The effects of amino acid replacements of glycine 121 on transmembrane helix 3 of rhodopsin.

Rhodopsin is a member of a family of G protein-coupled receptors with seven transmembrane (TM) helices. In rhodopsin, Gly121 is a highly conserved amino acid residue near the middle of TM helix 3. TM helix 3 is known to be involved in chromophore-protein interactions and contains the chromophore Schiff base counterion at position 113. We prepared a set of seven single amino acid replacement mutants of rhodopsin at position 121 (G121A, Ser, Thr, Val, Ile, Leu, and Trp) and control mutants with replacements of Gly114 or Ala117. The mutant opsins were expressed in COS cells and reconstituted with either 11-cis-retinal, the ground-state chromophore of rhodopsin, or all-trans-retinal, the isomer formed upon receptor photoactivation. The replacement of Gly121 resulted in a relative reversal in the selectivity of the opsin apoprotein for reconstitution with 11-cis-retinal over all-trans-retinal in COS cell membranes. The mutant pigments also were found to be thermally unstable to varying degrees and reactive to hydroxylamine in the dark. In addition, the size of the residue substituted at position 121 correlated directly to the degree of blue-shift in the λmax value of the pigment. These results suggest that Gly121 is an important and specific component of the 11-cis-retinal binding pocket in rhodopsin.

Rhodopsin, the photoreceptor molecule of the retinal rod cell, is a member of the family of G protein-coupled seven transmembrane (TM) 1 helix receptors (1). The photoreactive chromophore of rhodopsin is 11-cis-retinal, which is covalently bound in the interior of the protein as a protonated Schiff base. Photoisomerization of retinal causes receptor activation. Recently, a projection map of the TM helices has been obtained with 6-Å resolution in the plane of the membrane using electron microscopy on two-dimensional crystals of rhodopsin (2,3). A number of biochemical and biophysical studies indicate that TM helix 3 is crucial for the activation mechanism of rhodopsin. One critical residue on TM helix 3 is Glu 113 , which serves as the counterion to the protonated retinylidene Schiff base (4 -6).
Deprotonation of the Schiff base (7,8) and protonation of Glu 113 (9,10) occur in the formation of metarhodopsin II and are important in forming light-activated rhodopsin. Three conserved residues are located at the cytoplasmic end of TM helix 3, Glu 134 , Arg 135 , and Tyr 136 , which are involved in transducin binding (11,12). A conserved cysteine residue, Cys 110 , is located at the intradiscal end of TM helix 3 and forms a disulfide link to Cys 187 (13). In addition, changes in the environments of Glu 122 and Trp 126 have been shown to occur upon formation of metarhodopsin II (14,15).
In order to understand the mechanisms of spectral tuning and receptor photoactivation in the visual pigments, it is vital to gain additional information about the specific protein-protein and chromophore-protein contacts that define the chromophore-binding pocket in the ground state and how these contacts are affected by chromophore isomerization. There have been several models for how the retinal-protonated Schiff base interacts with Glu 113 on TM helix 3 (16 -18). In the most recent model, NMR constraints were used to position the chromophore in the interior of rhodopsin, which resulted in retinal situated between TM helices 3 and 6 with the ␤-ionone ring oriented toward TM helix 5 (19,20). This binding site model suggests close interactions of the retinal with Gly 121 , which is strictly conserved in all visual pigments (21).
We show that site-directed mutation of Gly 121 causes a decrease in thermal stability of the resulting pigment and an increase in reactivity with hydroxylamine. In addition, the size of the residue substituted at position 121 correlates remarkably well to the degree of blue-shift in the max value of the pigment. Furthermore, replacement of Gly 121 results in a relative change of opsin selectivity for reconstitution with 11-cisretinal over all-trans-retinal. In the following article (22), we show that the phenotypes of the Gly 121 mutants described in this report can be reverted by the mutation of a specific residue, Phe 261 , on TM helix 6.
Construction of Rhodopsin Mutant Genes-Site-directed mutagenesis was performed using restriction fragment replacement (25) in a synthetic gene of rhodopsin (26), which had been cloned into the expression vector as described previously (27). All TM helix 3 mutant genes were generated by substituting a 63-base pair SpeI-RsrII restriction fragment with a synthetic duplex containing the desired codon alteration. The nucleotide sequences of all cloned synthetic duplexes were confirmed by the chain terminator method for DNA sequencing of purified plasmid DNA using [ 35 S]dATP␣S.
Expression and Preparation of Rhodopsin Mutants-Opsin genes were expressed in COS-1 cells as described previously (23,27,28) except that a lipofection procedure was employed in place of the DEAEdextran transfection procedure. The cells were harvested and treated either with 11-cis-retinal followed by pigment purification in dodecyl maltoside as described (4) or were subjected to membrane preparation as described without chromophore regeneration (29). The pellet of COS cell membranes from a single 10-cm plate was resuspended in 0.5 ml of buffer and stored at Ϫ80°C in 80-l aliquots.
UV-visible Absorption Spectroscopy of Mutant Pigments-Spectroscopy was performed on a -19 Perkin-Elmer spectrophotometer at 25°C on purified samples unless otherwise specified. The molar extinction coefficient (⑀) of each mutant was determined by an acid denaturation method (4). Each ⑀ value was calculated from the formula, ⑀ ϭ (A/ A Rh )(A 440Rh /A 440 )⑀ Rh , where A was the absorbance at the max value, A 440 was the absorbance at 440 nm after acid denaturation, and ⑀ Rh was the molar extinction of rhodopsin (42.7 ϫ 10 3 M Ϫ1 cm Ϫ1 ). The spectral ratio, which is used as a measure of pigment yield and stability, is defined as A 280 divided by A at the visible max value after correction for any difference of the mutant pigment ⑀ value as described above.
Transducin Activation Assay-A radionucleotide filter-binding assay, which monitors the light-dependent guanine nucleotide exchange by transducin, was carried out at 10°C as described previously (30). For pigment samples purified in dodecyl maltoside, pigment concentration was determined before the assay by visible spectroscopy according to the ⑀ values given in Tables I and II. The assay mixture consisted of 4 M transducin, 3.0 nM purified pigment, and 20 M [ 35 S]GTP␥S in 50 l of assay buffer (50 mM Tris-HCl, pH 7.2, 100 mM NaCl, 4 mM MgCl 2 , 1 mM dithiothreitol). The activity assays of mutant opsins in COS cell membranes were performed as follows. Membrane aliquots were thawed at room temperature, and 1 l of 11-cis-retinal (7.5 mM) or all-trans-retinal (4.7 mM) ethanolic solution was incubated with 30 l of membrane suspension in the dark at room temperature for 1 h before assaying at room temperature. Opsin activity refers to any basal activity of a mutant in the absence of chromophore. Light activity refers to light-dependent transducin activation caused by samples incubated with 11-cis-retinal. All-trans-retinal activity refers to the light-independent transducin activation caused by opsin samples incubated with all-trans-retinal.
Reaction of Mutant Pigments with Hydroxylamine-The rates of hydroxylamine reaction with mutant pigments were determined in darkness at pH 7.0 as described previously (31). The conditions for the reaction at 20°C were 25 mM hydroxylamine, 50 mM Tris-HCl, pH 7.0, 100 mM NaCl, 0.1% dodecyl maltoside.
Regeneration Kinetics of Mutant Opsin G121L with Retinals in Membranes-The regeneration time courses of mutant G121L with 11-cisretinal or all-trans-retinal in COS cell membranes was measured by monitoring the initial rate of transducin activation at various periods after the addition of retinal to membrane aliquots. Retinal, 11-cis or all-trans (2 l of 25 M ethanolic solution), was mixed with 60 l of COS cell membrane suspension containing mutant opsin G121L in darkness and incubated at room temperature. At various times after retinal addition, aliquots (5 l) were removed and immediately assayed by the filter-binding method to determine transducin activity. Samples regenerated with 11-cis-retinal were illuminated to start the reaction. Samples regenerated with all-trans-retinal were assayed under dim red light. The initial rate (pmol of GTP␥S bound per min) of the transducin activation determined from a linear regression analysis was then plotted as a function of the time of incubation. Activities at time equals 0 were opsin activity without added chromophore. The experimental data were fitted by an exponential-rise function of the form, y ϭ a (1 Ϫ exp(Ϫbx)) ϩ c.

RESULTS
Spectral Properties of TM Helix 3 Mutants-Mutant opsin genes were prepared with single amino acid replacements at one of three sites in TM helix 3 of bovine rhodopsin, Gly 114 , Ala 117 , and Gly 121 (Fig. 1). Each of the mutant opsin genes was transiently transfected into COS-1 cells. Each expressed opsin was regenerated with the 11-cis-retinal chromophore, and the mutant pigment was purified after solubilization with dodecyl maltoside detergent according to an established immunoaffinity absorption procedure (4,31,32).
The set of Gly 121 mutants studied in detail included G121A, G121S, G121T, G121V, G121I, G121L, and G121W. The UVvisible spectrum of each of these mutants after purification in dodecyl maltoside is shown in Fig. 2. The spectral properties, including max value, molar extinction coefficient (⑀), and spectral ratio (absorbance at 280 nm versus absorbance at the visible max value), were determined by UV-visible spectroscopy for each purified mutant. The results of the spectral properties of the Gly 121 mutants are summarized in Table I. Replacement of Gly 121 by alanine or serine had little effect on the spectral properties of the resulting mutants when compared with those of rhodopsin. Mutant pigment G121T displayed a blue-shifted max value (483 nm) and an increased spectral ratio. The max value of G121V was further blue-shifted to 477 nm. The introduction of a residue with a larger side chain at position Gly 121 had dramatic effects on both max values and spectral ratios. The max values of both G121I and G121L were blue-shifted to 475 nm. In the G121I and G121W spectra shown in Fig. 2, the absorbance at 380 nm originated from free retinal since acid treatment did not shift the 380 nm peak to 440 nm (not shown). The G121W mutant did not form a stable pigment under the conditions of the purification. However, the max value of mutant G121W can be inferred to be 461 nm as described in Table I Table I. with the volume of the side chain introduced (Fig. 3).
All of the Gly 121 mutant opsins except G121W were expressed in transiently transfected COS cells at approximately the same levels as rhodopsin. G121W expression was about 70 Ϯ 4% of that of rhodopsin as judged by the A 280 value of immunoaffinity purified material from COS cells solubilized in dodecyl maltoside.
Sets of mutants with single amino acid replacements at Gly 114 or Ala 117 were prepared as controls for the Gly 121 mutants. Both Gly 114 and Ala 117 are predicted to be on the same helical face and are two and one helix turns toward the intradiscal end of TM helix 3, respectively (Fig. 1B). These residues should also be in close proximity to the retinal chromophore. In fact, a carboxylic acid group replacement at position 117 has been shown to be able to substitute for the Schiff base counterion at position Glu 113 (31,33). Of the set of Gly 114 mutants that included G114A, G114V, G114I, G114M, and G114W, only G114A was able to form a pigment in dodecyl maltoside. Any replacement with an amino acid side chain larger than the methyl group of alanine markedly decreased the level of expression in COS cells. In this context, it should be noted that rhodopsin mutation G114D has been reported to cause autosomal dominant retinitis pigmentosa (34). Most autosomal dominant retinitis pigmentosa phenotypes are thought to be associated with defective intracellular transport of the mutant opsin (35,36) Each of the Ala 117 single replacement mutants, which included A117G, A117V, A117I, A117M, and A117W, formed a stable pigment with a spectral ratio similar to that of rhodopsin prepared under identical conditions. The max values of these mutants were also close to 500 nm, ranging from a slight blue shift to 496 nm for A117G to a slight red shift to 504 nm for A117W (Table II). Whereas bulky side chain replacements were not well tolerated at positions 114 or 121, at position 117 even the replacement of Ala 117 by a tryptophan resulted in a stable pigment.
Reactivity of Mutant Pigments with Hydroxylamine-The Schiff base in rhodopsin is remarkably stable in the presence of hydroxylamine in darkness but reacts rapidly upon illumination. Amino acid replacements that affect the Schiff base environment have been shown to decrease the stability of mutant pigments in the presence of hydroxylamine (4, 28). For example, mutant pigment E113Q was shown to react with hydrox-ylamine even in the dark (4). Each of the TM helix 3 mutant pigments described above was treated with hydroxylamine in darkness to probe Schiff base stability (Fig. 4). Rhodopsin did not react with hydroxylamine under the conditions of the experiment during 60 min in the dark. Each of the Gly 121 mutant pigments reacted with hydroxylamine in the dark. However, the relative reactivity among the mutant pigments varied substantially. An inverse correlation between residue size at position 121 and reactivity is shown in Fig. 4, G121A was moderately reactive, whereas G121V reacted rapidly. Mutant pigment G121I reacted too rapidly to measure accurately. The experimental data points fit well to a single-exponential decay function. According to these values, mutant pigment G121L reacted with hydroxylamine at least 100-fold more rapidly than rhodopsin. Mutant pigment G121I was even more reactive than G121L. The remaining Gly 121 mutant pigments displayed in- There is an apparent linear correlation between the max values of the Gly 121 mutant pigments expressed in wave numbers and the volumes of the residues introduced. The max value of mutant G121W was determined as described in Table I. The average volume values and the horizontal error bars representing standard deviations are from Chothia (64). The standard errors of the measured max values are smaller than the symbols on this scale. For the regression line shown, the correlation coefficient is 0.95, and the slope is 10.9 cm Ϫ1 /Å 3 , which corresponds to 31 cal/mol/Å 3 .

TABLE I Spectral properties of the Gly 121 mutant pigments
The mutant pigments were prepared in dodecyl maltoside as described previously (23,31,50 (Fig. 4). A half-time of decay was calculated from the best fit to a single-exponential decay function.
d Rhodopsin pigment was stable during up to 2 h of hydroxylamine treatment. e The value of G121I could not be measured accurately due to the presence of free retinal, which is generated by gradual hydrolysis of Schiff base during the purification procedure (Fig. 2).
f Mutant G121W did not form a stable pigment in dodecyl maltoside upon incubation with 11-cis-retinal (Fig. 2). A max value for G121W was deduced by subtracting the max value of mutant pigment G121L/F261A from that of G121L and adding the result to the max value of mutant G121W/F261A (all values in wave numbers) (22). The max value of pigment G121W can also be estimated from the linear correlation between the max value and the size of the side chain at position 121 (excluding G121W) (Fig. 3). This approach results in a max value of 465 nm for G121W.
termediate reactivities that generally increased as the size of the residue at position 121 increased from alanine to leucine. The half-time of decay (t1 ⁄2 ) values for each Gly 121 mutant are presented in Table I. Transducin Activation by Purified Gly 121 Mutants-The ability of each of the Gly 121 mutants to catalyze guanine nucleotide exchange by transducin was assayed under a variety of conditions using a filter-binding assay method (30). For each mutant pigment reconstituted with 11-cis-retinal and purified in dodecyl maltoside detergent, light-dependent transducin activity was measured in solution. The activity values are presented in Table III relative to that of rhodopsin prepared and assayed in parallel. The level of light-dependent activity in Table III represents the value normalized for the amount of pigment based on visible spectroscopy and molar extinction as presented in Table I. Three of the mutant pigments were able to activate transducin at an approximately normal level. Three mutant pigments were as much as 15% defective in light-dependent transducin-activating ability in solution, G121S, G121T, and G121V.
Characterization of Gly 121 Mutant Opsin Activity in Membranes-The Gly 121 mutant opsins were assayed in COS cell membranes. These experiments were carried out to determine whether or not any of the mutant opsins was constitutively active (37). None of the Gly 121 mutant opsins displayed significant constitutive activity (Table III). Under the same assay conditions, mutant E113A, which is known to be constitutively active, displayed greater than 20% activity (22).

Activation of Gly 121 Mutant Opsins by All-trans-retinal in
Membranes-Certain mutant opsins, and even native opsin to a lesser extent, are able to be activated directly by all-transretinal in the dark (4,38,39). This activity depends on the ability of all-trans-retinal to enter the chromophore-binding pocket of the opsin or mutant opsin (40). Transducin activation by Gly 121 mutant opsins in COS cell membranes incubated with all-trans-retinal was determined. The time course of GTP␥S binding is shown for rhodopsin, G121A, G121V, and G121L (Fig. 5), and the relative rates are listed in Table III. Incubation with all-trans-retinal enhanced transducin activity depending on the amino acid replacement at position 121. The activity of each of the seven Gly 121 mutants in membranes incubated with all-trans-retinal was normalized to the percentage of its respective activity in light after regeneration with 11-cis-retinal of aliquots of the same membrane samples (Fig.  6). Even though none of the mutant opsins displayed constitutive activity, they were able to bind all-trans-retinal in membranes as judged by the relative amounts of transducin activity shown in Fig. 6. The transducin activation rates were assayed after incubation with all-trans-retinal for 1 h, which was shown to be a sufficient time for retinal binding to reach equilibrium (Fig. 7).
Regeneration Kinetics of Mutant Opsin G121L in Membranes-The time course of the regeneration of 11-cis-retinal into G121L in COS cell membranes was monitored by measuring the initial rate of light-dependent transducin activity as a function of time after the addition of 11-cis-retinal (Fig. 7). The kinetics of 11-cis-retinal regeneration with the G121L apoprotein followed an exponential rise with a rate constant of 0.04 min Ϫ1 . This rate constant is ϳ30 times smaller than the kinetics observed with native opsin. The kinetics of reconstitution of all-trans-retinal into G121L were also monitored (Fig.  7). The increase in activity as a function of time after the addition of all-trans-retinal to membranes containing G121L apoprotein can be approximated as a single-exponential rise with a rate constant of 1.1 min Ϫ1 . All-trans-retinal enters the chromophore-binding pocket to form an active species with a rate constant of approximately 30 times greater than that for 11-cis-retinal. In COS cell membranes, all-trans-retinal reconstitution with G121L reached completion almost immediately (within 2 min). In contrast, the half-time of reconstitution of G121L with 11-cis-retinal was approximately 19 min. Although at saturation, the level of activity of the all-trans-retinal reconstituted G121L mutant was lower (56%) than that of the 11cis-retinal reconstituted G121L mutant after photolysis, it was a Mutants G114V, Ile, Met, and Trp were expressed at very low levels and did not reconstitute in the presence of 11-cis-retinal to form pigments.

FIG. 4. Rates of hydroxylamine reaction with mutant pigments in darkness.
Absorbance at the max value of each mutant pigment is scaled to 1.0 at time equals 0, before the addition of hydroxylamine. The abscissa values represent the time after addition of hydroxylamine. The symbols represent experimental data points as relative absorption values normalized to their absorption at time equals 0. The curves represent the single exponential decay fits to the data. Rhodopsin (Rho) did not react with hydroxylamine under these conditions in darkness during the 60 min of the experiment, and the line represents the linear regression fit derived from the data points. Mutant pigment G121I reacted too rapidly to be measured accurately (i.e. t1 ⁄2 Ͻ 0.5 min) and is not shown. The half-time of decay (t1 ⁄2 ) values for each mutant are presented in Table I. significantly higher than that of rhodopsin (14%) measured in parallel (Table III). DISCUSSION The orientations and assignments of the seven TM helices of rhodopsin have been proposed based on electron microscopy of rhodopsin (2) and sequence alignment and comparison of relevant biochemical and mutagenesis studies from a large number of G protein-coupled receptors (41). These studies have provided a low resolution model for the receptor and a conceptual framework for considering specific residues in the primary structure for mutagenesis studies. Information about the structure of the retinal chromophore in rhodopsin and how it is situated with respect to the seven-helical bundle has also come from chemical, biophysical, and molecular biological approaches (16,17,19,(42)(43)(44)(45)(46)(47)(48). The salient point to emerge from these studies has been the model of a neutral chromophorebinding pocket (4, 6, 49) with significant chromophore-opsin interactions arising from amino acid residues on TM helices 3 and 6 (4 -6, 15, 50). In addition, NMR studies using isotopically labeled retinals in combination with molecular orbital calcula-tions have been used to position the retinal chromophore relative to Glu 113 in the interior of the rhodopsin structure (19). The carboxylate group of Glu 113 was found to be located ϳ3 Å from C 12 of the retinal (18).
This constraint on the retinal position in rhodopsin led us to investigate additional amino acid residues on TM helix 3 of rhodopsin that might be relevant to forming its retinal-binding pocket. In particular, we hypothesized that Gly 121 , which is conserved in all visual pigments, might provide a cavity in the protein interior that would allow the binding of 11-cis-retinal. This hypothesis was tested by preparing a series of single amino acid replacement mutants in which Gly 121 was substituted by residues with side chains of increasing size. The set of the seven Gly 121 mutants is shown in Fig. 2 and Table I. The mutant opsins were expressed in COS cells and studied using variations of established methods, including immunoaffinity purification and transducin activation assays.
COS cells expressing mutant opsins were treated with 11cis-retinal and solubilized in dodecyl maltoside buffer. The purified mutants displayed varying degrees of chromophore binding. For example, mutant G121A displayed an UV-visible spectrum very similar to that of recombinant rhodopsin pre- The opsin and all-trans activities, which were measured in COS cell membrane preparations at room temperature, are presented as percentage of the activity of each mutant measured in aliquot of the same preparations after incubation with 11-cis-retinal and illumination. Values are given as mean Ϯ S.E. (n). None of the mutant pigments was constitutively active.
b Light activity is reported as the percent of the light activity of rhodopsin measured under the same conditions, 427 Ϯ 61 (n ϭ 12) pmol of GTP␥S bound per min for a membrane sample containing 0.5 mg/ml total protein.
c Pigments were purified in dodecyl maltoside buffer and assayed at 10°C. The activity is reported as the percent of the light activity of rhodopsin measured under the same conditions, 135 Ϯ 1 (n ϭ 3) pmol of GTP␥S bound per min per pmol of pigment in a 100 l reaction. The amount of pigment is determined its absorption at max and the value given in Table I. NA, not applicable.

FIG. 5. Transducin activation by Gly 121 mutant pigments in COS cell membranes incubated with all-trans-retinal.
COS cell membranes containing the mutant opsin were prepared from transiently transfected COS cells. The ability of each mutant to activate transducin was evaluated by a GTP␥S filter-binding assay. The amount of GTP␥S bound to a filter is plotted as a function of time after the addition of membranes to the assay mixture. rho, rhodopsin. The data for all mutants are presented in Table III. FIG. 6. Transducin activation by Gly 121 mutant pigments in COS cell membranes incubated with all-trans-retinal. The activity of each Gly 121 mutant in membranes incubated with all-transretinal is normalized to the percentage of its respective light-dependent activity after regeneration with 11-cis-retinal. The mean and standard error of at least three independent measurements are represented. Rho, rhodopsin. The data for all mutants are presented in Table III. pared under identical conditions. At the other extreme, although the G121W opsin could be purified, it failed to form a stable visible-absorbing pigment in dodecyl maltoside. A lack of pigment in the immunoaffinity purification assay could indicate an inability of the mutant opsin apoprotein to bind 11-cisretinal or an instability of the mutant pigment under the conditions of the purification procedure. The latter explanation is more consistent with the overall data since the ability of each of the mutants in membranes to activate transducin in response to light after incubation with 11-cis-retinal was similar to that of rhodopsin (Table III). This indicates that 11-cis-retinal could enter the chromophore-binding pocket of each apoprotein in the COS cell membrane and form a covalent linkage to a similar extent as the native opsin, although the rate for ligand incorporation is reduced (Fig. 7).
The introduction of an amino acid residue with a bulky side chain at position 121 lowered the stability of the pigment in detergent. The mutant pigments G121V, G121I, and G121L were found to be significantly less stable than rhodopsin in detergent. At room temperature in the dark, the visible absorption bands of purified G121V, G121I, and G121L mutants decayed with concomitant increase of the 380-nm band, indicating the hydrolysis of the protonated Schiff base linkage (data not shown).
Hydroxylamine reactivity has also been used as a probe of pigment stability. As shown in Fig. 4 and Table I, there was also a significant trend toward increased dark reactivity of the pigment with hydroxylamine as the size of the side chain of the residue at position 121 was increased. The G121I pigment was the most reactive and decayed at least 1000-fold more rapidly than rhodopsin under identical conditions in the dark. In addition, it should be noted that the mutants with branched ␤ carbons at the 121-position (G121V and G121I) were less stable than G121L, even when the side chain volumes were smaller or comparable. The reactivity of a Schiff base with hydroxylamine is affected by the intrinsic pK a of the Schiff base as well as its accessibility to solvent and to reactant. The general result of dark reactivity of every Gly 121 replacement mutant with hydroxylamine and its size dependence indicates that the Gly 121 mutant pigments are unstable due to a sterically induced perturbation of chromophore-opsin structure, which alters the Schiff base environment.
The progressive blue-shift of the max values in Gly 121 mutants with bulkier side chains is consistent with the idea that a steric perturbation at position 121 can disrupt the normal interaction between the retinal chromophore and its counterion at Glu 113 . This conclusion is not unreasonable since Glu 113 is located approximately two and one-half helix turns below Gly 121 and given the unusual geometry of the Glu 113 -chromophore ion pair (18). The altered counterion-chromophore interaction can presumably render the Schiff base more accessible to the solvent and to hydroxylamine in the dark and also change its pK a (4,17). Therefore, given the fact that Gly 121 mutant opsins are able to bind 11-cis-retinal and to form functional pigments in membranes, we conclude that the instability of the Gly 121 mutant pigments purified in detergent is a result of greater susceptibility of the protonated Schiff base linkage to hydrolysis.
Bovine opsin may become constitutively active secondary to mutation of a number of individual amino acid residues, including Glu 113 , Glu 134 , and Lys 296 (37,51), which have also been shown to be involved in the mechanism of rhodopsin photoactivation (40). The mutation presumably perturbs the equilibrium of the apoprotein population between inactive and active conformations and shifts it toward the active state (37,52). This model explains the increased binding of all-transretinal, the equivalent of an agonist ligand in the biogenic amine receptor, to the constitutively active mutant opsins and the inhibition of the constitutive activity by the binding of the antagonist 11-cis-retinal. However, it has been shown recently that the opsin-all-trans-retinal complex activates transducin by a different mechanism than that of the native photolyzed pigment (39).
A unique and interesting phenotype of Gly 121 mutant opsins (in membranes) is the significant binding of all-trans-retinal and the activation of transducin in the dark (Fig. 5), in the absence of constitutive activity. When the activity in the presence of all-trans-retinal is presented as a percentage of the light-dependent activity after 11-cis-retinal incubation, it is clear that the mutant opsins G121S, G121I, G121L, and G121W (presumably in their inactive conformations) have a significant defect in their abilities to discriminate between the two isomeric forms of the chromophore. Only the G121S mutant does not fit a general trend of increasing activity with side chain size, possibly due to the presence of a polar hydroxyl group.
Taken together, the results of the spectral studies of the Gly 121 mutants purified in dodecyl maltoside and the activity studies carried out in membranes strongly suggest that the replacement of the conserved glycine by any residue tested affects the architecture of the chromophore-binding pocket. The effects include a decrease in pigment stability and a loss of strict preference for the 11-cis-retinal isomer over all-transretinal. The effects are generally progressive with the size of the residue substituted for Gly 121 . This size effect is most striking in the case of the data presented in Fig. 3. The behavior of the Gly 121 mutants is specific and is not observed with mutations at positions Gly 114 or Ala 117 .
These results imply that Gly 121 plays an important role in defining the conformation and specificity of the chromophorebinding pocket in rhodopsin. Considering the strong conservation of a glycine residue at position 121 in all visual pigments, it is intriguing to speculate that a glycine at this position creates a packing space for either the chromophore or for another amino acid side chain that contributes to the integrity of , the initial rate of the light-dependent transducin activation at a given time after the addition of 11-cis-retinal to membranes containing mutant G121L apoprotein in darkness was measured. For all-trans-retinal (ATR) (solid circles), the initial rate of transducin activation in darkness at a given time after the addition of all-trans-retinal was measured. The experimental data points were fitted to an exponential-rise function of the form, y ϭ a (1 Ϫ exp(Ϫbx)) ϩ c. The rate constants (b) for the rise of activity as a function of time after 11-cis-and all-trans-retinal addition were 0.04 and 1.1 min Ϫ1 , respectively. the retinal-binding pocket structure. Glycine residues are common in transmembrane helices (53,54) and are known to be critical residues that allow good van der Waals contacts in helix-helix packing (55)(56)(57). It is also interesting to note that Gly 121 is one of several residues with small side chains on TM helix 3 that is highly conserved. In particular, Gly 114 and Ala 117 lie on the same face of TM helix 3 as Gly 121 . However, substitution of Ala 117 with residues having larger side chains does not significantly influence the binding of 11-cis-retinal or the other properties tested. In the following paper, the interactions of Gly 121 are further explored by introducing secondsite mutations (22).
The present results may also be reconciled with previous findings in a variety of other G protein-coupled receptors. In the human D2 dopamine receptor, the substituted-cysteine accessibility method was employed to map the ligand bindingsite crevice (58,59). Several residues on TM helix 3 were shown to be the most reactive to methanethiosulfonate derivatives and were assigned to face a water-accessible crevice, Val 111 , Asp 114 , Val 115 , and Cys 118 . These four residues, which correspond to Gly 114 , Ala 117 , Thr 118 , and Gly 121 in rhodopsin, form a cluster at the extracellular half of TM helix 3. In the 5-HT 2A receptor, Ser 159 , which corresponds to Gly 121 in rhodopsin, was found to interact with its ligand (60). In the human NK-1 receptor, Val 118 , also equivalent to Gly 121 in rhodopsin, has been identified as a key residue to confer species selectivity for several non-peptide antagonists (61)(62)(63). The residue on TM helix 3 at a position corresponding to Gly 121 in rhodopsin plays a role in regulating the specificity and affinity of ligand binding in many G protein-coupled receptors.