(Received for publication, July 28, 1994; and in revised form, December 7, 1994)
From the
The interaction of the bovine opsin apoprotein with transducin
in rod outer segment membranes was investigated using a guanyl
nucleotide exchange assay. In exhaustive binding experiments, opsin
activates transducin, with half-maximal exchange activity occurring at
0.8 mol of opsin/mol of transducin. The opsin activity was
light-insensitive, hydroxylamine-resistant, unaffected by
stoichiometric concentrations of retinaloxime, and more heat-labile
than rhodopsin. The t of transducin activation
in the presence of excess opsin was 8.5 min, compared with 0.7 min for
metarhodopsin(II). The second-order rate constants were determined to
be 0.012 pmol of guanosine 5`-(
-thio)triphosphate (GTP
S)
bound per min/nM opsin and 0.35 pmol of GTP
S bound per
min/nM metarhodopsin(II). Opsin was able to activate more than
one transducin, although there appeared to be a turnover-dependent
inactivation of the apoprotein. Opsin showed a broad pH range
(5.8-7.4) for optimal activity, with no activity in buffers of pH
>9, whereas metarhodopsin(II) exhibited activity at pH >9.
Regulation of opsin activity by stoichiometric amounts of retinal was
observed, with inhibition by 11-cis-retinal and stimulation by
all-trans-retinal. A model for opsin activity is proposed.
Visual transduction in the rod cell is mediated by the
photoreceptor, rhodopsin (for a recent review, see Hargrave and
McDowell(1992)). The rhodopsin holoprotein consists of an
11-cis-retinylidene chromophore covalently attached to the
apoprotein, opsin, in a protonated Schiff base with the -amino
group of Lys
. Upon absorbing a photon, the chromophore
isomerizes from an 11-cis to an all-trans form, and
rhodopsin undergoes a series of conformational changes, characterized
by several photostationary intermediates, the most stable of which is
metarhodopsin(II) (
= 380 nm). Accompanying
the conformational changes are the deprotonation of the Schiff base,
followed by the hydrolysis and release of the chromophore, yielding the
apoprotein and all-trans-retinal (for a recent review, see
Birge(1990)). Under bright illumination of the retina, the apoprotein
is a major form of the photoreceptor. When the lighting conditions
change from light to dark, the holoprotein is regenerated from the
apoprotein by the binding of 11-cis-retinal (Dowling, 1960).
Thus, there is a continual cycling of rhodopsin among the holoprotein,
the metastable photointermediates, and the apoprotein.
Rhodopsin is a member of the family of G-protein-coupled transmembrane receptors and interacts with the retinal G-protein, transducin, in a light-dependent manner (reviewed in Hargrave and McDowell(1992)). Although there are a number of conformational states of light-activated rhodopsin, several lines of evidence suggest that metarhodopsin(II) is a key catalyst of transducin activation in the rod outer segment. First, the kinetics of formation of the metarhodopsin(II) coincide with the kinetics for interaction of transducin with metarhodopsin(II) containing membranes (Vuong et al., 1984). Second, the amount of metarhodopsin(II) relative to metarhodopsin(I), an earlier formed and less stable intermediate, is increased in the presence of transducin and decreased when transducin release is stimulated by addition of GTP (Hofmann et al., 1983). Third, conditions which stabilize or promote the formation of metarhodopsin(II) stimulate the light-dependent activation of transducin (Bennett et al., 1982; Emeis et al., 1982; Bornancin et al., 1989; Longstaff et al., 1986; Kibelbek et al., 1991). Thus, the main active conformation of rhodopsin, as defined by its ability to activate transducin and initiate the photoreceptor response to light, appears to be metarhodopsin(II).
It is not known whether other conformations of rhodopsin can activate transducin or regulate the cell's responsiveness. Physiological studies of bleaching adaptation have suggested that a non-metarhodopsin(II) conformation, possibly opsin, plays a role in controlling the sensitivity of the cell to light (Brin and Ripps, 1977; Pepperberg et al., 1978; Catt et al., 1982; Clack and Pepperberg, 1982; Jones et al., 1989; Corson et al., 1990; Cornwall et al., 1990; Jin et al., 1993). Biochemical studies of transducin activation by non-metarhodopsin(II) conformations have produced conflicting reports. Several groups have concluded that only metarhodopsin(II) is able to activate transducin (Hofmann et al., 1983; Longstaff et al., 1986; Kibelbek et al., 1991). These workers studied rhodopsin-transducin interactions after treatment of metarhodopsin(II) with hydroxylamine, a compound which causes the release of retinal from the apoprotein (Wald and Brown, 1953). Complete inhibition by hydroxylamine was found in experiments using either light-scattering, centrifugation, or transducin assays (Hofmann et al., 1983; Bornancin et al., 1989; Calhoon and Rando, 1985; Pepperberg et al., 1987; Kibelbek et al., 1991). On the other hand, Okada et al.(1989) have reported a hydroxylamine-resistant form, which they termed ``post-metarhodopsin(II),'' which stimulated GTP hydrolysis by transducin to levels similar to metarhodopsin(II). In more recent experiments, unregenerated opsin produced in transfected COS cells exhibited some ability to activate transducin at acidic pH (Cohen et al., 1993). Since different properties of the rhodopsin-transducin interaction are measured in the various assays, and the reaction conditions vary significantly, it is not apparent how to reconcile these findings.
In order to better define the conformations able to activate transducin, we have prepared the bovine opsin apoprotein in rod outer segment membranes and studied its interaction with transducin, using a guanyl nucleotide binding assay. We have found that the apoprotein is capable of stimulating the exchange of guanyl nucleotides by transducin in a light-independent manner. Furthermore, we have characterized and compared the opsin activity with the activity of metarhodopsin(II) and found significant differences in the rate, extent, sensitivity to hydroxylamine, and pH. We have also found that the opsin activity can be modulated by 11-cis- and all-trans-retinal and propose a model for the opsin structure to account for the observed activity.
Figure 1: UV-visible absorption spectra of opsin. Bovine opsin apoprotein containing membranes were prepared from bleached rod outer segments. Spectra of opsin (solid line) and unbleached rhodopsin (broken line) were determined after the membranes were solubilized with 1% dodecyl maltoside. Removal of retinal chromophore is indicated by the absence of absorbance in the visible wavelengths.
Pigments were regenerated by
incubating 5 µM opsin with 5-fold molar excess of either
11-cis-retinal, 11-cis-locked retinal (Bhattacharya et al., 1992), or all-trans-retinal, at 22-24
°C for 1 h in 10 mM sodium phosphate, pH 7.0. The
chromophores were added from ethanol stock solutions, and the final
ethanol concentration during regeneration was 2%. A UV-visible spectrum
was taken after solubilization in 1% dodecyl maltoside. In the case of
11-cis-retinal, dark-light difference spectra were obtained
after bleaching the sample for 1 min with projector light ( >
515 nm) to determine the extent of regeneration.
Transducin was
purified from frozen bovine retinae (Bubis and Khorana, 1991). The
purified transducin was dialyzed against 5 mM Tris-HCl, 100
mM NaCl, 5 mM MgCl, 50% glycerol (w/v),
10 mM 2-mercaptoethanol, and stored at -20 °C.
Protein concentrations were determined by a modified Lowry assay using
bovine serum albumin as standard (Peterson, 1977). The purified
transducin bound between 0.9-1.0 mol of
GTP
S(
)/mol of protein, as determined by exhaustive
reaction with [
S]GTP
S in the presence of
excess light-activated rhodopsin (see below).
Phospholipids were extracted from 50 bovine retinae according to the procedure of Weigand and Anderson(1982), except that retinal was first removed from the rod outer segment membranes by bleaching in the presence of 50 mM hydroxylamine followed by a washing step before extraction. Vesicles were made by solubilizing the phospholipids on ice in 2% CHAPS followed by overnight dialysis against 10 mM Tris-HCl, pH 7.0.
Figure 2:
Titration of transducin activation by
opsin. Opsin containing membranes, having either no detectable
chromophore ((A/A
= 25, closed triangles) or residual retinaloxime (A
/A
= 8, open
triangles), were incubated with 60 pmol of transducin and 500 pmol
of [
S]GTP
S (1100 cpm/pmol) in 250 µl
for 2 h. Also, rhodopsin was assayed both in the light (open
circles) and in the dark (closed circles). Transducin
activation was determined by measuring the bound
[
S]GTP
S using a nitrocellulose filtration
assay. The solid lines are sigmoidal fits to the data using
the Hill equation. Concentrations of opsin and metarhodopsin(II)
required to activate 50% of the transducin were 41 ± 7 and 6.6
± 0.7 pmol, respectively. The Hill coefficient was 1.9 for opsin
and 1.2 for light-activated rhodopsin. The background activity from
transducin alone, which was 6% of the maximal activity, was subtracted
from all data shown.
The pH profile for opsin was generated by fitting the data to the
equation below which represents the sum of two titratable groups with
pK values of k
and k
,
respectively,
where B is the maximum value of the
activity, and n
and n
are
parameters related to the steepness of the titration curve. The pH
profile for metarhodopsin(II) was fitted by adding a third ionizable
group to the above equation to yield the following
equation.
The initial rate data for both opsin and metarhodopsin(II) were
fitted to single parameter exponential decay equations. These equations
yielded values for the t for the reaction. The
second-order rate constant was determined from the slope of the linear
portion of the fit for the opsin or metarhodopsin(II) concentration in
the linear range for transducin activation.
Figure 3:
Kinetics of transducin activation by
opsin. Left panel, excess opsin (900 pmol, triangles)
or metarhodopsin(II) (150 pmol, open circles) was added to 150
pmol of transducin and 750 pmol of [S]GTP
S
in 750 µl. At the indicated times reactions were terminated by
dilution and aliquots were assayed for bound nucleotide. The inset shows early kinetic points. The time required to activate half the
transducin was 8.6 ± 0.1 min for opsin and 0.67 ± 0.1 min
for rhodopsin, as determined from a single parameter exponential fit (solid lines). Right panel, initial rate of
transducin activation by limiting concentrations of opsin (0.2
µM). The time required to activate half the transducin was
0.012 pmol of transducin/min/nM opsin, as determined from a
single parameter fit (solid line). The closed triangles represent opsin with A
/A
= 25, and open triangles represent opsin with A
/A
=
8.
Stimulation of
guanyl nucleotide exchange on purified transducin by opsin, measured by
the binding of [S]GTP
S to transducin, was
used to assay the interaction between opsin and transducin
(Wessling-Resnick and Johnson, 1987a). The reaction of opsin with
transducin was compared in two complementary sets of experiments. In
the first set, an exhaustive binding reaction was performed using a
fixed concentration of transducin, in order to measure the total number
of interactions that occur. The opsin apoprotein caused a
concentration-dependent stimulation of nucleotide exchange on
transducin (Fig. 2). The concentration dependence was sigmoidal,
with half-maximal activity occurring at 41 ± 7 pmol, which is
0.80 ± 0.1 mol of opsin/mol of transducin. Opsin preparations
with residual retinaloxime (A
/A
=8, Fig. 2, open triangles) gave results that were
identical to preparations having undetectable retinaloxime (closed
triangles). Metarhodopsin(II) also stimulated transducin (Fig. 2); however, the half-maximal activity occurred at 6.6
± 0.7 pmol which was 0.13 ± 0.01 mol of rhodopsin/mol of
transducin. This specific activity is similar to the activities found
earlier using different reaction conditions: limited bleaching of
rhodopsin in reconstituted phospholipid membranes (Fung et
al., 1981), limited GTP
S (Wessling-Resnick and Johnson,
1987a), and in detergent-lipid micelles (Knox and Khorana, 1991). At
the highest concentrations of opsin used, 84% of the transducin was
activated, whereas metarhodopsin(II) was able to activate all of the
transducin. The least active species tested was rhodopsin in the dark,
for which significant transducin activation was observed only at the
highest amounts (>200 pmol). The activity observed in the dark for
rhodopsin may reflect the intrinsic activity of dark rhodopsin or may
be due to artifacts arising from the presence of either unregenerated
opsin or a minor fraction of rhodopsin that was exposed to light during
isolation.
Even in reaction mixtures containing excess transducin, the opsin-induced nucleotide exchange was finished by 1 h at room temperature. The cessation of nucleotide exchange was not due to inactivation of transducin or depletion of nucleotide during the assay (data not shown). Unlike metarhodopsin(II), which decays at room temperature (Hofmann et al., 1983) and thus exhibits a decay in the activation of transducin, opsin did not lose the ability, when incubated at room temperature, to activate transducin (data not shown). Furthermore, preincubation of opsin at room temperature in the presence of transducin before addition of nucleotide, only caused a 10% decrease in subsequent nucleotide exchange assays (data not shown). Thus, the cessation of opsin-stimulated nucleotide exchange appears to require interaction and activation of transducin.
Figure 4:
Transducin activation by opsin is
insensitive to hydroxylamine. Opsin (50 pmol, closed
triangles) and rhodopsin (5 pmol, open circles), were
incubated for 1 min with hydroxylamine at the indicated concentrations
at pH 7.4 in the light ( > 515 nm). Transducin (85 pmol) and
[
S]GTP
S (500 pmol) were then added to a
final volume of 250 µl, and the reaction was carried out for 2 h.
Light-dependent transducin activation by rhodopsin is severely
inhibited by hydroxylamine, with 50% inhibition occurring at 0.4
mM, whereas opsin activity was inhibited less than 10% at 5
mM hydroxylamine. The line for the rhodopsin data is an
exponential fit, whereas the line for the opsin data is a linear
regression.
In order to determine whether the activity induced by the apoprotein depended on a specific three-dimensional conformation, the heat stability of opsin was investigated. Earlier studies had found that opsin and rhodopsin thermally denature at different temperatures, 56 and 72 °C, respectively (Miljanich et al., 1985; Khan et al., 1991). Therefore, opsin and rhodopsin were preincubated in the dark at 0, 70, or 100 °C and then used to activate transducin in an exhaustive binding assay at 25 °C (Table 2). No transducin activation was observed in heated opsin samples, whereas rhodopsin displayed 100% light-dependent activity when preincubated at 70 °C. Thus, the opsin conformation responsible for interaction with transducin is disrupted by heat. This experiment shows that residual rhodopsin does not contribute to the observed activity and also indicates that the opsin activity is not merely due to the activation of transducin by the cytoplasmic loop residues in a manner similar to that observed by the addition of synthetic peptides (Okamoto et al. 1991; Hamm et al., 1988). Rather, it suggests that a specific arrangement of the loops, necessary for interaction with transducin, occurs in opsin.
Figure 5:
Turnover of transducin by opsin. 50 pmol
of opsin (closed triangles) or metarhodopsin(II) (open
circles) and 1000 pmol of [S]GTP
S was
assayed with varying amounts of transducin for 2 h in 250 µl. At
the highest transducin concentrations tested, 2.4 mol of transducin
were activated per mol of opsin. Linear regression (solid
lines) gave slopes of 0.7 for opsin and 1.0 for metarhodopsin(II)
induced activity.
Figure 6:
pH profile of transducin activation by
opsin. Opsin (30 pmol, closed triangles) and metarhodopsin(II)
(3.8 pmol, open circles) were assayed in six different
buffers, pH 4-10, in 250 µl containing 500 pmol of
[S]GTP
S and 100 pmol of transducin. The pH
activity profiles of opsin shows a maximum around pH 6.5 with a steep
decline above pH 8.0. The activity of transducin alone, which has been
subtracted from the data, rises below pH 6.0, and at pH 5.0 about 20%
of the transducin has bound [
S]GTP
S in 2
h.
In contrast, the profile for metarhodopsin(II) was
complex, with activity observed at pH >8.5 (Fig. 6). The
reduced extent of nucleotide exchange at high pH was caused by a slower
rate of transducin activation at pH 9.7 compared with pH 6.5 (the ratio
of the rates was found to be 0.64 (data not shown)). The distinct drop
in activity around pH 8.0 followed by a recovery at higher pH was
observed in three buffers. The metarhodopsin(II) profile in Fig. 6was fitted assuming that three titratable groups underwent
changes in their ionization state. The data in the region below pH 8.0
were fitted by two groups, with pK values of 5.3
and 7.7, similar to the values found for opsin. For data in the region
of alkaline pH, the fit with a pK
of 8.2 was not
as good. In summary, pH profiles present another property in which the
activities of opsin and metarhodopsin(II) differ.
Figure 7:
Retinal
modulates the opsin activity. Opsin was incubated in the dark with
5-fold excess of 11-cis-retinal, 11-cis-locked
retinal or all-trans-retinal. A, 20 pmol of
regenerated pigment was assayed with 60 pmol of transducin and 500 pmol
of [S]GTP
S in 250 µl for 2 h in the
dark. Both 11-cis- and 11-cis-locked retinal inhibit
transducin activation by opsin in the dark. B, 4 pmol of
regenerated material or metarhodopsin(II) was assayed as in A.
All-trans-retinal caused a dramatic enhancement of the opsin
activity in the dark, to levels comparable with
metarhodopsin(II).
In contrast, regeneration of opsin with all-trans-retinal results in a dramatic stimulation of nucleotide exchange (Fig. 7B). The activity was comparable with metarhodopsin(II) and could be measured immediately after the addition of stoichiometric amounts of all-trans-retinal. Previously, stimulation of opsin activity by all-trans-retinal has been reported (Yoshizawa and Fukada, 1983; Cohen et al., 1992) although at significantly lower levels than found here. Thus, it appears that a metarhodopsin(II)-like conformation is formed upon the addition of all-trans-retinal to the apoprotein. In summary, 11-cis-retinal inhibits the activity of the apoprotein, whereas all-trans-retinal enhances it.
Using opsin in rod outer segment membranes and purified
transducin, we have found that the bovine opsin apoprotein activates
nucleotide exchange on transducin in a light- and
hydroxylamine-insensitive manner. From these experiments, as well as
the unique characteristics of the activity, we have shown that the
activity is not due to contaminating chromophore containing
photoproducts. It is possible to reconcile various other reports and
our main findings. First, Okada et al.(1989) have reported a
hydroxylamine-resistant stimulation of GTP hydrolysis on transducin by
a post-metarhodopsin(II) species. The rates of transducin activation
were comparable with that of metarhodopsin(II), in contrast to those
here, in which opsin had a 10-fold slower rate. The discrepancy may be
due to saturating concentrations of post-metarhodopsin(II) which were
used, their more indirect and slower assay of GTP hydrolysis (Vuong and
Chabre, 1990), or residual all-trans-retinal in the
preparations. Second, although opsin activates transducin, the binding
interaction of these two proteins appears substantially weaker than
that between transducin and metarhodopsin(II), since transducin remains
in the supernatant following centrifugation with opsin membranes (data
not shown; Pepperberg et al., 1987). This may also explain why
light-scattering experiments, which require tight association, failed
to detect apoprotein interaction with transducin (Hofmann et
al., 1983; Bruckert et al., 1988). Third, activation of
transducin by post-metarhodopsin(II) products was observed by Kibelbek et al.(1991) who attributed this activity to the reversibility
of the metarhodopsin(II)-metarhodopsin(III) transition. However in our
experiments, these photoproducts were not present, since we have
removed the chromophore. Furthermore, the activity is insensitive to
hydroxylamine. In addition, several reports contain data in which
apparent opsin activity was attributed to background nucleotide
exchange (Wessling-Resnick and Johnson, 1987b) or to the retinaloxime
in the preparations (Yoshizawa and Fukada, 1983; Fukada et
al., 1982). The experiments reported here clearly eliminate these
explanations. Finally, the lipid may affect the opsin activity, since
Rando and colleagues (Calhoon and Rando, 1985; Longstaff et
al., 1986) failed to observe apoprotein activity with opsin
reconstituted into egg phosphatidylcholine. The ability of
metarhodopsin(II) to activate transducin is very sensitive to the
hydrophobic environment ()(Knox and Khorana, 1991), and it
additionally appears that the apoprotein may exhibit similar
sensitivity. (
)Therefore the lipid environment may be
crucial to apoprotein structure and function. This may help to explain
why a loss in activity was observed when retinaloxime-containing
preparations were extracted with organic solvents, which would alter
the lipid composition of the membrane (Yoshizawa and Fukada, 1983;
Fukada et al., 1982). This point may have relevance to the
observations of Cohen et al. (1993) in which no apoprotein
activity was observed at pH 6.7, and some activity was observed at pH
6.1. In those experiments, COS cell membranes containing transfected
opsin were used for assay, which might have altered properties when
compared with opsin in rod outer segment membranes. Therefore, we
conclude that opsin is able to activate transducin under physiological
conditions, but with a significantly lower activity than
metarhodopsin(II).
One of the striking differences between
transducin activation by opsin and metarhodopsin(II) is the dependence
of transducin activation on pH. Opsin shows a profile with peak
activity occurring in the range 6.0-7.4 and a decline in activity
above this range. By pH 9, no activity can be detected. On the other
hand, metarhodopsin(II) exhibits a more complex behavior, having a
profile similar to opsin below pH 7.5, but retaining up to 70% of the
maximal activity at higher pH. Rhodopsin expressed in COS cell
membranes appears to be less active at high pH than rhodopsin in rod
outer segment membranes (Cohen et al., 1992; Fahmy and Sakmar,
1993). For both opsin and metarhodopsin(II), the effects of high pH are
fully reversible. Thus, the active conformation of both opsin and
metarhodopsin(II) may contain one or more titratable groups that
control the transition from active to less active. However, the
sensitivity to pH is dramatically different, and the nature of the
amino acid residue(s) that respond differently to pH changes in opsin versus metarhodopsin(II) are not known. The fact that opsin is
inactive at higher pH, and metarhodopsin(II) is not, appears
to rule out artifacts due to surface membrane effects. A number of
workers have pointed to the importance of charges located at or near
Lys, the site of retinal attachment, for transducin
activation (Longstaff et al., 1986; Arnis and Hofmann, 1993;
Cohen et al., 1993; Fahmy and Sakmar, 1993). Although the
major difference between opsin and metarhodopsin(II) involves the
removal of the chromophore, it is not clear whether any significant
changes in protonation state, accounting for the activity difference,
occur at Lys
as the pH is varied from pH 7 to 9. There
have been several reports concerning pH-sensitive conformations of
rhodopsin. Koutalos(1992) has shown that there is a spectral blue shift
in the dark in rhodopsin at high pH, leading to a proposal that
chromophore-protein interactions are sensitive to pH. Our experiments
suggest that there may be similar conformational changes associated
with high pH occurring in metarhodopsin(II). In fact, Arnis and
Hofmann(1993) have proposed two conformations of metarhodopsin(II) that
are related by the protonation of an unidentified residue and may have
different abilities to interact with transducin.
It appears that the binding of transducin to opsin is the rate-limiting step in activation, rather than other steps that occur after opsin and transducin interact. In control experiments, preincubation of opsin and transducin, in the absence of guanyl nucleotide, for 2 h at room temperature, had little effect on the subsequent activation of transducin (data not shown). Furthermore, we were unable to isolate an opsin-transducin complex by centrifugation (data not shown; Pepperberg et al., 1987).
The nature of the binding site for transducin on metarhodopsin(II)
has been studied by site-directed mutagenesis (Khorana, 1992) and by
peptide competition (Konig et al., 1989). From these studies,
a structural model has emerged in which the transducin binding site on
rhodopsin is created by three cytoplasmic loops. This interaction leads
to high affinity binding, and subsequent activation of transducin. In
order to explain the activation of transducin by opsin, we propose that one of the three loops involved in the interaction is in a
different relative position in opsin compared with metarhodopsin(II).
According to this hypothesis, there will be a lower interaction energy
of opsin with transducin and hence a lower apparent affinity leading to
fewer transducin molecules activated. This model can also explain the
regulation of opsin activity by retinal. The experiments presented here
show that when 11-cis-retinal binds to the apoprotein,
reforming rhodopsin, a less active conformation is formed. In the
model, the binding of 11-cis-retinal to opsin disturbs the
relative position of the two loops, contributing to the transducin
binding site, and further reduces the binding energy, effectively
preventing transducin activation in the dark. A different effect was
observed with all-trans-retinal, which enhanced opsin activity
to the level observed for metarhodopsin(II). Binding of
all-trans-retinal to opsin could move the loop to the position
occupied in metarhodopsin(II), reforming the high affinity binding
site. Although the model explains the basic observations, certain
properties of opsin regenerated with all-trans-retinal are
different from metarhodopsin(II) (Cohen et al., 1992; Hofmann et al. 1992), ()suggesting that the conformations
are distinct. Future work will be directed to determining the
cytoplasmic loops involved in the opsin activation of transducin.
Although the activation of transducin by opsin has structural implications, as discussed in the model, the physiological relevance of an active apoprotein remains uncertain. Numerous groups (Barlow, 1964; Brin and Ripps, 1977; Pepperberg et al., 1978; Catt et al., 1982; Clack and Pepperberg, 1982; Jones et al., 1989; Corson et al., 1990; Cornwall et al., 1990; Jin et al., 1993) have implicated the apoprotein in bleaching adaptation (Rushton, 1961). The results presented in our work provide several possible explanations for this process. In one scheme, opsin could desensitize the photoreceptor by triggering the same adaptation mechanisms as metarhodopsin(II), through transducin. This would explain both the excess adaptation beyond the loss of quantum catch and recovery of the photoreceptor when opsin recombines with retinal or its analogs. In another scheme, opsin could affect photoreceptor sensitivity by competing for the available transducin molecules, thus limiting metarhodopsin(II) interaction with transducin. In both schemes, the control of apoprotein activity by retinal would provide a means for cycling through the various sensitivities observed in bleaching adaptation.