPGR in the pathogenesis of RP is now
recognized (3), the physiological function of RPGR and the molecular
basis of disease caused by its mutations remain elusive. To date, few
reports have appeared on the analyses of the native RPGR protein from
the retina. In a previous study, we found RPGR to be concentrated in
the connecting cilia of rod and cone photoreceptors. Furthermore,
analyses of mice lacking RPGR indicated a role for RPGR in maintaining
the polarized distribution of proteins across the connecting cilia (5).
In an attempt to delineate the biochemical pathway in which RPGR is a
component, we used a protein interaction screen to identify proteins
that may physiologically interact with RPGR. With the RCC1 homology domain of RPGR as a bait, we have isolated an interacting protein, RPGRIP, and demonstrated its co-localization with RPGR in the connecting cilium. We found a significant difference between RPGR and
RPGRIP in their association with the cilium. RPGR has both a ciliary
pool and a sizable cytosolic (or loosely associated) pool. In contrast,
RPGRIP is exclusively and stably associated with the ciliary
axoneme. Because its ciliary localization is unchanged in RPGR knockout
mice and because it remains bound to the ciliary axoneme when RPGR is
fully solubilized, we conclude that RPGRIP is targeted independently to
the cilium, whereas RPGR is anchored here through binding to RPGRIP.
Given that the connecting cilium is a minute structure with a limited
number of resident proteins, their co-localization in this cellular
compartment and the finding that the expression of RPGRIP is
specific to the photoreceptors provide strong evidence for the
functional significance of their interaction.
The RPGRIP described here is the murine ortholog of the human and
bovine RPGRIP proteins reported recently (6, 7). Differences between
our work and the two reports are noted in several aspects. First, the
reported human sequences are shorter by up to 700 residues compared
with the murine sequence reported here. Analysis of the human RPGRIP
genomic sequence in GenBankTM with the GenScan program
predicted 5' coding exons that are not included in the two reports (6,
7) and that are highly homologous with the murine RPGRIP. These
putative upstream exons could be identified by reverse
transcription-polymerase chain reaction using human retinal
mRNA as a template.3
Therefore, the reported human RPGRIP sequences may not be complete at
the 5' end. Second, we found no evidence in murine tissues for
substantial alternative processing in the coding region of the RPGRIP
transcript. We examined such a possibility extensively with Northern
blot analysis. Initial Northern blotting data indicated the possible
presence of a variant encoding a much smaller protein; this raised the
interesting possibility that a soluble RPGR interacting protein
(without the coiled coil domain) could sequester RPGR in a different
cellular compartment, which would have functional implications. Further
examination with single-stranded probes indicated that the smaller
transcript is derived from an overlapping gene. Data base search also
indicates that this overlapping gene organization is conserved between
the mouse and human genomes. Third, one of the reports found
co-localization of RPGR and RPGRIP in a different subcellular
compartment, i.e. the outer segment (7), whereas we have
concluded that RPGRIP localizes exclusively in the connecting cilium
and is presumably responsible for concentrating RPGR in the cilium. The
reason for this discrepancy is not yet clear, but we suggest it could
be accounted for by the differences in the procedure for tissue
fixation. In particular, we observed that aldehyde fixation prior to
embedding and sectioning was detrimental for staining both RPGRIP and
RPGR at the light microscopy level. This appears to limit access by the
antibodies rather than destroy the antigenic epitopes, because
dissociated photoreceptors which would have lost much of the materials
surrounding the cilia are much less sensitive to aldehyde fixation.
Ultrathin frozen sections, which would have antigenic sites more fully
exposed, could also withstand pre-embedding fixation.
The photoreceptor is a highly polarized cell in which a nonmotile
connecting cilium links two morphologically and functionally distinct
parts, the inner and outer segment. The ciliary axoneme originates from
a basal body located at the distal end of the inner segment, and its
core structure is composed of nine outer microtubule doublets but no
central microtubule singlet, forming what is usually referred to as a 9 + 0 arrangement. The structural organization is analogous to the
transition zone of motile cilia (26, 27). Unlike motile cilia, however,
the connecting cilium daily transports a prodigious amount of proteins
including rhodopsin (see Ref. 28 for a recent review) and other
components of phototransduction. The inner segment contains all of the
organelles for biosynthetic and metabolic functions. The outer segment
is a specialized organelle where the phototransduction cascade takes
place. The outer segment is continually renewed throughout life by
shedding older portions at the tip and adding new membranes at the
base. As the sole physical link between the inner and outer segments,
the connecting cilium is critically important for the directional
transport of nascent proteins destined to the outer segment, against a
steep concentration gradient. Because the inner and outer segments have
very different protein composition, the connecting cilium also acts as
a diffusion barrier against redistribution (29). Maintenance of the
outer segment is essential for efficient phototransduction, as well as
for the long term survival of the photoreceptor cell as demonstrated by
studies in animal models. Interestingly, a number of proteins also
exhibit light-dependent rapid movement across the cilium, although its physiological significance remain undefined (30). Thus the
finding that RPGRIP anchors RPGR in the connecting cilium is consistent
with their putative function in regulating protein transport in the
connecting cilium.
In transfected COS cells, RPGRIP exhibits a mixed punctate and
filamentous pattern. This does not appear to result from binding to an
existing cytoskeletal structure because in double labeling experiments
RPGRIP staining does not fully overlap with microtubule or intermediate
filaments. These data indicate that RPGRIP expressed in cultured cells
polymerizes into high molecular weight complexes that are resistant to
detergent solubilization. The coiled coil domain is likely to play a
part in mediating the oligomer formation.
In the cilium, RPGRIP also appears to exist in higher molecular weight
complexes. Judging by data from chemical cross-linking and electron
microscopy, the RPGPIP complex does not appear to be in extensive
contact with or integrated into the microtubule arrays. Two
possibilities can be envisioned. RPGRIP may form filamentous structures running parallel to the axonemal microtubules, although no
ultrastructural evidence currently exists for such a structure. Alternatively, RPGRIP may be a component of the
microtubule-membrane cross-linkers. These are the Y-shaped structures
seen in transverse sections that project from each microtubule doublet
at the junction between the A and B tubules to the adjacent plasma
membrane (12, 31). These structures co-purify with the ciliary axoneme
and are resistant to detergent extraction. Their location is confined to the connecting cilium, matching that of RPGRIP. One caveat to this
hypothesis is that a morphologically equivalent structure is also
present in the transition zone of all motile cilia (32), and our data
indicate that RPGRIP expression is confined to photoreceptors. These
differences could be reconciled if related protein(s) are responsible for reconstituting the cross-linkers in motile cilia. Although its exact structural organization remains undefined, the
localization of RPGRIP between the external wall of the microtubule doublet and the plasma membrane that surrounds it should bring it into
close contact with soluble and membrane-bound proteins trafficking
through or along the connecting cilium, either by intraflagellar
transport or by movement along the plasma membrane (33-35). Thus,
RPGRIP is well suited to serve as a scaffold to anchor RPGR and
possibly other regulatory complexes, which in turn would control the
directional transport of proteins to or from the outer segment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage
vector. Multiple overlapping clones covering the full-length coding
region were obtained. The extreme 5' untranslated sequence not
represented in the phage clones were obtained by performing the RACE
(rapid amplification of cDNA ends) procedure using reagents from
CLONTECH. Sequences from the RACE product and from
the phage cDNA clones were joined to yield the final cDNA
sequence of RPGRIP (GenBankTM accession no. AY008297).
-tubulin, and acetylated
-tubulin (clone 6-11B-1) were obtained from Sigma. Antibody against
a mouse tektin (tektin-t) (9) was kindly provided by Dr. Yoshitake Nishimune (Osaka University). This antibody recognizes an 86-kDa tektin
specific in testes as well as a 60-kDa tektin-like protein in
lungs.2 For immunoblotting,
samples were separated on SDS-polyacrylamide gels and electroblotted to
polyvinylidene difluoride membranes. Membranes were incubated with
primary antibodies, followed by peroxidase-conjugated secondary
antibodies, and were developed by ECL or color substrates. Detection of
RPGR and RPGRIP in the retina by immunofluorescence microscopy was
performed on frozen sections. Optimal labeling was obtained only if the
mouse eyes was unfixed before freezing and sectioning. Freshly cut
sections, 5-10 µm in thickness, were post-fixed on slides for 1-2
min with 2% formaldehyde/PBS, blocked in 5% normal goat serum/PBS,
and incubated with primary and Cy3-conjugated secondary antibodies. After the final wash, slides were stained with the nuclear dye Hoechst
33342 and mounted in aqueous mounting media. Dissociated photoreceptor
cells were prepared and stained as described (10). To perform
immunofluorescence microscopy on transiently transfected cells, cells
grown on coverslips were fixed in methanol at
20 °C for 5 min,
washed with 1% Triton X-100/PBS, blocked in 5% goat serum/PBS, and
incubated with primary antibodies overnight at 4 °C. After washing,
cells were incubated with the secondary antibody conjugated to Alexa488
(Molecular Probes) for 1 h at room temperature. Coverslips were
washed as above and mounted on slides in ProLong Antifade (Molecular
Probes). In experiments utilizing immunostaining, pre-immune serum or
non-immune normal rabbit IgG was included, as appropriate, for negative controls.
- and acetylated
-tubulins and tektin filaments in our preparation validates its designation as axoneme-enriched (see Fig. 7). Pellets were extracted for 30 min at room temperature in the dilution buffer
supplemented with one of the following reagents: 0.5% SDS, 1 M NaCl, 6 M urea, 0.5% saponin, 10 mM Mg2+-ATP, or 2.5 mM AMP-PNP.
Samples were centrifuged again at 170,000 × g for 30 min, and the supernatants and pellets were analyzed by SDS-PAGE and immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of
rod cGMP phosphodiesterase (PDE
), which had previously been reported (13). The remaining clones contained overlapping cDNAs apparently derived from the same gene. To obtain the full-length coding sequence, we screened a second mouse retinal cDNA library constructed in a
phage vector using one of the original cDNA isolates as a probe. The longest among the second set of cDNA clones contained an
open reading frame of 1331 amino acid codons (Fig.
1). The conceptually translated
polypeptide has a predicted molecular weight of 152 kDa, is rich in
glutamic acid residues, resulting in a rather acidic isoelectric point
(pI = 4.8), and is highly hydrophilic with no predicted membrane
spanning regions. This protein is referred to as RPGRIP, for
RPGR-interacting protein, following the designation given in two recent
reports (6, 7). Search of the data base identified a BAC clone
(GenBankTM accession no. Al135744) that encodes the human
ortholog of RPGRIP. This places the human RPGRIP on chromosome 14q11.
The next closely related sequence to RPGRIP in the data base is a human
gene of unknown function (KIAA1005; GenBankTM accession no.
AB023222). A comparison of the overlapping cDNA clones from the
initial yeast two-hybrid screen localized the RPGR binding domain
within the C-terminal 300 residues. A 90 amino acid linker region
consisting mostly of glutamic acid residues separates the RPGR-binding
domain from the rest of the polypeptide. A stretch of 340 amino acid
residues near the N terminus (amino acid 210-550) is predicted to form
a coiled coil structure (14). This region also exhibits sequence
similarity to the coiled coil region of several motor proteins, a
microtubule-binding protein (CLIP-170/restin), and intermediate
filament binding proteins (desmoplakin I and II). Downstream from the
coiled coil region lies a C2-like domain, found in several protein
families with a role in phospholipid binding (15).
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Fig. 1.
Upper panel, conceptually translated
amino acid sequence of RPGRIP. Lower panel, structural
features of RPGRIP. The rectangle represents the full-length
coding sequence. The horizontal lines at either end indicate
untranslated regions. The positions of the coiled coil domain, the C2
domain, and the RPGR-binding domain are indicated. The
lines underneath the structure diagram denote the
regions of the probes used in the Northern blotting experiments shown
in Fig. 3.
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Fig. 2.
A, the 1331-residue open reading frame
transiently expressed in COS cells (lane 1) has the same
apparent molecular mass as the native RPGRIP from retina (lane
2). B, GST pull-down assay for RPGR and RPGRIP binding.
The GST-RPGRIP fusion protein was incubated with varying amounts of
mouse brain lysate (as a source for native RPGR) followed by pull-down
with glutathione-Sepharose. Bound proteins were analyzed by
immunoblotting. The first lane on the left
shows the total amount of RPGR present in 10 µl of brain lysate
(loaded on the gel directly). The last lane shows a negative
control in which a GST protein, instead of the GST-RPGRIP fusion
protein, was incubated with the brain lysate.
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Fig. 3.
Tissue-specific expression of RPGRIP.
A, Northern blot analysis demonstrates the RPGRIP transcript
is ~10 kb in size and is detected in photoreceptors only from among
the tissues examined. Probes used for the four blots are indicated
below each blot. The 4.4-kb band is derived from an
unrelated gene. The testis alone expresses a 3-kb variant. The 18 S
ribosomal RNA is shown as a control for loading. B, Multiple
tissue immunoblot illustrates photoreceptor-restricted expression of
the RPGRIP protein among the tissues examined. The 210-kDa band
corresponding to RPGRIP is present both in the wild type
(WT) and the RPGR knockout (KO) retina.
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Fig. 4.
Immunofluorescence microscopy.
A, retinal sections stained with antibodies for RPGRIP
(left) or RPGR (right). Genotypes of mice are
indicated. RPGRIP staining is similar in both genotypes, but RPGR
staining is abolished in the RPGR knockout mouse.
RPE, retinal pigment epithelium; OS, outer
segment; ONL, outer nuclear layer; INL, inner
nuclear layer; IPL, inner plexiform layer; GC,
ganglion cell layer; WT, wild type. B, staining
for RPGRIP in dissociated rod photoreceptors. DIC,
differential interference contrast optics. RPGRIP staining is largely
confined to the cilium (black arrowheads). -Tubulin
staining indicates the position of the basal bodies (white
arrowheads). C, schematic diagram of a rod
photoreceptor. D, staining for RPGRIP in the cone-dominant
ground squirrel retina indicates localization of RPGRIP in the
connecting cilia of cones as well. An arrowhead indicates
the position of CC.
-tubulin, a marker for the
basal body (10), on photoreceptors that had been mechanically
dissociated. These preparations contained mostly shaken-off rod outer
segments attached to the connecting cilia. Comparison of the
immunofluorescence and Nomarski images indicated that RPGRIP
localization is well defined; staining is limited to the connecting
cilium and ends abruptly at the junction where the cilium joins the
outer segment. Only trace amount of staining appear to be in the basal
bodies (Fig. 4B). A schematic diagram of a photoreceptor
cell is shown in Fig. 4C to help illustrate the subcellular
localization pattern. The staining pattern of RPGRIP strongly
resembles that of RPGR in dissociated photoreceptor cells (5).
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Fig. 5.
Ultrastructural localization of RPGRIP in
transverse sections. RPGRIP in the connecting cilium
(CC) is revealed by the 5-nm gold particles, which appear
external to the profiles of the nine microtubule doublets.
Left, two photoreceptor profiles are shown. The
upper one was sectioned at the plane of the connecting
cilium and the apical inner segment of the same cell. The
lower one was sectioned at the level of the basal body
(BB), as indicated by its enclosure within the inner
segment. Note lack of labeling in the basal body. Upper
right, a profile of connecting cilium at a higher magnification.
Lower right, a profile of connecting cilium with silver
enhancement following immunogold labeling. M, mitochondria;
IS, inner segment. Bar = 0.1 µm.
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Fig. 6.
Analyses of RPGRIP transiently expressed in
COS cells. A, immunofluorescence of COS cells
expressing an untagged RPGRIP. Note the punctate and filamentous
pattern of RPGRIP immunoreactivity. At the right is an
untransfected cell. B, chemical cross-linking of RPGRIP.
Cells were incubated in situ with the indicated amount of
DMP and analyzed by immunoblotting with the RPGRIP polyclonal antibody.
Putative dimers were generated with limited cross-linking, which
co-existed with monomers and higher molecular weight species
(arrowheads). C, homo-oligomer formation is also
demonstrated by reciprocal immunoprecipitation. Each tag-specific
antibody could precipitate both its cognate protein as well as the
other tagged protein from COS cells co-transfected with EGFP- and
Myc-tagged RPGRIP. The two differently tagged RPGRIPs could be
distinguished by the difference in their sizes on immunoblots probed
with the polyclonal RPGRIP antibody.
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Fig. 7.
Stable association of RPGRIP with the ciliary
axoneme. A, RPGRIP is present exclusively in the
cilium-enriched fraction. Lane 1, cytosolic fraction of
photoreceptor inner and outer segments; lane 2,
detergent-soluble fraction; lane 3, insoluble fraction.
Shown are immunoblots probed with antibodies against RPGRIP, RPGR, and
-tubulin, respectively, and the matching Coomassie-stained gel.
RPGRIP is present exclusively in the insoluble fraction, whereas RPGR
is present in both the cytosolic and the insoluble fractions.
Enrichment for
-tubulin shown in lane 3 confirms this
fraction is enriched for the ciliary axoneme. B, RPGRIP
complex exhibits greater stability than RPGR as well as other known
cytoskeletal components of the ciliary axoneme. P, pellet;
S, supernatant. C, chemical cross-linking of the
axonemal fraction (lane 3 in panel A) with the
indicated amount of DMP, analyzed by immunoblotting with the RPGRIP
polyclonal antibody. Putative dimers and higher oligomers were
generated with limited cross-linking (arrowheads). The major
band at ~210 kDa is the RPGRIP monomer.
-tubulin. Tektins are
present as a stable protofilament in the basal bodies as well as in
cilia and flagella and are thought to be integral to the outer axonemal
microtubule doublets (23-25). Using specific antibodies for these
proteins, to examine the different fractions from the same experiments,
we found that all were partially solubilized by high salt treatment.
Thus the RPGRIP complexes appear to be less soluble. The
axoneme-enriched fraction was also subjected to chemical cross-linking,
and the results are shown in Fig. 7C. Limited cross-linking
produced molecular species consistent with dimers and trimers of
RPGRIP. Immunoblotting with the acetylated
-tubulin antibody did not
reveal any cross-linking between RPGRIP and tubulin, indicating that
RPGRIP is not extensively in contact with or integral to microtubules
but might exist as large complexes composed of multiple subunits of
RPGRIP. This finding is consistent with results from COS cell
expression in which RPGRIP was found to exist as dimers or higher order
oligomers. These data in toto indicate that RPGRIP is a
structural component of the ciliary axoneme.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
---|
We thank Michael Scimeca for technical assistance, Dr. Changwan Lu for constructing the cDNA libraries, and Drs. Virgil Muresan and Dorothy Roof for discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY10309, the Foundation Fighting Blindness (Baltimore, MD), and the Chatlos Foundation (Longwood, FL).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.
Recipient of a career development award from Research to Prevent
Blindness. To whom correspondence should be addressed: Berman-Gund Lab., Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA
02114. Tel.: 617-573-3904; Fax: 617-573-3216; E-mail:
tli@meei.harvard.edu.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M009351200
2 Y. Nishimune, personal communication.
3 D. -H. Hong, G. Yue, M. Adamian, and T. Li, our unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
RP, retinitis
pigmentosa;
RPGR, retinitis pigmentosa GTPase regulator;
RPGRIP, RPGR-interacting protein;
RCC1, regulator of chromatin condensation;
RACE, rapid amplification of cDNA ends;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
DMP, dimethyl
pimelimidate;
AMP-PNP, adenosine 5'-(,
-imino)triphosphate;
kb, kilobase pair(s);
EGFP, enhanced green fluorescent protein.
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