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J Biol Chem, Vol. 275, Issue 3, 1937-1943, January 21, 2000

Mutation of the Fourth Cytoplasmic Loop of Rhodopsin Affects Binding of Transducin and Peptides Derived from the Carboxyl-terminal Sequences of Transducin  and  Subunits^*

Oliver P. Ernst^§, Christoph K. Meyer^§, Ethan P. Marin^§¶, Peter Henklein, Wing-Yee Fu^¶**, Thomas P. Sakmar^¶**, and Klaus Peter Hofmann^§§

From the  Institut für Medizinische Physik und Biophysik,  Institut für Biochemie, Charité, Medizinische Fakultät der Humboldt Universität zu Berlin, Schumannstr. 20-21, 10098 Berlin, Germany and the ^** Howard Hughes Medical Institute, ^¶ Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10021

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G_t), is not well defined. We developed a novel assay to measure the ability of G_t, or G_t-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the  subunit of G_t, or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the  subunit of G_t, are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the -derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G_t (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the  subunit of G_t. We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the  subunit of G_t and plays a role in the regulation of subunit binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G protein-coupled receptors transmit extracellular signals to the cell's interior via heterotrimeric G proteins and effector enzymes or ion channels (1, 2). Rhodopsin is one of the archetypes of the G protein-coupled receptor superfamily. It triggers the biochemical amplification machinery of the visual cascade in the rod photoreceptor cell, which comprises the G protein transducin (G_t)¹ and the effector, a cyclic GMP-specific phosphodiesterase (3, 4). The transduction of a light signal begins with the photochemical cis-trans isomerization of the chromophore, 11-cis-retinal. Protein conformational changes are transmitted from the ligand-binding site to the cytoplasmic surface of the receptor where catalytic activation of G_t occurs. This intramolecular conversion from inactive (rhodopsin) to active (R*) states mediated by chromophore isomerization has been termed "signal transmission" (5). Key structural correlates of the transition to the active state include deprotonation of the retinylidene Schiff base (6) with concomitant protonation of the Glu¹¹³ counterion (7, 8) and the protonation of the cytoplasmic surface of rhodopsin (9, 10) mediated by the highly conserved Glu¹³⁴ residue at the cytoplasmic border of transmembrane (TM) helix 3. Movements of TM helices have been proposed to accompany the signal transmission process, with a change in the orientation of TM helices 3 and 6 relative to each other as the most prominent feature (11-13).

The cytoplasmic surface of rhodopsin comprises four loops and a carboxyl-terminal tail. The first (C1), second (C2), and third (C3) cytoplasmic loops connect adjacent TM helices. The fourth cytoplasmic loop (C4) is bounded by TM helix 7 at its amino terminus and two palmitoyl groups inserted into the membrane bilayer at its carboxyl terminus. The palmitoyl groups are attached to Cys³²² and Cys³²³ via thioester linkages. A schematic of the structure of rhodopsin is presented in the preceding paper (14). A variety of experimental approaches, including proteolysis, chemical modification, peptide competition, and site-directed mutagenesis in combination with biochemical and biophysical assays, have been employed to map the sites of rhodopsin responsible for the binding and activation of G_t. The salient results have indicated that loops C2 and C3 are involved in G_t binding and activation (15, 16). In addition, recent studies indicate a role for the loop C4 in G_t activation (14, 17, 18). Despite these studies and the availability of a high-resolution crystal structure for the G_t holoprotein (19), there is little information concerning: 1) the key functional intramolecular interactions on the cytoplasmic surface of rhodopsin that form and regulate the catalytic site for G_t, 2) G_t subunit specificity for binding to particular cytoplasmic regions of rhodopsin, 3) the molecular mechanism of rhodopsin-catalyzed nucleotide release by G_t.

In the preceding paper (14), we identified a region at the amino terminus of loop C4 of rhodopsin that most likely interacts with the  subunit of G_t, G_t. Here, we studied the interaction of site-directed mutants of rhodopsin with G_t and peptides derived from the carboxyl-terminal sequences of G_t and G_t (20-22). We used a novel assay in which the all-trans-retinal in metarhodopsin II (MII) is photoconverted to the cis configuration using blue light. The flash-induced photoregeneration of rhodopsin from MII can be followed spectroscopically with millisecond time resolution (20). Since stabilization of R* by G_t or G_t-derived peptides inhibits the rate of photoregeneration, the assay can be used to monitor the interaction of R* with G_t (21, 22). We show that peptide (340-350), corresponding to the carboxyl-terminal undecapeptide of G_t, and peptide (50-71)-far, corresponding to the carboxyl-terminal cysteinyl-thioetherfarnesyl peptide of G_t, stabilized R*. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group of the G_t-derived peptide prevented stabilization of R*. G_t failed to stabilize mutant rhodopsins with alterations of the amino terminus of loop C4 near the TM helix 7 border. The G_t-derived peptide also failed to stabilize these mutants, suggesting that loop C4 comprises part of a binding site for the carboxyl-terminal tail of G_t.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Rhodopsin and G_t-- Purified bovine rhodopsin was prepared from hypotonically washed rod outer segment membranes essentially as described (23). The recombinant mutant pigments used in this study are shown in Fig. 1A. The construction, expression, and purification of these samples were reported (14). Following purification, the samples were concentrated ~10-fold using Centricon-30 filtration devices (Amicon). G_t holoprotein was purified from rod outer segment preparations essentially as described previously (24).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences of recombinant rhodopsins and Gt-derived peptides. A, amino acid sequence of the loop C4 of wild-type rhodopsin (WT Rho) and mutant opsins (CTr1, CTr2, CTr4, and CysXV). In bovine rhodopsin, this region extends from position 310 at the intracellular junction of the TM helix 7 to Cys322 and Cys323, which are palmitoylated and inserted into the membrane (40-42). Changes from the wild-type sequence are highlighted in gray. In CTr1, CTr2, and CTr4, portions of the C4 loop have been replaced with analogous sequences from the 2-AR. In CysXV, the palmitoylation sites are replaced by serine residues. B, amino acid sequences of peptides were derived from the carboxyl termini of bovine Gt and Gt subunits. The (340-350) peptide corresponds to the native sequence of Gt, and the (50-71)-far corresponds to the native sequence of Gt, which is post-translationally modified by cysteinyl thioether farnesylation. Peptides with alterations of primary structure, or a lack of carboxyl-terminal farnesylation, were used as controls. The carboxyl termini of the Gt-derived peptides were amidated. The carboxyl termini of the Gt-derived peptides, and the amino termini of all the peptides, were unmodified.

Peptide Synthesis-- The synthetic peptides used in this study are listed in Fig. 1B. Peptides were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy with HBTU activation (Fastmoc, 0.1 mmol small-scale cycles) on an ABI Model 433A peptide synthesizer. Farnesylation of G_t-derived peptides was carried out by dissolving pure peptide (60 mg) in 5 ml of a solution of 50% (v/v) 1-propanol containing 35 mmol of sodium carbonate. The resulting solution was saturated with nitrogen and 0.6 ml of a freshly prepared 10% (v/v) farnesyl bromide solution in 1-propanol was slowly added under vigorous stirring while pH was adjusted to >9. The solution was flushed with nitrogen again and incubated for 24-48 h with shaking. The farnesyl peptides were purified by reverse phase high performance liquid chromatography.

Instrumentation-- Time-resolved absorption traces were recorded on a custom-built single-wavelength absorption photometer. Light from a 150-W halogen light source passes through a Jobin-Yvon HR460 monochromator (focal length 460 mm, 1200 lines/mm, slit width set to 1 mm) tuned to 543 nm, and from there through the cuvette (4-mm optical path length) and a band-pass interference filter onto a large surface PIN photodetector. The output is nulled and amplified twice, filtered with a 500-µs electronic low-pass filter and recorded using a modified Nicolet 2090-IIIA digital oscilloscope.

Photoregeneration Experiments-- The rhodopsin photoregeneration assay was performed as reported (20) with adaptation for recombinant pigments as follows. All samples contained 2 µM pigment in a volume of 0.26 ml of 200 mM Na₂HPO₄ (pH 8.0), 10 mM NaCl, and approximately 0.03% (w/v) dodecyl maltoside (DM). Due to the concentration procedure required for recombinant samples, the final DM concentration was not precisely known, but was estimated not to exceed 0.035% (w/v). After equilibrating the sample cuvette to 13 °C, the sample was illuminated for 30 s with a green HeNe laser (543.5 nm, 5 mW, Melles Griot) to cause quantitative formation of MII. Absorption at 543 nm was recorded continuously. After 50 ms, a flash of blue light (412 ± 7 nm, about 20-µs duration) was applied to the sample and formation of photo-regenerated pigment was measured at 543 nm for an additional 200 ms. Discharge of the flashlamp affected the sensitive electronics of the detector, causing a brief artifactual negative deflection. Four records induced by four separate flashes were collected from each sample with 30-s intervals between the recordings. Starting with the initial illumination, each experiment took approximately 140 s. The four records were averaged to produce experimental data traces as presented in Fig. 2. The experimental photoregeneration signal traces are depicted as absorbance changes at 543 nm versus time (i.e. a rising signal indicates a proportional increase of absorbance due to reprotonation of the Schiff base).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Photoregeneration of rhodopsin in the presence of increasing concentrations of Gt or Gt-derived peptides. The presence of Gt, (340-350), or (50-71)-far causes a dose-dependent reduction in photoregeneration. Each trace shows a representative individual experiment and the time-dependent increase in absorbance at 543 nm. The photoregeneration signal is initiated by a flash at 50 ms. Fits to the slow phase of the signal are shown as dotted lines. Insets show the initial slope of the slow phase of photoregeneration versus added concentration of Gt or peptide as indicated. These plots were fit using a hyperbolic function with additive offset. IC50 values and errors are derived from the fits to the dose-response data and presented in the insets. A, photoregeneration of rhodopsin in the presence of increasing concentrations of Gt (IC50 = 2.56 ± 2.0 µM). B, photoregeneration of rhodopsin in the presence of increasing concentrations of (340-350) peptide (IC50 = 49.5 ± 6 µM). C, photoregeneration of rhodopsin in the presence of increasing concentrations of (50-71)-far peptide (IC50 = 285 ± 74 µM).

Numerical Fitting Procedures and Determination of Initial Slope Values-- The photoregeneration signal comprises a fast phase, which is not resolved, and a slow phase, which is monitored for 200 ms (see Fig. 2). Data points obtained 4.5-7.0 ms after the blue flash were averaged and used as an estimate for the amplitude of the fast phase. The relative amplitude of the fast phase of the photoregeneration of the recombinant pigments was the same as that of rhodopsin. The initial slope of the slow phase of a photoregeneration trace was determined from the numerical fit of a simple exponential-rise function offset by the amplitude of the fast phase. Values for relative slope are presented (Table I) to demonstrate the effect of G_t or G_t-derived peptides on the initial slope of the photoregeneration signal. Relative slope is defined as the ratio of the initial slope of the slow phase of the photoregeneration trace in the presence of G_t or G_t-derived peptides versus the initial slope in their absence. A relative slope of 1.0 indicates no effect, whereas a slope of <1.0 indicates inhibition of photoregeneration. The relative slope for an experiment with rhodopsin and G_t (3 µM) was typically about 0.7.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Effect of G_t and G_t-derived Peptides on the Photoregeneration of Rhodopsin-- A photoregeneration assay was employed that measures the kinetics of photoconversion of MII to rhodopsin (20). The assay takes advantage of the conformational coupling of the cytoplasmic surface of the active state of rhodopsin, R*, to the chromophore-binding pocket in the membrane-embedded domain of the receptor. The slow phase of the photoconversion kinetics essentially monitors the reprotonation of the retinylidene Schiff base as a function of time after a sample of R* is subjected to a blue flash. Photoconversion of R* (_max = 380 nm) to rhodopsin (_max = 500 nm) is inhibited if R* is bound to G_t or certain G_t-derived peptides. R* that is not bound in a stabilizing complex with G_t or G_t-derived peptides is more readily photoconverted.

The effect of G_t on the photoconversion of R* was studied first (Fig. 2A). The change in absorbance at 543 nm is plotted as a function of time. The blue flash is applied to the sample at 50 ms. Superimposed upon an initial rapid change in amplitude, the slow phase of the trace represents the back conversion of R* to rhodopsin. The experiment was repeated with identical rhodopsin samples in the presence of increasing concentrations of G_t (0, 0.5, 1.0, 2.0, 3.0, and 5.0 µM). The traces are superimposed to show a clear dose-dependent inhibition of the photoregeneration reaction by G_t. The experimental traces were fit to an exponential function in order to calculate values for initial slopes. The calculated fits are shown as dashed lines in Fig. 2. The initial slopes of the photoregeneration traces are plotted as a function of G_t concentration in the inset. A satisfactory hyperbolic fit yielded an effective concentration at 50% inhibition (IC₅₀) value of 2.56 ± 2.0 µM. This value effectively represents a binding constant for the interaction between R* and G_t under the conditions of the assay.

The effects of two peptides corresponding to the carboxyl-terminal regions of G_t and G_t on the photoconversion of R* were studied next (Fig. 2, B and C). The amino acid sequences of (340-350) and (50-71)-far are presented in Fig. 1B. The (50-71)-far peptide carries the post-translational isoprenylation that is characteristic of G_t. Fig. 2B shows six superimposed photoregeneration traces obtained from identical rhodopsin samples in the presence of increasing concentrations of (340-350) (0 to 1000 µM). The traces show a clear dose-dependent inhibition of the photoregeneration of R* by (340-350). The initial slopes of the photoregeneration traces are plotted as a function of (340-350) concentration in the inset to Fig. 2B. A satisfactory hyperbolic fit yielded an IC₅₀ value of 49.5 ± 6.0 µM for the interaction between R* and (340-350).

Fig. 2C shows six superimposed photoregeneration traces obtained from identical rhodopsin samples in the presence of increasing concentrations of (50-71)-far (0 to 1000 µM). The traces show a clear dose-dependent inhibition of the photoregeneration of R* by (50-71)-far. Plotting the initial slopes of the photoregeneration traces as a function of (50-71)-far concentration (inset to Fig. 2C) permitted a satisfactory hyperbolic fit that yielded an IC₅₀ value of 285 ± 74 µM for the interaction between R* and (50-71)-far.

Specificity of the Effects of (340-350) and (50-71)-far Peptides-- The specificity of the effect of the G_t-derived peptides was studied by performing control experiments with altered peptides. The peptide sequences are shown in Fig. 1B. One control peptide, (340-350) K341R/L349A, was studied to evaluate the specificity of the carboxyl-terminal sequence of G_t in R* interaction. This peptide failed to show peptide-R* interaction (25, 26) and the substitution of Leu³⁴⁹ by alanine in G_t was reported to abolish coupling to active rhodopsin (27, 28). As shown in Fig. 3, the (340-350) K341R/L349A peptide failed to inhibit the photoregeneration of R*. Similarly, the requirements for length, primary structure, and farnesylation of the G_t-derived peptide were tested. Peptides (60-71)-far and (50-71)-far both inhibited photoregeneration similarly (Fig. 3). Positions 64 and 67 in G_t have been reported to be critical for interaction with MII, as observed with the altered peptide (60-71)-far F64T/L67S (29). The (60-71)-far F64A/L67A peptide did not inhibit photoregeneration (Fig. 3). In addition, the (60-71) peptide, which lacked cysteinyl farnesylation, did not inhibit photoregeneration (Fig. 3). This finding is consistent with earlier results showing that lack of farnesylation prevented MII stabilization by G_t-derived peptides (30, 31). In other control experiments, the 1D4 peptide, corresponding to the carboxyl-terminal 18 amino acids of rhodopsin, did not affect photoregeneration, nor did it affect the inhibition of photoregeneration by G_t and the G_t-derived peptides (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Altered peptides do not inhibit photoregeneration. Photoregeneration experiments were performed as described in the legend to Fig. 2 in the presence of altered peptides (amino acid sequences are shown in Fig. 1). Concentrations were 200 µM for the Gt-derived peptides and 500 µM for the Gt-derived peptides. The inverse of intensity change at the detector in arbitrary units is plotted as a function of time. Data collected in the absence of peptide were normalized to a value of 1.0 at 250 ms; data collected in the presence of peptides were scaled to data collected with the same rhodopsin sample in the absence of peptide. An arbitrary unit is approximately 0.15 mOD units at 543 nm. Inhibition of photoregeneration is abolished by the conservative substitution of two amino acids in the (340-350) or (60-71)-far peptide, or by removal of the farnesyl moiety in the (60-71) peptide. The longer (50-71)-far peptide shows functional identity to (60-71)-far.

It has been reported that detergent concentration has an influence on the activation rate of G_t by R* (16, 32). The effect of varying concentrations of DM on the photoregeneration kinetics of rhodopsin and on the inhibition of photoregeneration by G_t (3 µM), (340-350) (200 µM), or (50-71)-far (500 µM) was studied. Varying DM concentrations from 0.01 to 0.10% (w/v) had no effect on the photoregeneration kinetics of rhodopsin, and no effect on the inhibition of photoregeneration by (340-350) (data not shown). However, the inhibition of photoregeneration by G_t and (50-71)-far was reduced by increasing DM concentrations from 0.01% to 0.10% (data not shown). This effect mirrors the reduction in the rate of G_t activation by R* in the presence of increasing [DM], as previously reported (16, 32). The final DM concentration under the standard conditions of the photoregeneration assay using heterologously expressed and purified mutant pigments is estimated to be 0.01-0.035%, a range in which detergent effects were found to be modest. Furthermore, the final DM concentration in assays of each of the recombinant samples is virtually identical.

Photoregeneration Assay of Recombinant Rhodopsin and Loop C4 Mutants-- Photoregeneration assays were carried out on wild-type recombinant rhodopsin and four mutant rhodopsins (Fig. 1A). Representative photoregeneration traces are presented in Fig. 4. In each panel, the change in absorbance at 543 nm is plotted as a function of time. The blue flash is applied to the sample at 50 ms. Superimposed upon an initial rapid change in amplitude, the slow phase of the trace represents the back conversion of R* to rhodopsin. The black trace shows the result with pigment alone. The red trace shows the result with pigment in the presence of G_t (3.0 µM), (340-350) (200 µM), or (50-71)-far (500 µM) as indicated. The behavior of COS cell-expressed rhodopsin was similar to that of bovine rhodopsin in the photoregeneration assay. Fig. 4 shows typical experimental traces obtained with purified recombinant rhodopsin in the presence of G_t, (340-350), and (50-71)-far. Typical traces obtained with bovine rhodopsin are presented in Fig. 2. These results confirm that the photoregeneration assay can be employed to study recombinant pigments prepared in relatively small quantities in a heterologous expression system.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   The effect of Gt, (340-350), and (50-71)-far on photoregeneration of heterologously expressed rhodopsin and C4 loop mutants. Experiments were carried out as described in the legend to Fig. 2. Concentrations of Gt, (340-350), and (50-71)-far were 3, 200, and 500 µM, respectively. The vertical scale bar, which depicts 0.075 mOD at 543 nm, applies to all traces. Black traces show photoregeneration of pigment alone. Red traces show photoregeneration of each pigment in the presence of Gt (Transducin), (340-350), or (50-71)-far as indicated at the top of each column of panels. The sample used in each row is indicated in the labels at left. Each pair of traces is representative of at least two sets of experiments performed on different samples. WT, CTr1, and CysXV show distinct effects of Gt or Gt-derived peptides. Photoregeneration of CTr4 is not influenced by Gt or Gt-derived peptides, and CTr2 shows an effect with (50-71)-far, a minor effect with Gt holoprotein and no effect with (340-350).

Four mutant pigments with alterations of the amino acid sequence of the C4 loop were prepared (Fig. 1A). Mutants CTr1, CTr2, and CTr4 are essentially chimeric receptors in which parts of the C4 loop of rhodopsin are replaced by sequences from the ₂-adrenergic receptor (₂-AR). Mutant CysXV (C322S/C323S) was designed to evaluate the effect of receptor palmitoylation on the photoregeneration kinetics. The mutant opsin genes were expressed in COS cells, treated with 11-cis-retinal and purified in DM detergent solution. The levels of palmitoylation of the expressed mutant pigments CTr2 and CTr4 were similar to that of the wild-type receptor expressed in parallel (14); CysXV was not palmitoylated (14, 33). The ability of each of the mutant pigments to activate G_t was evaluated using a fluorescence G_t activation assay (Table I). Mutant pigments CTr2 and CTr4 were significantly defective in their ability to activate G_t (14).

In the absence of G_t or G_t-derived peptides, the photoregeneration kinetics of rhodopsin and the four C4 mutants were essentially identical (Fig. 4, black traces). This result suggests that the C4 loop mutations do not affect the photoregeneration reaction. The effects of G_t or G_t-derived peptides on the photoregeneration of the mutant pigments are shown in Fig. 4 (red traces). The effects can be conveniently evaluated by comparing relative slopes (Fig. 5A). The relative slope is defined as the initial slope of the slow phase of the photoregeneration trace in the presence of G_t, (340-350), or (50-71)-far divided by the initial slope of the trace in the absence of G_t or G_t-derived peptide. The relative slope provides a quantitative measure of the effect of G_t or a G_t-derived peptide on the slow phase of the photoregeneration kinetics. A relative slope of 1.0 indicates no inhibition of photoregeneration, and relative slopes of <1.0 indicate a progressive inhibition of photoregeneration. Average values for relative slopes are presented in Table I.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Quantitation of the effects of Gt, (340-350), and (50-71)-far on the photoregeneration of recombinant rhodopsin and rhodopsin mutants. The slow phases of the photoregeneration traces were fit with exponential-rise equations, and the initial slopes were determined. A, relative slopes. Bars represent the average ratio of the initial slope of the slow phase of photoregeneration of pigment in the presence of Gt (Transducin), (340-350), or (50-71)-far to the initial slope with pigment alone. The error bars display the standard errors. The photoregeneration of mutants CTr2 and CTr4 is unaffected by Gt and (340-350). Only mutant CTr4 photoregeneration is insensitive to (50-71)-far. B, normalized inhibition. The vertical bars indicate the ability of Gt and Gt-derived peptides to inhibit photoregeneration of mutant pigments relative to their effect on wild-type rhodopsin (WT). The normalized inhibition values were determined from the following equation: (1  mean relative slope)mutant/(1  mean relative slope)rho-dopsin. The error bars depict the propagated standard errors from the determination of relative slope. Numerical values are given in Table I. This analysis allows for direct comparison of the effects of each peptide and Gt on each mutant. The effect of Gt and Gt-derived peptides on CTr1 and CysXV are similar to their effect on rhodopsin. The photoregeneration of CTr4 does not show an effect of Gt or either Gt-derived peptide. This behavior is consistent with a failure of CTr4 to bind Gt, (340-350), and (50-71)-far. Replacement of the entire C4 loop in CTr2 shows a graded effect: CTr2 is essentially insensitive to inhibition by (340-350) and Gt, but shows effects with (50-71)-far nearly identical to those of wild-type rhodopsin.

Photoregeneration of mutant CTr1 was inhibited by G_t and the G_t-derived peptides. The degrees of inhibition were identical to those seen with wild-type rhodopsin. The behavior of mutants CTr2 and CTr4 was different from that of rhodopsin. G_t and the peptide (340-350) did not inhibit photoregeneration of CTr2. This result is best appreciated in Fig. 5A, where the relative slopes for CTr2 in the presence of G_t and (340-350) are ~1.0. However, the (50-71)-far peptide was able to inhibit photoregeneration of CTr2 to the same extent observed with rhodopsin. The photoregeneration of mutant CTr4 was not affected by G_t, (340-350), or (50-71)-far. This result is best seen in Fig. 5A, where the relative slopes for CTr4 in the presence of G_t and the G_t-derived peptides are ~1.0. Photoregeneration of mutant CysXV was inhibited by G_t and the G_t-derived peptides. However, the inhibition by peptide (50-71)-far was relatively more pronounced for CysXV than for rhodopsin.

Each of the peptides and G_t inhibit the photoregeneration of rhodopsin to different degrees. Therefore, the ability of each peptide and G_t to affect a particular mutant cannot be compared directly using relative slopes. However, by normalizing the relative slope of a mutant to that of rhodopsin, such a comparison can be made. This expression, termed the normalized inhibition, is obtained from the following equation: (1  mean relative slope)_mutant/(1  mean relative slope)_rhodopsin. The normalized inhibition for rhodopsin is defined to be 1.0. A value of zero indicates no inhibition of the photoregeneration of the mutant pigment by a particular ligand. Data are plotted in Fig. 5B and listed in Table I. Mutant CTr1 is similar to rhodopsin with respect to inhibition of photoregeneration by G_t, (340-350), and (50-71)-far. Mutant CTr2 shows essentially normal interaction with (50-71)-far, but fails to be affected by G_t and (340-350). Mutant CTr4 is unaffected by G_t and both G_t-derived peptides. The photoregeneration of mutant CysXV is inhibited by (340-350) and G_t normally, but displays an enhanced sensitivity to (50-71)-far.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several lines of evidence suggest that the conformation of the cytoplasmic surface of the active state of rhodopsin, R*, is coupled to the conformation of the chromophore-binding pocket in the membrane-embedded domain of the receptor (3, 4). In analogy to G protein-coupled receptors with diffusible ligands in which G protein binding stabilizes a receptor conformation with a high affinity ligand-binding site, MII is stabilized at the expense of its tautomeric forms by the binding of G_t or G_t-derived peptides. This stabilization of MII is the basis of the "extra-MII" assay (34, 35). This assay, however, can only be applied under conditions of a dynamic equilibrium between metarhodopsin I and MII, which is exquisitely sensitive to membrane environment, pH, temperature, ionic strength, etc. Therefore, an assay was developed that measures the kinetics of photoconversion of MII to rhodopsin in detergent solution (20-22). The assay uses the fact that photoconversion of MII (_max = 380 nm) to rhodopsin (_max = 500 nm) following a blue actinic flash is inhibited if the MII molecule is bound to G_t (20), or certain G_t-derived peptides (21, 22) as a result of the coupling between the conformation of the cytoplasmic surface and that of the chromophore-binding pocket.

The initial step in photoregeneration, the photoisomerization of the retinal to its cis conformation, may be compared with loading a spring that subsequently drives the protein, including its cytoplasmic domain, back to the ground state conformation (20). The product formed in this initial step, termed "reverted meta (RM)," is characterized by a MII-like protein conformation and a cis-retinal with a deprotonated Schiff base; it is spectrally indistinguishable from MII. RM rapidly converts to a rhodopsin-like species characterized by a rhodopsin-like protein conformation and a cis-retinal with a protonated Schiff base. The presence of G_t does not affect RM formation, indicating that the isomerization of the retinal itself is unaffected. However, bound G_t prevents RM from converting to rhodopsin, by stabilizing the MII-like conformation of RM. Dissociation of G_t from RM by GTPS treatment allows RM to revert to rhodopsin quantitatively (20). The effects, and presumably the mechanism of action, of certain G_t-derived peptides on photoregeneration are similar to those of G_t itself (22).

Photoregeneration Is Sensitive to Interactions with G_t and Certain G_t-derived Peptides-- G_t interacts with R* to stabilize the active signaling state such that photoregeneration to rhodopsin is effectively blocked (20). Recently, a peptide corresponding to the carboxyl terminus of G_t and a peptide analogue related to the carboxyl terminus of G_t were demonstrated to mimic the effect of G_t by inhibiting photoregeneration of R* (22). Here we showed that synthetic peptide (340-350) could cause the same effect as G_t (Fig. 2). In addition, the effect of (340-350) was specific to its primary structure since a mutant peptide had no effect (Fig. 3). Synthetic peptide (50-71)-far also inhibited photoregeneration of R* (Fig. 2). The effect was specific to its primary structure and to the presence of cysteinyl thioether farnesylation (Fig. 3). Using single peptides that represent small regions of G_t provides a powerful probe of subunit- and domain-specific interactions.

The potencies of the (340-350) and (50-71)-far peptides are about 20- and 100-fold less than that of G_t, respectively (Fig. 2). This finding is reasonable considering that the tertiary structure of a short peptide is less defined, so that a higher binding energy, and thus concentration, is needed for the "induced fit." Also, the cytoplasmic surface domain of R* comprises multiple interaction sites for G_t binding, including the loops C2 and C3 (15, 16, 36). G_t also has at least two, and probably more, sites that interact with the receptor during binding and activation. Peptide (340-350) showed a clear inhibition of photoregeneration with an almost complete suppression at saturating concentrations (Fig. 2B). The peptide (50-71)-far showed a lower efficacy to inhibit photoregeneration. Although the inhibitory effect saturated at high concentrations with a normal first-order binding isotherm, there was not a complete suppression of the photoregeneration effect (Fig. 2C). The binding of (50-71)-far to R* is likely to be quite complex due to specificity which arises from both the farnesyl moiety and the peptide sequence (Fig. 3). The carboxyl-terminal region of G_t was also studied using a MII difference spectroscopy assay with similar findings (30, 31).

The Role of a Conserved Region at the Amino Terminus of Loop C4 of Rhodopsin in G_t Binding-- We used the photoregeneration assay to probe the effects of G_t and G_t-derived peptides on expressed rhodopsin and rhodopsin mutants. The rhodopsin loop C4 mutations did not significantly affect the signal transmission path itself, as is seen from the similar kinetics of the photoregeneration signals in the absence of G_t and G_t-derived peptides (Fig. 4). The results in Figs. 2 and 3 show that G_t, and peptides (340-350) and (50-71)-far are specific probes of rhodopsin signaling. Therefore, inhibition of photoregeneration by G_t or G_t-derived peptides is interpreted as the specific interaction of these reagents with the intracellular surface. A lack of inhibition due to alteration of either the peptide or the C4 loop is interpreted as a disruption of interaction. An advantage of the photoregeneration assay is that only productive binding interactions that stabilize specific conformations of the protein are reported.

In theory, a particular mutation might have the effect of uncoupling the conformation of the cytoplasmic surface from that of the chromophore binding pocket. For example, an E134Q mutant has been shown to assume a partially activated conformation at the cytoplasmic surface while the chromophore and surrounding structures remain in the dark, inactive conformation (12). This type of mutant might give misleading results, as photoregeneration (monitored by structural rearrangements surrounding the chromophore) could proceed unhindered, even as G_t or peptides bound normally to the cytoplasmic surface. The rhodopsin loop C4 mutants showed no evidence of any uncoupling between the chromophore-binding pocket and cytoplasmic surface conformations. All of the mutants showed similar photoregeneration kinetics in the absence of peptide (Fig. 4, black traces). This suggests that the effects of the mutations are localized to the cytoplasmic surface, and do not affect the photoregeneration process itself. However, the mutant E134R/R135E photoregenerated with kinetics that were distinctly different from that of rhodopsin (data not shown), and therefore was not considered further in this study. Of course, mutants could exist that would foil all assays that rely on the detection of binding events at the intracellular surface resulting from conformational changes that are induced elsewhere in the protein. The extra-MII assay commonly used for rhodopsin, and the GTP-induced agonist affinity shift assay extensively used for other G protein-coupled receptors have the same potential limitations and are much less sensitive and specific.

Taken together, the results in the preceding paper (14) and the biophysical analysis of selected rhodopsin mutants herein strongly suggest that the amino terminus of C4 plays an important role in G_t binding and activation. Both G_t and (340-350) binding are disrupted when a tripeptide in this region is replaced by a sequence from the ₂-AR (Fig. 5). The most straightforward interpretation of the data is that the amino-terminal region of loop C4 directly influences or is part of a direct binding site for the carboxyl-terminal tail of G_t.

It is surprising that CTr4, in which residues 310-312 are replaced with analogous sequence from the ₂-AR, binds neither (340-350) nor (50-71)-far. Can the two peptides bind to overlapping sites on the receptor, each of which includes the 310-312 region? This seems unlikely, because the carboxyl termini of G_t and G_t are located at a significant distance apart from each other in the structure of the heterotrimer (19). Two potential explanations, which are not mutually exclusive, arise: 1) the peptides bind to different sites on the receptor and the sites are allosterically coupled; 2) G_t undergoes a large conformational change on contact with R* to bring the carboxyl termini of G_t and G_t into close proximity with the interaction domain near residues 310-312. A conformational switch in G_t, induced by the contact with R*, is an element of the "sequential fit" model (22), and it may be identical to the switch in G_t that was suggested earlier (31).

Possible Role of G_t-farnesyl in Docking of G_t to the Active Receptor-- The relevance of the data to the binding site of G_t is more subtle. The observation that CTr2, but not CTr4, binds (50-71)-far highlights the complexity of the binding interaction between (50-71)-far and rhodopsin. Further evidence of this complexity, as noted above, is that (50-71)-far fails to fully inhibit the photoregeneration reaction, even at saturating concentrations. This behavior contrasts with that of (340-350) (Fig. 2). In addition, both the farnesyl moiety and the peptide itself are required for binding to R* (Fig. 3). Each is likely to have a distinct binding site that may be differently altered in the mutants studied. The binding of (50-71)-far to CTr2, but not CTr4, suggests that the structural integrity of the fourth loop is disrupted by substitution of 310-312 with ₂-AR sequence, but that the substitution of the entire loop restores the structural determinants required for (50-71)-far binding. In this scenario, the tertiary, but not necessarily the primary structure of C4 would be critical for (50-71)-far binding. We have recently provided direct evidence, based on monolayer expansion measurements, that both the farnesylated carboxyl terminus of G_t and the myristoylated amino terminus of G_t are involved in the membrane interaction of G_t (37). G_t-far plays a role in both membrane and receptor interactions of G_t. We hypothesize that opening of the fourth loop structure could guide the farnesylated carboxyl terminus of G_t toward the receptor, thus ensuring the docking of G_t. By definition, such a process proceeds through predominantly hydrophobic interactions and avoids the need for G_t to "jump" onto the receptor via a transiently soluble intermediate state, which would slow the catalytic interaction (38, 39). Assuming a fundamentally similar G protein activation mechanism for rhodopsin and the ₂-AR, it is conceivable that the intact tertiary fourth loop structure in the CTr2 mutant has the capability to form a docking site for the -peptide from G_t.

In summary, we developed a novel biophysical assay to probe the G_t-binding domain of rhodopsin and expressed rhodopsin. G_t and peptides corresponding to the carboxyl-terminal regions of G_t and G_t specifically bind to R* and stabilize the active state of the receptor. We conclude that the amino-terminal region of loop C4 acts as part of the binding site for G_t and modulates the G_t-binding domain of R*. Future work is underway to reconcile the various models for allosteric regulation of the G_t-binding surface of R*, especially concerning the binding of G_t. This work will require the use of additional rhodopsin mutants and expressed G protein subunits.

	ACKNOWLEDGEMENTS

We thank Oleg Kisselev for helpful discussions and Cliff Sonnenbrot for oligonucleotide synthesis.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Training Grant GM07982 and Medical Scientist Training Program Grant GM07739 (to E. P. M.), Deutsche Forschungsgemeinschaft Grant Sfb 449 (to K. P. H. and O. P. E.), and the Fonds der Chemischen Industrie (to K. P. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Contributed equally to the results of this study.

Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence may be addressed: Box 284, Rockefeller University, 1230 York Ave., New York, New York, 10021. Tel.: 212-327-8288; Fax: 212-327-7904; E-mail: sakmar@rockvax.rockefeller.edu.

^§§ To whom correspondence may be addressed: Institut für Medizinische Physik und Biophysik, Charité, Medizinische Fakultät der Humboldt Universität zu Berlin, Ziegelstr. 5-9, 10117 Berlin, Germany, Tel.: 030-2802-6141; Fax: 030-2802-6377; E-mail: klaus_peter.hofmann@charite.de.

	ABBREVIATIONS

The abbreviations used are: G_t, transducin; AR, adrenergic receptor; C1, first cytoplasmic loop of rhodopsin; C2, second cytoplasmic loop of rhodopsin; C3, third cytoplasmic loop of rhodopsin; C4, fourth cytoplasmic loop of rhodopsin; DM, dodecyl--D-maltoside; G_t, subunit of transducin; G_t, heterodimer subunit of transducin; G_t, subunit of transducin; MII, metarhodopsin II; R*, active conformation of rhodopsin; RM, reverted metarhodopsin; TM, transmembrane.

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