(Received for publication, June 30, 1995)
From the
Visual arrestin (48 kDa) plays a role in the deactivation of rhodopsin by binding to the light-activated, phosphorylated form of the receptor. In bovine rod outer segments that were prepared in the presence of protease inhibitors, two faster migrating forms of arrestin, with apparent molecular masses of 46 and 44 kDa, were observed by Western blot analysis. The 46-kDa form was more evident in rod outer segments of eyes kept in the light than those placed in darkness and was found to be identical to that generated by in vitro proteolysis of arrestin by pure retinal calpain II. In vitro analysis showed that arrestin was proteolyzed only when bound to rhodopsin; soluble arrestin was not significantly cleaved by calpain. Proteolysis involves sequential cleavage at two, possibly three sites, resulting in the removal of 27 amino acids from the COOH terminus. The remaining 46-kDa protein was resistant to further proteolysis by calpain. Unlike intact arrestin, the 46-kDa truncated arrestin was not readily released from the receptor after the receptor had lost its chromophore, nor was it released upon the addition of 11-cis-retinal to regenerate the receptor. Truncated arrestin was found to inhibit receptor dephosphorylation to the same extent as intact arrestin. In conclusion, these results provide evidence that a 46-kDa form of arrestin in rod outer segments is a product of selective proteolysis by calpain. Furthermore, they suggest that this proteolysis may provide a mechanism for prolonging the phosphorylated state of the visual receptor.
The Ca-activated, neutral cysteine proteases,
known as calpains, appear to play roles in a large variety of cellular
processes (see Murachi(1989), Suzuki(1990), and Suzuki and Ohno (1990)
for reviews). Biochemically, two different classes of calpain have been
described, based on different concentrations of Ca
required for activation in vitro; calpain II requires
more Ca
than calpain I (Mellgren, 1980). Calpains are
abundant in neural tissues, where calpain II seems to be the
predominant isozyme (Murachi et al., 1981; Nixon et
al., 1986; Kawashima et al., 1988). Recently, we
demonstrated the presence of calpain II in rat and bovine rod
photoreceptor outer segments (Azarian et al., 1993).
The photoreceptor outer segment is an extremely specialized organelle, devoted to the absorption and transduction of light. Phototransduction begins with the absorption of a photon of light by rhodopsin. The chromophore of rhodopsin is isomerized from 11-cis-retinal to all-trans-retinal, inducing a conformational change in the receptor. The photoexcited rhodopsin activates the G protein, transducin, thus triggering an enzymatic cascade that results in the hydrolysis of cGMP and the closure of the cGMP-gated channels (see Hargrave and McDowell(1992) and Lagnado and Baylor(1992) for reviews). Rhodopsin is deactivated by phosphorylation of serine and threonine residues in its carboxyl tail and the subsequent binding of arrestin (also known as S-antigen) (Wilden et al., 1986; Wilden, 1995).
The binding of arrestin to rhodopsin inhibits dephosphorylation of the receptor and thus maintains rhodopsin in a deactivated state (Palczewski et al., 1989a). When all-trans-retinal is reduced by retinol dehydrogenase (Ishiguro et al., 1991) and removed from rhodopsin, arrestin is released (Hofmann et al., 1992). Following the release of arrestin, the phospho-opsin can be dephosphorylated by a phosphatase 2A (Palczewski et al., 1989b; Fowles et al., 1989). Regeneration with 11-cis-retinal then returns the dephosphorylated receptor to its dark state, in which it can be activated by the absorption of another photon of light. Any modification of arrestin that affects its ability to be released from rhodopsin should therefore influence how long rhodopsin remains deactivated.
Arrestin contains a PEST sequence (Mangini and Garner, 1991); i.e. a domain rich in proline, glutamate, serine, threonine, and aspartate residues, present in many substrates of calpain (Rogers et al., 1986; Wang et al., 1989). Although there is some question about whether such a sequence actually affects substrate susceptibility (Molinari et al., 1995), its presence in arrestin makes this protein a possible candidate for regulation by calpain. In the present study, we noted the presence of two additional, more mobile forms of arrestin in bovine rod outer segment preparations. We found that in vitro proteolysis of arrestin by retinal calpain II generates a product that is identical to one of the additional forms of arrestin detected in rod outer segment preparations. This result suggests that this in vitro event mimics a physiological one. Additional in vitro experiments were performed to characterize calpain proteolysis of arrestin and gain some insight into its potential function.
Preparation of Rod
Outer Segments-Rod outer segments (ROSs) were purified from
fresh bovine retinas on continuous sucrose gradients as described
(Azarian et al., 1993), with modifications. Unless otherwise
stated the eyes, which came from light-adapted cattle, were placed in
light-sealed plastic containers (PGC Scientific) on ice, immediately
after their removal from the animal, and were thus transported to the
laboratory (2 h). Subsequent preparation of the ROSs was performed
under dim red light. Retinas were removed and vortexed for 30 s with 10
ml/10 retinas of 20% sucrose in buffer A containing 20 µM leupeptin and 0.2 mM PMSF. The homogenate was filtered
through a nylon mesh (Sargent-Welsh, number 100) and distributed among
six 23-ml sucrose gradients (25-55% sucrose in buffer A). The
gradients were centrifuged for 90 min at 100,000 g (Beckman SW28), and the ROS band was carefully siphoned off with a
Pasteur pipette. The pooled ROSs were diluted with 1 volume of 100
mM KCl in buffer A and centrifuged at 10,000
g for 5 min (Sorvall SS-34). ROSs were resuspended in buffer B.
To purify ROSs from frozen bovine retinas (J. and A. Lawson,
Lincoln, NE) for some of the urea-stripped ROS membrane preparations,
the procedure of Wilden and Kühn(1982) was used.
With both procedures, purified ROSs contained 0.4-0.5 mg/retina
of rhodopsin and had an A/A
ratio of 2.2-2.4 in 3% lauryldimethylamine N-oxide
(Calbiochem). Aliquots of purified ROSs were flushed with argon,
covered with aluminum foil, and snap-frozen in liquid nitrogen for
storage at -80 °C.
To test for the presence of p46 in fresher ROSs, a retina was removed quickly from an eye less than 5 min post-mortem and placed in ice-cold buffer A, containing 130 mM NaCl, 0.2 mM PMSF, 0.1 mM calpeptin, and 40 µM leupeptin (this procedure was carried out at a small local slaughterhouse). A crude but rapid preparation of ROSs was obtained by vortexing the retina for 20 s and then after 1 min removing the suspended crude ROSs by pipette and placing them directly in sample buffer for SDS-PAGE and Western blotting.
Bovine retinal arrestin was purified as described (Buczylko
and Palczewski, 1993), with modifications. Briefly, 40-60
dark-adapted bovine retinas were homogenized with 2 volumes of buffer C
containing 2 mM PMSF. The homogenate was centrifuged for 20
min at 10,000 g (Sorvall SS-34). The supernatant was
loaded on a 2.5
5-cm DEAE-cellulose column equilibrated in
buffer C. The column was washed with buffer C and eluted with 0.1 M NaCl in buffer C. The eluate was loaded on a 2.5
5-cm
heparin-Sepharose column equilibrated with 0.1 M NaCl in
buffer C. The column was washed with 0.15 M NaCl and 20
µM phytic acid (Sigma) in buffer C before elution with 1
mM phytic acid and 0.15 M NaCl in buffer C. The
eluate was adjusted to 0.5 M NaCl and passed through a 2.5
5-cm phenyl-Sepharose column (Kasp et al., 1987). The
effluent (containing arrestin) was diluted to 0.1 M NaCl in
buffer C and loaded on a second heparin-Sepharose column. The column
was washed with 0.2 M NaCl in buffer C and eluted with 0.3 M NaCl in buffer C. The latter eluate was concentrated on
Centricon-30 cartridges (Amicon) and stored frozen at -80 °C
or in 50% glycerol at -20 °C. This preparation of arrestin
was homogeneously pure as judged by a Coomassie-stained SDS-PAGE and by
arrestin binding assays (cf. Fig. 1A, lane 1).
Figure 1: Different forms of arrestin in ROS extracts. Bovine ROSs (200 mg of protein), obtained from dark-adapted retinas, were lysed and extracted with hypotonic buffer and then washed several times with buffer containing 0.5 M KCl, as described under ``Experimental Procedures.'' One percent of each extract was electrophoresed in a 10% SDS-polyacrylamide gel, transblotted, and immunolabeled with an arrestin antibody (C10C10). Lane 1, hypotonic extract; lanes 2-4, sequential washes with 0.5 M KCl. Molecular mass standards are indicated on the left (in kDa), and the apparent molecular masses for arrestins on the right.
The catalytic subunit of phosphatase 2A was partially purified from
bovine ROSs, without contamination by arrestin. The soluble fraction of
dark-adapted ROSs (prepared as in previous section) was dialyzed
overnight against buffer C and filtered to remove particulate matter.
The filtrate was loaded on a heparin-Sepharose column (2.5 5
cm) equilibrated in buffer C, and phosphatase 2A activity was eluted
with 0.1 M NaCl in buffer C (Erdõdi et al., 1992). Arrestin (Buczylko and Palczewski, 1993) and
phosphatase type 1 (Erdõdi et al., 1992)
remain bound to heparin in the presence of 0.1 M NaCl. The
phosphatase 2A activity was concentrated in Centricon-30 cartridges
(Amicon) and precipitated in 80% EtOH at room temperature (Brandt et al., 1974). The pellet was extracted twice with buffer
C,and the insoluble material was collected from the pooled extracts by
centrifugation. The final supernatant was stored on ice and was used as
ROS phosphatase.
To isolate p46 generated by in vitro calpain proteolysis of arrestin, ROS membranes, containing arrestin-phosphorhodopsin complexes, were incubated with calpain and then washed with high salt buffer (as above). The supernatant, which contained primarily 46-kDa arrestin, was Western blotted, and the 46-kDa arrestin was excised, destained, and washed as above.
Some of each of the samples, along with identically
prepared 48-kDa arrestin, was digested by trypsin and the resulting
peptides were separated by C reversed-phase HPLC. The
peptide corresponding to the COOH-terminal of each p46 species was
identified as the peak present in a p46 sample, but not in a 48-kDa
arrestin sample. Sequencing and laser desorption mass spectrometry of
this peptide was carried out by the Harvard Microchemistry Facility.
The remainder of each of the samples was used by this facility in an
attempt to obtain NH
-terminal sequence.
Figure 2: Conditions for proteolysis of arrestin by calpain. Purified arrestin (20 pmol, as determined by Bradford assay) was incubated without (lanes 1 and 2) or with (lanes 3-14) different forms of rhodopsin or opsin (200 pmol) in stripped ROS membranes for 5 min in the dark at 30 °C, then 5 min in the light (white boxes) or dark (black boxes). Samples were incubated for an additional 40 min without (A) or with (B) 2 units of purified retinal calpain II (final volume, 20 µl) in the dark, then centrifuged through a sucrose cushion. Supernatants (S) and pellets (P) were electrophoresed in a 10% SDS-polyacrylamide gel and visualized with Coomassie Blue. Preparation of the different forms of rhodopsin is described under ``Experimental Procedures.'' The presence of arrestin in the pellet indicates its binding to rhodopsin. Proteolysis of arrestin is manifest by the presence of a 46-kDa form, which is evident only in the pellet. On the right, the apparent molecular masses are indicated in kDa; the position of the receptor is indicated by r. Rh, unbleached rhodopsin; Rh-P, phosphorylated Rh; Op, opsin; Op-P, phosphorylated Op.
Time Course of Arrestin Proteolysis by Calpain in Vitro- Fig. 3shows the proteolysis of purified arrestin incubated with phosphorylated, photoactivated rhodopsin in urea-stripped ROS membranes. SDS-PAGE analysis of rhodopsin-bound arrestin after incubation with calpain for different lengths of time showed that arrestin is first truncated to yield an intermediate form with an apparent molecular mass of 46.5 kDa (Fig. 3, lanes 2-5). At times less than 2 min, the 46.5-kDa form could be detected as the only truncated form (not shown). This product is replaced by the 46-kDa form, which appears to be relatively stable; even addition of fresh calpain after 60 min did not result in any further proteolysis in the ensuing 20 min (Fig. 3, lane 8). With light-adapted ROS membranes that had been washed once with hypotonic buffer (and thus contained endogenous bound arrestin) and then incubated with calpain, the final product of arrestin was also found to be the 46-kDa form.
Figure 3: Time course of proteolysis of arrestin by calpain. Arrestin (200 pmol) was incubated with phosphorylated rhodopsin (2 nmol) in stripped ROS membranes for 5 min in the dark at 30 °C. The mixture was illuminated for 5 min and an aliquot (200 pmol of rhodopsin) was transferred to Laemmli sample buffer at t = 0 min (lane 1). Retinal calpain II (18 units, final volume of 180 µl) was then added, and aliquots (200 pmol of rhodopsin) were quenched in sample buffer at the indicated intervals. Protein was separated in a 10% SDS-polyacrylamide gel and visualized with Coomassie Blue. The initial product of proteolysis (46.5 kDa), which is first evident after 2.5 min (lane 2), is slightly less mobile than the final product (46 kDa), which is most evident after 80 min (lane 7). Addition of fresh calpain after 60 min and incubation for 20 min did not result in any further proteolysis (lane 8). The apparent molecular masses (kDa) are indicated on the left.
Figure 4: Immunological analysis of truncated arrestins. Western blots of arrestin and truncated arrestins labeled with different antibodies: polyclonal antibody (pAb) and monoclonal antibodies that recognize epitopes of residues 42-48 (a, 5C6.47), 288-296 (b, C10C10), 375-380 (c, S2.4.C5), and 375-386 (d, A9C6). The apparent molecular masses (kDa) of arrestin and its truncated products are indicated on the left. A, arrestin was partially proteolyzed (2.5 min) with calpain II in vitro (as in Fig. 3, lane 2). mAb A9C6 (d) recognized only nonproteolyzed arrestin (lane 4). The polyclonal antibody (pAb) and mAbs 5C6.47 (a) and S2.4.C5 (c) recognized arrestin and both of its truncated products (46.5 and 46 kDa) (lanes 1-3). B, high salt extracts of ROS membranes (as in Fig. 1, lane 2). mAb C10C10 (b) recognized 48-kDa arrestin and both p46 and p44 (lane 1). mAb S2.4.C5 (c) recognized only 48-kDa arrestin and p46 (lane 2). mAb A9C6 (d) recognized only 48-kDa arrestin (lane 3). C, high salt extract of ROS membranes (lane 1), 46-kDa in vitro proteolytic product (lane 2), and half each of lanes 1 and 2 added together (lane 3), labeled with mAb C10C10 (b). D, diagram of arrestin, indicating its PEST sequence (PEST) and the epitopes recognized by the mAbs used in A-C (from Donoso et al.(1990) and Dua and Donoso(1993)). Using the PEST-FIND program (Rogers et al., 1986), the PEST score for the sequence shown was determined to be +13. PEST scores may range from -45 to +50; scores of +5 or greater indicate potential PEST sequences (Rogers et al., 1986). Bold italic letters in the PEST sequence represent the PEST amino acids. Numbers indicate the residues of each site. Domains I and II refer to the calpain generated fragments isolated by HPLC and sequenced in Fig. 5.
Figure 5:
Sequence analysis of calpain-proteolyzed
arrestin. A, reversed-phase HPLC profiles of trypsin-digests
of arrestin and p46-arrestin. Traces represent an acetonitrile gradient
of 0-40% (v/v) in 90 min. Peptide elution was monitored at 210
nm. The asterisk indicates a peak present in the p46 sample,
but not the intact arrestin sample. The peptide corresponding to this
peak in different p46 samples was sequenced and its mass was determined
by laser desorption mass spectrometry. B, reversed-phase HPLC
profile of products released by calpain cleavage of 60 µg of
arrestin. Peptide elution was monitored at 214 nm. Arrestin peptide peaks I (12% CHCN) and II (19%
CH
CN) are indicated as well as two other major peaks (asterisk), which were found to contain no determinable
protein sequences. The observed and calculated masses of peptides I and
II, as determined by mass spectrometry, are indicated. C,
amino acid sequences of the two termini of intact bovine arrestin (from
Shinohara et al.(1987)) and 46-kDa arrestin. The sequences of
the peptides released by in vitro proteolysis of arrestin by
calpain and isolated in peaks I and II (see B) are also shown
and aligned with the sequence of arrestin.
The same antibodies were used to label Western blots of high salt extracts of bovine ROS membranes that had been prepared in the presence of protease inhibitors and washed with hypotonic buffer. Fig. 4B shows that mAb C10C10 recognized 48-kDa arrestin, plus the two faster migrating forms, p46 and p44 (lane 1). mAb S2.4.C5 recognized p46, but not p44 (lane 2). mAb A9C6 did not recognize either p46 or p44 (lane 3). It is likely that p44 is the variant described in two recent reports (Palczewski et al., 1994; Smith et al., 1994). This variant is formed by alternative mRNA splicing and is identical with 48-kDa arrestin except that the COOH-terminal 35 residues(370-404) are replaced by a single alanine (Palczewski et al., 1994; Smith et al., 1994). On the other hand, p46 possesses the same immunological properties as that generated in vitro by calpain proteolysis, both are recognized by mAb S2.4.C5, but not by mAb A9C6.
The mobilities of p46 and the 46-kDa product of arrestin cleaved by calpain in vitro were compared on Western blots, following SDS-PAGE. Fig. 4C shows a Western blot labeled with mAb C10C10. It contains hypotonically washed ROS membranes (lane 1; as in Fig. 4B, lane 1), 46-kDa truncated arrestin generated from in vitro calpain proteolysis (lane 2; as in Fig. 3, lane 5), and half each of lanes 1 and 2 added together (lane 3). This analysis showed that p46 and the product of calpain-cleaved arrestin in vitro have the same mobility in SDS-PAGE.
Different samples of p46 were
subjected to NH-terminal sequencing. In three separate
attempts, no sequence data could be obtained from either the in
vitro or in vivo p46, or from intact arrestin, which was
prepared in parallel with the p46 samples. It appears that the NH
terminus of p46 (from both sources) remains intact and blocked,
like that of intact arrestin which is acetylated (Shinohara et
al., 1987).
For COOH-terminal analysis, the samples of p46 and
samples of intact arrestin were first completely digested with trypsin
and the products were separated by HPLC. In comparing the resulting
HPLC profiles, there was one peak that was repeatedly present in the
p46 samples, but not in the intact arrestin samples, and would
immunoreact with mAb S2.4.C5, but not mAb A9C6 (asterisk in Fig. 5A). The sequence and the mass of the peptide from
this peak was the same from both sources of p46. The sequence was
ESFQDENFVF, which correspond to residues 368-377 of arrestin (cf. Shinohara et al.(1987)). The masses obtained
from laser desorption mass spectrometry of the peptide also indicated
truncation at Phe. They were consistently
25 Da
larger than the calculated mass of this peptide (1261 Da), which is
probably due to a sodium adduct (Roepstorff, 1994). Truncation after
the next residue, which is a glutamic acid in bovine arrestin
(Shinohara et al., 1987), would give an extra mass of 129 Da
(over the 1261 Da). Termination with the residue, Phe, indicates that
the carboxyl terminus of the peptide could not have been generated by
trypsinolysis.
Together with the immunological results above, these data indicate that the p46 obtained from ROS preparations and the p46 generated by in vitro calpain proteolysis of arrestin are both formed by removal of the last 27 amino acids from arrestin. That both forms of p46 are identical indicates that p46 is generated in situ by calpain (which is present in photoreceptors) and that in vitro analysis of the proteolysis of arrestin by pure calpain is physiologically relevant.
Peptides released by calpain from stripped ROS
membranes, containing
arrestin-[P]phosphorhodopsin complexes, were
isolated by HPLC for sequencing and mass spectrometry. After incubation
with calpain for 40 min (as above), samples were centrifuged and the
supernatants removed. The amount of
P detected in the
supernatant indicated that only 1.3% (±0.3% S.E.; n = 8) of phosphorylated residues were released into the
supernatant. Therefore, although the COOH terminus of rhodopsin is very
sensitive to proteolysis by a variety of proteases
(Kühn et al., 1982), calpain cleavage of
the phosphorylated COOH terminus of arrestin-bound rhodopsin appears to
be negligible. Amino acid sequences could be obtained from two of the
four major peaks isolated by HPLC (Fig. 5B). Both of
these sequences corresponded to partial internal sequences of arrestin.
One (from peak I; elution at 12% CH
CN) corresponded to
residues 381-385; the other (from peak II; elution at 19%
CH
CN) corresponded to residues 386-404 (Fig. 5C). Calculated and observed masses for the peak
I peptide were 600 and 601.9 ± 0.6, respectively, for the peak
II peptide, 2185 and 2186.9 ± 2.2, respectively. Given that a
peak containing peptides I and II conjugated together was not obtained,
these data suggest that calpain cleaves the COOH terminus of arrestin
first between residues 385 (Leu) and 386 (Lys). This is consistent with
the immunological results presented in Fig. 4. It appears that
there are two more cleavage sites, between residues 380 (Phe) and 381
(Ala) and then between residues 377 (Phe) and 378 (Glu), to generate
p46. However, it is possible that only the latter occurs, with residues
378-380 subsequently being lost from the released peptide. We
know from the sequence of the COOH terminus of p46 that residues
378-380 are indeed removed, although we did not isolate and
obtain the sequence of a released peptide containing these residues.
The two peaks from which we were unable to obtain amino acid sequence
data might have contained blocked NH
-terminal fragments
from other proteins in the ROS membranes or from calpain itself; the
NH
termini of both the large and small subunits of calpain
are cleaved by autolysis (Suzuki et al., 1981; Mellgren et
al., 1982; Hathaway et al., 1982).
Figure 6:
Light dependence of 46-kDa arrestin in situ. Western blot of crude ROSs labeled with mAb S2.4.C5,
which labels intact arrestin (48 kDa) and 46-kDa arrestin (46 kDa). Lane 1, crude ROSs (50 µg of protein) from eye placed
in darkness for 30 min after enucleation. Lane 2, crude ROSs
from the other eye of the same animal, kept in light for 30 min after
enucleation (half as much protein as in lane 1). Lane
3, same as lane 1, except that half as much protein was
loaded. Lane 3 contains less intact arrestin than lane
2, because there is less arrestin in ROSs in the dark than in the
light (even in crude ROSs, which contain more inner segment proteins).
However, even when the total ROS protein from the retina kept in the
dark is doubled (lane 1), and the amount of intact arrestin is
similar to that in lane 2, no 46-kDa arrestin is evident.
Apparent molecular masses are indicated in kDa on the left.
Figure 7:
Release of arrestin from rhodopsin.
Arrestin (20 pmol, as determined by absorbance at 280 nm) was incubated
with phosphorylated rhodopsin (200 pmol) in stripped ROS membranes for
5 min in the dark and then 5 min in the light at 30 °C. Samples
were incubated for an additional 10 min with or without 2 units of
retinal calpain II (total volume, 20 µl) for partial proteolysis
and quenched with 100 µM leupeptin. After a 10-min
incubation with or without 2 mM NHOH (final
volume, 22 µl), the mixtures were centrifuged through a sucrose
cushion. Supernatants (S) and pellets (P) were
resolved in a 10% SDS-polyacrylamide gel and stained with Coomassie
Blue. Most of the intact arrestin (lane 3) and most of the
46.5-kDa truncated arrestin (lane 7) were released into the
supernatant by treatment with NH
OH. However, most of the
46-kDa truncated arrestin remained in the pellet, even after
NH
OH treatment (lane 8). The numbers on
the right indicate apparent molecular masses
(kDa).
To test if the p46 would be released from opsin upon its regeneration, we added excess 11-cis-retinal to previously stripped ROS membranes, containing phospho-opsin bound to arrestin that had been proteolyzed by calpain in vitro. Aliquots were removed after various intervals, centrifuged, and the supernatants and pellets were analyzed by SDS-PAGE. The arrestin was not detectable in the supernatant, even after 4 h of incubation. In another experiment, hypotonically washed ROS membranes, containing p44 and p46, were incubated with excess 11-cis-retinal. In this case, neither truncated arrestin was detected in the supernatant.
Figure 8:
Inhibition by bound arrestin of
dephosphorylation of rhodopsin. 200 pmol of P-labeled Rh-P
in stripped ROS membranes was incubated without (control; sample 1) or
with (samples 2, 3, and 4) 200 pmol of arrestin for 5 min in the dark
and then 5 min in the light at 30 °C. Forty units of retinal
calpain II (sample 4) or buffer (samples 1, 2, and 3) were added, and
samples were incubated for 20 min (final volume, 200 µl). Samples
were incubated for an additional 10 min with 2 mM NH
OH and 3 mM EDTA (samples 1, 2, and 4) or 3
mM EDTA (sample 3). ROS phosphatase, obtained from eight
bovine retinas, was then added to each sample. Aliquots (20 pmol of
Rh-P) were taken at indicated times and the acid-soluble
P
label was counted and subtracted from the background. Thus the extent
of rhodopsin dephosphorylation as a function of time is illustrated. In
sample 4 of the experiment shown in this figure, no more than 1% of the
phosphorylated COOH termini of rhodopsin molecules was proteolyzed by
calpain. Inset, an aliquot of the samples at the end of the
time course was centrifuged through a sucrose cushion and the entire
supernatant and pellet of each was visualized in a Coomassie
Blue-stained SDS-polyacrylamide gel (only pellets of samples 3 and 4
are shown). No intact arrestin was evident in the pellet containing
truncated arrestin (sample 4). Most of the arrestin was soluble in the
sample of intact arrestin that had been treated with NH
OH
(sample 2); compare supernatant (S) with pellet (P).
Apparent molecular masses (kDa) are indicated on the right.
In this experiment, sample 1 contained
phosphorhodopsin, and samples 2, 3, and 4 contained phosphorhodopsin
with arrestin bound. Sample 4 was then treated with calpain to cleave
the arrestin, and samples 1, 2, and 4 were incubated with 2 mM NHOH for 10 min. The samples were then incubated with
ROS phosphatase. During this incubation, as shown in the inset of Fig. 8, which is an SDS-PAGE analysis of the arrestin
after centrifugation: sample 1 contained phospho-opsin only; sample 2
contained phospho-opsin with intact arrestin, most of which was
soluble; sample 3 contained phospho-opsin with intact arrestin bound;
and sample 4 contained phospho-opsin with p46 arrestin bound (no intact
arrestin was detectable).
Sample 1 was used as a control standard;
addition of the NHOH to phosphorhodopsin in this sample had
no effect on receptor dephosphorylation. Samples 3 and 4, containing
bound intact and truncated arrestin, respectively, inhibited
dephosphorylation of the receptor to the same extent (60-70%
inhibition, compared with the control), when dephosphorylation was
linear with time (R
0.95 for all curves) (Fig. 8).
This level of inhibition of dephosphorylation is comparable with that
described in a previous report (Palczewski et al., 1989a). In
sample 2, release of most of the intact arrestin into the supernatant
by NH
OH permitted dephosphorylation that was only
20-30% (range) less than that of the control (Fig. 8).
We have shown that purified bovine ROSs contain two truncated arrestins, p46 and p44, in addition to 48-kDa arrestin. Both forms were found to be bound tightly to ROS membranes (requiring high salt to be eluted), making it unlikely that they represent soluble contaminants released from other cells when the retina was disrupted to release the ROSs. During the course of the present study, p44 has been shown to be an alternatively spliced variant of arrestin (Palczewski et al., 1994; Smith et al., 1994), and the presence of p46 has been reported in human and rabbit retinas, as well as bovine retinas (Smith, 1995). Our studies indicate that p46 is the same as that generated by calpain proteolysis of arrestin in vitro, providing strong evidence that p46 is formed by calpain proteolysis of arrestin and that in vitro analysis of the proteolysis ispertinent.
In vitro analysis showed that arrestin is selectively proteolyzed by retinal calpain II when arrestin is bound to rhodopsin. Proteolysis of arrestin occurs at the COOH terminus. First, a 19-amino acid peptide is removed to give rise to a 46.5-kDa intermediate. The 46-kDa product is then generated by the removal of an additional 8 residues in a one- or possibly two-step process. This final product is resistant to further proteolysis by calpain. The consequence of arrestin proteolysis is that the remaining product maintains a high binding affinity for the receptor after the removal of its chromophore; in contrast to intact arrestin, which is released from the receptor after the removal of all-trans-retinal (Hofmann et al., 1992). The presence of truncated arrestin on opsin inhibits its dephosphorylation. It is unclear how p46 arrestin might be released from phospho-opsin; incubation of the complex with 11-cis-retinal did not elicit release.
An
alternative possibility to explain the selective proteolysis of
arrestin could be that calpain might be only active at the ROS disk
membrane. Consistent with this suggestion, the active form of calpain I
has been localized at the membrane in situ (Saido et
al., 1993), and certain phospholipids have been shown to lower its
Ca requirement for activity (Saido et al.,
1992). However, the conditions of the in vitro assays used in
the present study included sufficient Ca
for maximal
proteolysis of a soluble substrate, casein (cf. Azarian et
al.(1993)). Indeed, casein was proteolyzed by calpain in the
presence of soluble arrestin, which resisted proteolysis.
The binding of arrestin to rhodopsin has two functions. First, it enhances the decoupling of phosphorylated rhodopsin from transducin (Wilden et al., 1986; Bennett and Sitaramayya, 1988). Second, it inhibits the dephosphorylation of rhodopsin by a phosphatase 2A (Palczewski et al., 1989a). We are a long way from understanding the physiological function of arrestin proteolysis; however, our in vitro results provide some insight. We have shown that the binding of 46-kDa truncated arrestin to rhodopsin inhibits receptor dephosphorylation to the same extent as intact arrestin. Because this truncated product is not released from the receptor upon loss of the chromophore, the dephosphorylation of phospho-opsin, as well as phosphorhodopsin, is inhibited. Therefore, by retarding the release of arrestin from the receptor, the effect of calpain proteolysis of arrestin would be to prolong the phosphorylated state of the receptor, thus inhibiting the return of the receptor to the state in which it can be activated again by the absorption of another photon of light.
It is not clear what lighting conditions might be optimal for promoting arrestin cleavage. Investigation of this question will require experiments with retinas that are better suited than bovine retinas for physiological studies. It is noteworthy that when frog ROSs are exposed to dim conditioning illumination prior to a bright flash of light, the subsequent receptor dephosphorylation is inhibited (Biernbaum et al., 1991); inhibition of receptor dephosphorylation is the predicted consequence of arrestin cleavage.
An apparent inconsistency with calpain proteolysis of arrestin
occurring in the light is that cytosolic Ca levels of
ROSs have been reported to decrease upon illumination (Ratto et
al., 1988; Gray-Keller and Detwiler, 1994). However, as noted
above, arrestin cannot be proteolyzed until it binds rhodopsin, so that
the light dependence of its proteolysis is determined by its own
conformation. Moreover, especially in subsaturating light, the ROS
Ca
concentration in the light is reported to be still
rather high (no less than 325 nM in light sufficient to induce
a photoresponse that is 70% of maximal; Gray-Keller and Detwiler, 1994)
in comparison with that in other cell types. Nevertheless, how does ROS
calpain II function in a submicromolar concentration of
Ca
, when, like other calpain IIs, it requires
hundreds of µM Ca
for half-maximal
caseinolytic activity in vitro (Azarian et al.,
1993)? (
)This question, as it applies to calpain II
generally, has been discussed extensively elsewhere (e.g. Croall and DeMartino(1991)). The most important consideration
stems from studies showing that some protein activators (Pontremoli et al., 1988, 1990) and lipids (Coolican and Hathaway, 1984;
Saido et al., 1992) lower the Ca
requirement
for in vitro calpain activity. In the cell, the concentration
of lipid and protein activators are orders of magnitude higher than
they are in in vitro studies. Because such activators increase
the affinity of calpain for Ca
, in vitro Ca
requirements do not reflect Ca
requirements in the cell. For example, another
Ca
-regulated enzyme, protein kinase C, is not
dependent on any Ca
for activity in the presence of
sufficiently high concentrations of lipid (Mosior and Epand, 1994).
Of
the many proteins considered as putative calpain substrates, most are
cytoskeletal proteins, enzymes, or membrane proteins (Takahashi, 1990;
Saido et al., 1994). Interestingly, arrestin is none of these,
suggesting that the role of calpain might be broader than previously
anticipated. Given the similarity in structure and function among the
arrestin family (Lohse et al., 1992), it is plausible that
non-visual arrestins might be also substrates of calpain. Consistent
with this notion, we have found, using the PEST-FIND program of Rogers et al.,(1986), that most arrestins have PEST sequences.
Indeed, some forms of -arrestin have particularly strong PEST
sequences. Human
-arrestin-1 (Parruti et al., 1993),
bovine
-arrestin (Lohse et al., 1990), and rat
-arrestin-1 (Attramadal et al., 1992) have PEST scores of
+21 for sequences near their carboxyl tail; the score for the
bovine visual arrestin PEST sequence is +13.