Structural and Functional Role of Helices I and II in Rhodopsin
A NOVEL INTERPLAY EVIDENCED BY MUTATIONS AT GLY-51 AND GLY-89 IN THE TRANSMEMBRANE DOMAIN*
Laia Bosch,
Eva Ramon,
Luis J. Del Valle and
Pere Garriga
From the
Centre de Biotecnologia Molecular, Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, Colom 1, 08222 Terrassa, Catalonia, Spain
Received for publication, February 6, 2003
, and in revised form, March 24, 2003.
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ABSTRACT
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The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.
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INTRODUCTION
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Rhodopsin is the photoreceptor protein in rod cells of the vertebrate retina that serves as signal transducer from a photon of light to a neural response (1, 2, 3). The role of rhodopsin in visual phototransduction is central to the complex process of vision (4). Rhodopsin is an integral membrane protein that belongs to the large superfamily of G-protein-coupled receptors (GPCR),1 characterized by a seven-transmembrane helical motif (5, 6, 7). Because of the structural similarity among its members, rhodopsin has been used as a model for the GPCR superfamily. This visual receptor, however, has unique structural features, such as having its ligand (11-cis-retinal) covalently bound through a protonated Schiff base linkage to Lys-296 in the seventh helical segment of the protein. Retinal isomerization, upon light absorption, generates a signal in the helical cluster that is transmitted to the cytoplasmic domain where the interaction with the different proteins of the phototransduction cascade takes place. A recent landmark in rhodopsin studies has been the report of its crystal structure (8, 9, 10). Although important information on structural properties of the protein has been derived from the crystal structure (11, 12, 13), the details of the conformational rearrangements after retinal photoisomerization are far from being completely understood. Recently, the presence of rhodopsin in native disk membranes organized as rows of dimers has been reported (14). This specific arrangement would be responsible for the high efficiency in light absorption by retinal photoreceptors. Dimerization is thought to be a universal phenomenon for modulation of receptor function in GPCR (15).
Retinitis pigmentosa (RP) is a group of inherited degenerative retinopathies genetically and clinically heterogeneous (16, 17). In the past decade, more than 150 mutations have been discovered in the opsin gene associated with autosomal dominant retinitis pigmentosa (ADRP). These are mainly point mutations and a few deletions. Mutations associated with ADRP are found spread all over the opsin gene in the three domains of the receptor: intradiscal, transmembrane, and cytoplasmic.
We started a detailed characterization of ADRP mutants in the transmembrane region of rhodopsin with a 2-fold interest, (i) elucidation of the molecular mechanism of the disease and (ii) structural and functional information, from the study of these mutations that might be relevant to other members of the GPCR superfamily. The recent crystal structure together with the available biochemical data on rhodopsin mutants has shed more light into the molecular mechanism of RP associated with rhodopsin mutations (18, 19). Previous studies show that conservative mutations in helix III at Leu-125 (site of the ADRP mutation L125R) led to misfolding, and it was proposed that the folding of the intradiscal and transmembrane domains are coupled (20, 21, 22). These studies were further extended to dissect the effect of mutations at Leu-125 in the folding and function of rhodopsin (23).
G51V and G89D naturally occurring ADRP mutations were first reported in 1991 (24, 25). The G51A mutation was reported in 1993 (26). A preliminary characterization of G51V (27) and G89D (28) mutants together with other ADRP mutations was carried out in 293S cell membranes. In another study these mutants were expressed in COS-1 cells and purified (29). Later, the mutants G51A, G51V, and G89D were studied in the context of the folding and packing of the transmembrane domain together with ADRP mutations in the other helices of rhodopsin (22). These studies showed that G51V forms chromophore like wild-type rhodopsin (WT), whereas G89D could only form it partially (22). We took these previous studies as a starting point for a detailed characterization of the helical environment of Gly-51 and Gly-89 in transmembrane helices I and II of rhodopsin by analyzing a series of mutants at these positions. In particular, one of the questions we wanted to answer is what is the biochemical defect associated with Type I mutations (like G51V) which allow the protein to fold but still cause the disease. It was stated that the nature of this defect remained enigmatic (27).
Helices I and II have not received much attention in structural and functional studies because it was thought that they were not involved as major players in the process of rhodopsin activation. It seems clear, however, from the high degree of specificity in helix-helix interactions in rhodopsin that the wealth of information derived from mutagenesis studies is unraveling, making it necessary to address in detail the structural and functional effects of mutating amino acid residues of these helices.
In the present work, a series of mutations at the sites of the mutants associated with ADRP, G51A, G51V (helix I), and G89D (helix II) have been constructed and characterized (Fig. 1; Table I). We find that mutations at position 51 are essentially affecting the optimal packing of the helices, with a clear destabilizing effect due to the size of the amino acid substitution. Furthermore, the G51V mutant is defective in signal transduction as a result of an unstable Meta II conformation. This suggests that mutations classified as Type I can be defective in dark state stability and in signal transduction. Mutations at position 89 are, in turn, more affected by the charge of the ionizable residues introduced in the transmembrane domain. The G89D mutant is also defective in transducin (Gt) activation. Altered photointermediate formation, including faster metarhodopsin II (Meta II) decay and increased formation of other non-functional conformations of rhodopsin, like metarhodopsin III (Meta III), is proposed to be physiologically relevant for the mutations associated with ADRP. Overall our results provide evidence for the presence of specific interhelical interactions, in the region delimited by helices I, II and VII in the transmembrane domain of rhodopsin, that are important for the stability and the function of the receptor.

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FIG. 1. A secondary structure model of bovine rhodopsin. The amino acids substituted in this work are circled, and the mutations studied are indicated. Cys-110 and Cys-187 form a disulfide bond (indicated by the dashed line), and Cys-322 and Cys-323 are palmitoylated. Horizontal lines approximately define the borders of the seven membrane-embedded helical segments (indicated by IVII).
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EXPERIMENTAL PROCEDURES
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MaterialsBovine retinas were from J. A. Lawson Co. (Lincoln, NE). GTP
S was purchased from Roche Molecular Biochemicals. 11-cis-retinal was a gift of Prof. P.P. Philippov (University of Moscow). Dodecyl maltoside (DM) was from Anatrace. Cyanogen bromide-activated Sepharose 4B was from Sigma. Anti-rhodopsin monoclonal antibody rho-1D4 was purified from myeloma cell lines (30) and coupled to cyanogen bromide-activated Sepharose 4B as described (31).
Construction of Opsin MutantsMutations were introduced into the synthetic bovine opsin gene (32) by replacement of a BclI-HindIII restriction fragment by synthetic DNA duplexes containing the required codon changes in the case of the mutants at position 51. For the mutants at position 89 the restriction fragment replaced was BglII-NcoI. The mutant genes were cloned in the pMT4 vector (33) as described (20, 21, 22). Mutations at position 89 were carried out using a pSK vector (29) derived from the vector pCMV5 (34). The correct sequences of the mutations introduced was confirmed by the dideoxy chain-terminated method.
Expression and Purification of WT and Mutant RhodopsinsWT and mutant opsin genes were expressed in transiently transfected monkey kidney cells (COS-1) as described (35). After the addition of 5 µM 11-cis-retinal in the dark, the transfected COS-1 cells were solubilized in 1% DM, and the proteins were purified by immunoaffinity chromatography. The purification procedure was carried out as described in detail (21). The expressed proteins were treated to obtain the correctly folded (11-cis-retinal binding) fractions. This was carried out by differential elution from the anti-rhodopsin 1D4-Sepharose resin essentially as previously described (20, 21, 22, 36). The folded fractions of these mutant rhodopsins were the ones used throughout the present study. Purity of the immunopurified rhodopsins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining.
UV-Visible Absorption Spectra of WT and Mutant RhodopsinUV-visible spectra were performed with a PerkinElmer Life Sciences
-7 spectrophotometer equipped with water-jacketed cuvette holders connected to a circulating water bath. All spectra were recorded with a bandwidth of 2 nm, a response time of 1 s, and a scan speed of 240 nm/min at 20 °C. The molar extinction coefficients were determined as described (37). For photobleaching experiments samples were illuminated with a 150-watt fiber optic light (Fiber Lite A-200; Dolan-Jenner, Woburn, MA) equipped with a >495-nm long-pass filter for 10 s, and the corresponding bleached spectrum was recorded immediately after illumination. In the case of mutant G51L, which showed abnormal bleaching, illumination for different lengths of time was carried out after the initial 10-s illumination.
Rate of Meta II Decay as Measured by Retinal ReleaseThe rate of retinal release, which parallels the Meta II decay of the protein in the case of WT under the conditions used, was studied using fluorescence spectroscopy essentially as described (38). Typically 2 µg of pigment in a volume of 200 µl of 2 mM NaH2PO4, pH 6.0, 0.05% DM (buffer A) was used. For the assay the excitation and emission wavelengths were 295 nm (slit, 0.25 nm) and 330 nm (slit, 12 nm), respectively. The samples were bleached for 30 s, and the fluorescence increase was measured. The assay was also performed in parallel using the same conditions except that the protein was measured in the Gt activation buffer (10 mM Tris-HCl, pH 7.1, 100 mM NaCl, 2 mM MgCl2, and 0.012% DM (Gt assay buffer)) because the assay is known to be sensitive to DM concentration and to pH. This was done to better correlate the Meta II decay data with the Gt activation results. Spectra were recorded at 20 °C. Spectra obtained were normalized and fit to single or double exponential functions using SigmaPlot (Jandel Scientific).
Gt Activation AssayGt was prepared from bovine rod outer segments as described (39). Gt activation was measured by means of fluorescence spectroscopy. The fluorescence technique has been applied to the study of rhodopsin mutants (40, 41, 42). The excitation and emission wavelengths were 295 nm (slit, 2 nm) and 340 nm (slit, 12 nm), respectively. Samples were continuously stirred and maintained at 20 °C. For the assay, rhodopsin (WT or mutants) was photobleached for 30 s with light >495 nm and added to a final concentration of 5 nM to a 400 nM solution of Gt concentration of 5 nM to a 400 nM solution of Gt in Gt assay buffer. After 5 min, GTP
S was added to a final concentration of 5 µM (at t = 0). The relative initial rates of Gt activation were obtained from the slope of the fluorescence increase in the first 60 s after GTP
S addition and normalized to the value of a WT sample used as a control.
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RESULTS
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Expression and Characterization of Mutations at Position 51 in Helix IMutants G51A and G51V, associated with ADRP, and the synthetic mutant G51L, expressed in transiently transfected COS-1 cells and purified by immunoaffinity chromatography with rho-1D4 antibody, showed an expression level similar to that of WT. The SDS-PAGE pattern for these mutants was comparable with that of WT, showing a main opsin band at around 40 kDa and the absence of lower bands (not shown), usually associated with misfolded proteins (20, 21, 22, 36, 43). The reconstituted mutant rhodopsins eluted in buffer A formed normal chromophore and UV-visible absorption spectra like WT (Fig. 2), with A280/A500 ratios of 1.61.7. The absorption spectra for the mutants showed small wavelength shifts in their corresponding visible absorption maxima. G51V had an absorbance maximum slightly red-shifted to 502 nm, whereas G51L had it blue-shifted to 497 nm (Table II). Although the purified mutant proteins folded correctly to a conformation that was able to bind 11-cis-retinal-like WT, thermal stability of the mutants in the dark was severely reduced (Table III). Furthermore, the secondary structure of the proteins was also less stable as measured by circular dichroism (data not shown).

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FIG. 2. UV-visible absorption spectra of WT and mutants at position 51 and 89. Mutant genes corresponding to WT and mutant opsins were expressed in transiently transfected COS-1 cells, reconstituted with 11-cis-retinal, solubilized in DM, and immunopurified. The purified proteins were in buffer A. Spectra were recorded in the dark at 20 °C. A, WT from COS-1 cells; B, G51A (ADRP) mutant; C, G51V (ADRP) mutant; D, I, G51L mutant; D, II, G51L-photobleaching behavior. The sample was illuminated for different times with light of > 495 nm. 1, non-illuminated dark sample; 2, 10 s; 3, 30 s; 4, 1 min. E, G89A; F, G89L mutant; G, G89F mutant; H, G89D (ADRP) mutant; I, G89K mutant.
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The photobleaching behavior of G51A mutant was normal, like that of the WT (data not shown), but that of G51L was clearly altered (Fig. 2, panel D, II). After 10 s of illumination with light >495 nm, in the case of mutant G51L, the 497-nm species was not fully converted to the 380-nm species. Instead, two bands of similar intensity were detected, one at 380 nm and the other one at about 470 nm (trace 2 in Panel D, II, Fig. 2). Further illumination for 30 s or 1 min (traces 3 and 4 in Panel D, II, Fig. 2) only caused a slight decrease of the 470-nm band with a concomitant small increase in the 380-nm band. A very similar behavior was previously seen for G51V (22).
Meta II decay, as measured by fluorescence spectroscopy, was faster with increasing size of the amino acid side chain at position 51 (Fig. 3). Thus, G51A and G51V decay curves could be fit to single exponential functions with t
of 23.2 and 15.7 min, respectively (Table IV). G51L decay, however, could not be fit to a single exponential but to a double exponential function with a very fast component (t
, 0.7 min) and a slow component (t
, 19.8 min). Meta II decay was also measured for the rhodopsin mutant samples in Gt assay buffer (Table IV). Under these experimental conditions, t
for the decay of G51A was now slightly faster, 18.7 min as compared with 23.2 min in buffer A. However, the decay for the G51V mutant could no longer be fit to a single exponential, and it had to be fit to a double exponential function, with two components, one very fast (t
, 1.9 min) and the other slow (t
, 30.2 min). G51L did not show any significant change in the Meta II decay values obtained in the Gt assay buffer.

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FIG. 3. Fluorescence Meta II decay of the WT and the Gly-51 mutants. The fluorescence Meta II decay assays were carried out in buffer A as described under "Experimental Procedures." All data are the average of two different experiments.
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Regarding the ability of these mutants to function normally in signal transduction, G51A could activate Gt to essentially the same level as WT. Mutant G51V, however, showed a very reduced level of Gt activation, which was dependent on the time after bleaching when the protein was added to the Gt reaction mixture (Fig. 4). When the G51V mutant was added to the reaction mixture immediately after bleaching (time 0 in Fig. 4), the maximum activation corresponded to an initial rate about 0.2 that of WT (maximal Gt activation for this mutant, 100% in Fig. 4). When the bleached G51V protein was added after several minutes, the initial rate of the Gt activation was reduced, and after 12 min it was essentially abolished. The curve obtained in Fig. 4 has a t
of about 2 min, which correlates nicely with the t
of the fast component of the fluorescence Meta II decay in Gt assay buffer. The G51L mutant did not show any significant degree of activation under any experimental conditions, even upon addition of the protein immediately after bleaching.

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FIG. 4. Gt activation of G51V mutant at different times after bleaching. Gt initial rates were plotted for reactions started at different times after bleaching of the mutant protein sample. All data are the average of two different experiments. Temperature, 20 °C.
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Expression and Characterization of Mutations at Position 89 in Helix IISeveral mutants at position 89, the site of the naturally occurring G89D mutation, were constructed, expressed, and characterized. The mutants studied were G89A, G89L, G89F, G89K, and G89D. The mutant proteins were expressed at similar levels to that of WT in the case of the hydrophobic replacements G89A, G89L, and G89F. Replacements where an ionizable, presumably charged side chain was introduced (G89K and G89D) were expressed at a much lower level, about one-fifth that of the WT. The electrophoretic pattern for the correctly folded fraction of these mutants was WT-like, with the main opsin band at 40 kDa and the characteristic trailing smear (not shown).
UV-visible absorption spectra of the mutant proteins eluted in buffer A showed A280/A500 ratios of 1.61.8 (Fig. 2). It should be noted that G89A, G89L, and G89F could form chromophore to a similar extent as WT upon elution in phosphate-buffered saline, pH 7.2. The substitutions involving an ionizable amino acid side chain had significantly higher A280/A500 ratios, in the 2.52.8 range, indicating that they were eluted as mixtures of folded and misfolded proteins. The separated folded fractions showed A280/A500 ratios of 1.71.8 (Fig. 2). UV-visible absorption spectrum in the dark, corresponding to the G89K mutant, showed a clear definite UV-absorbing band centered at 350 nm in addition to the normal chromophoric band at 500 nm. All the other mutants showed a WT-like band in the visible region at 500 nm, and no wavelength shifts were detected.
The Meta II decay of G89A, G89L, G89F, and G89K was slightly faster than the WT in normal assay conditions. In the Gt assay buffer the decays for these mutants were only slightly faster when compared with the values obtained in buffer A. G89D showed a different behavior in this assay. Its decay in buffer A was slow with a t
of 29.1 min (single exponential fit); but in Gt assay buffer the fluorescence curve was best fit to a double exponential function, with a fast component (t
, 2.9 min) and a slow component (t
, 38.5 min) (Table III). This behavior, specific to the ADRP G89D mutant, was not seen in any other of the mutants at position 89.
Gt activation of mutant G89A was similar to that of WT. G89F showed a higher initial rate of Gt activation, whereas G89L had about 30% reduction in the rate. The two mutants that showed lower Gt activation were G89K and G89D. In particular, the G89D mutant rhodopsin, associated with ADRP, showed more than a 50% reduction in the initial Gt activation rate (Table IV).
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DISCUSSION
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Mutations at Position 51 in Helix IGly-51 in the transmembrane helix I of rhodopsin is part of a GXXXN motif conserved in the GPCR superfamily. The naturally occurring mutations G51A and G51V are found in ADRP patients. It was reported that conservative mutations in the transmembrane region of rhodopsin cause misfolding of the mutant proteins (21). However, in those cases that the mutant proteins are able to fold to form the correct retinal binding pocket, it is intriguing as to why they are still deleterious. We constructed and expressed the synthetic G51L mutant to explore the effect of the size of the amino acid side chain in the packing of the helices and in signal transduction.
The correctly folded fractions of the mutant rhodopsins were separated and characterized. The folded fractions of the three mutants gave UV-visible absorption spectra with A280/A500 ratios of 1.61.7. The SDS-PAGE pattern was like that for the WT protein with no lower bands, characteristic of mutants with altered phenotype. Although these features would point to mutants having a similar phenotype to that of the WT, other parameters studied indicate that these mutant proteins are destabilized, both thermally, in the dark, and also after photo-activation, when compared with WT. The mutant proteins in the dark are less stable than WT, with the stability decreasing with increasing size of the side chain. A number of factors are shown to affect the thermal stability of dark-state rhodopsin, including retinal disease-causing mutations (44) or zinc binding (45). Other factors mediating the stability of dark-state rhodopsin are proposed, like a conserved ion-pair interaction in the intradiscal loop E-2 of rhodopsin (46). A destabilizing effect is also observed for the active intermediate Meta II of the G51 mutants formed upon photobleaching. The decrease in the ability of the mutants to activate Gt can also be correlated with the increase in size of the side chain at position 51 and point to a disruption of the interhelical packing due to the mutations. The fact that an increase in one or two carbon atoms makes such a significant difference indicates a tight steric coupling with another amino acid side chain. From the reported crystal structure (8), this interaction is probably with Val-300 in transmembrane helix VII (Fig. 5A). The substitution of glycine at position 51 by the bulkier valine and leucine side chains would result in steric clash with the Val-300 side chain (Fig. 5, B and C) and perturbation of the local helical environment of Pro-303 (involved in the pronounced kink of helix VII) included in the conserved NPXXY motif of the GPCR superfamily. This motif or amino acids within it are thought to be important in receptor signaling (47, 48, 49, 50, 51). For example, interaction between Met-257 in helix VI and this motif has been suggested in the case of rhodopsin from a site-directed mutagenesis study (52). It is noted that for G51V there is a relationship between the time after bleaching and the ability of the mutant protein to activate Gt. Mutation to alanine in the G51A mutant can be tolerated, but G51V and G51L show a clearly altered behavior upon illumination and are clearly defective in signal transduction. The impaired ability of these mutants to activate Gt is, thus, correlated with the decreased stability of the active conformation Meta II. That was also the case in the C110A/C187A double mutant that could form chromophore but had a destabilized Meta II species (53).

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FIG. 5. Model of helices I, II, and VII of rhodopsin. Model in the region of interest around G51 showing helices I, II, and VII. This model is based on the coordinates from 1L9H
[PDB]
(10). A, model for the WT with native G51. The proximity of G51 in helix I and Val-300 in helix VII can be observed. The proximity of D83 in helix II to Val-300 (coming from behind) is also shown. This model was generated with the program DS ViewerPro (Accelrys Inc.). B, model for mutant G51A with the introduced alanine coming closer to Val-300 in helix VII. C, model for mutant G51V (ADRP). Note the increased contact between Val-51 and Val-300. The models in B and C were prepared using Insight II (Accelrys Inc.). In these models the eighth cytoplasmic helix after the region of Val-300 in helix VII is shown. This helix is known to be important in Gt activation. Mutation at Gly-51 would perturb interaction with Val-300 in helix 7, and this change could be transmitted to the cytoplasmic helix VIII, thus affecting Gt activation.
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Fluorescence Meta II decay results can be best fit to double exponential functions, reflecting a fast component and a slow component (Table IV). These double exponential curves could be explained by assuming that two different species with different kinetics are formed. Thus, in the case of the mutants reported, the fluorescence increase observed could be interpreted as reflecting the direct decay of Meta II to free all-trans-retinal and formation of a non-active rhodopsin conformation that could be the Meta III species (54). The fast Meta II decay correlates with the Gt activation values obtained for mutants G51V and G51L (Table IV). The decrease in the Gt activation rate at different times after bleaching obtained for mutant G51V has a t
of about 2 min for the process, which correlates with the t
for the fast component of the fluorescence decay curve (in Gt assay buffer), proposed to reflect decay to opsin and all-trans-retinal.
Mutations at Position 89 in Helix IIIn this case the size of the side chain does not seem to be as important as the charge of the introduced side chain. Of all the mutants at this position, only the G89D mutant rhodopsin had abnormal photobleaching (22). This mutant also showed a slow rate for retinal release, in agreement with previous studies (22). However the G89D mutant in Gt assay buffer showed the biphasic behavior observed for the G51V and G51L mutants. Gly-89 is next to Gly-90 in transmembrane helix II. The G90D mutant is associated with the retinal disease congenital night blindness, and the aspartate in the mutant would be in the retinal binding pocket next to the Schiff base (55). In the same region, another mutation related to congenital night blindness, T94I, is reported to have very unusual thermal and conformational properties (44). Gly-89 forms a cavity together with glycine 90, and a water molecule is located very close to the carbonyl group of Gly-89 at hydrogen-bonding distance (10). A functional role for the water molecules associated to rhodopsin has been recently proposed (10). In this regard, the Gly-89 to Lys mutation, which also decreases the rate of Gt activation, may disrupt the Gly-89-water interaction and perturb a fine electrostatic network in the vicinity of the retinal Schiff base. In the case of G89K, this mutant showed a UV-absorbing species at about 350 nm in the absorption spectrum in the dark, which was not previously observed in any other mutant studied so far. This species corresponds to a Schiff base-linked chromophore because it shifts to 440 nm upon acidification of the sample (not shown). Interestingly, Drosophila UV pigments have lysine at a homologous position in the Gly-89/Gly-90 region in human rhodopsin (56, 57). A lysine in the second transmembrane helix of the invertebrate UV pigments was proposed to affect wavelength regulation via a charge interaction (58).
For the other mutations, G89A shows similar Gt activation to the WT. However, G89F and G89L show opposite effects; G89F slightly increases the activity, whereas G89L decreases it. Because phenylalanine is a bulky residue, the observed difference in activation may be related to the stereochemistry of the side chains, planar for Phe versus tetrahedral for Leu rather than to the actual molar volume of the corresponding side chains.
Structural Consequences of Mutations at Gly-51 and Gly-89We have shown that position 51 in helix I of rhodopsin is very sensitive to the size of the introduced side chain. The G51V mutant associated with ADRP is defective in signal transduction and has an unstable Meta II active conformation. For mutations at position 89 (located closer to the retinal Schiff base), although some role can be assigned to the size of the side chain, the results obtained for G89D and G89K suggest that charge effect is more important for mutations at this position. The other mutation associated with ADRP, G89D, is also shown to be defective in signal transduction. Transmembrane helix II was not previously implicated in conformational rearrangements (involving helix movements) during the Gt activation process. However, very recently a structural change in helix II of the angiotensin II type 1 receptor has been reported (59). Furthermore, the interaction between helix II and helix VII has also been postulated (60). In rhodopsin, the highly conserved Asp-83 in helix II is close to Val-300 in helix VII and may be hydrogen-bonded to the amide nitrogen of this amino acid through a water molecule (10). Mutations at position 89, although in a region closer to the retinal Schiff base than to the Gly-51 region, could cause a helical rearrangement that would result in changes at Asp-83 in the same helix, disrupting the interaction with Val-300 in helix VII. A model showing the proximity of Gly-51 in helix I, Val-300 in helix VII, and Asp-83 in helix II is shown in Fig. 5. Our results indicate that helix I and helix II are important in the stability and function of rhodopsin and suggest a novel interplay between these helices and helix VII in the structure and in the conformational rearrangements ensuing photoactivation of rhodopsin.
Physiological Relevance with Regard to RPMutations G51A, G51V, and G89D have been found in RP patients and cause retinal degeneration. Because of extreme genetic and clinical heterogeneity of RP (17) it is difficult to correlate the molecular defect caused by a given mutation in rhodopsin and the severity of RP. Despite this heterogeneity, an effort was made to correlate the disease progression in patients with ADRP and rhodopsin mutations (61). In this study, clinical features regarding patients with G51V and G89D mutations are presented (61). These data indicate that G51V mutation results in a more benign clinical phenotype than G89D. G89D is especially defective in electroretinogram amplitude (indicating significant loss of retinal function). Our biochemical and spectroscopic data show that these two mutants show altered photobleaching and altered functionality. The two mutants show biphasic retinal release curves with a significant amount of the slow component being formed that may be interpreted as reflecting formation of non-active species. Interestingly, mutant G89D, which shows a more severe clinical phenotype than G51V (61), shows a higher amount of this slow species formed (82% for the former compared with 61% for the latter). In the case of mutants G51A and G89D, these were reported to be partially misfolded (22). However, in the study of the folded fraction of the G51A mutant reported here the only difference observed with regard to the WT properties is its reduced thermal stability in the dark. Unfortunately no clinical data are available for this mutant. The results obtained for G51V and G89D mutants associated with ARDP suggest that in addition to a structural effect, reduced stability and subsequent failure in Gt activation can also have implications in RP caused by mutations in rhodopsin.
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FOOTNOTES
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* This work has been supported by Dirección General de Enseñanza Superior e Investigación Científica Grant PM980134 and in part by grants from Fundación Lucha contra la Ceguera and Dirección General de la ONCE. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 34-93-7398044; Fax: 34-93-7398225; E-mail: pere.garriga{at}upc.es.
1 The abbreviations used are: GPCR, G-protein-coupled receptor; Meta II, metarhodopsin II; Meta III; metarhodopsin III; DM, n-dodecyl-
-D-maltoside; RP, retinitis pigmentosa; ADRP, autosomal dominant retinitis pigmentosa; Gt, transducin; WT, wild-type rhodopsin; GTP
S, guanosine 5'-3-O-(thio)triphosphate. 
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ACKNOWLEDGMENTS
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We thank Francesc Corcho for generating models B and C in Fig. 5. We also thank Xun Liu for initial communication of the thermal stability in the dark state of Gly-51 mutants.
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REFERENCES
|
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- Hargrave, P. A. (2001) Investig. Ophthalmol. Vis. Sci. 42, 3-9[Free Full Text]
- Sakmar, T. P., Menon, S. T., Marin, E. P., and Awad, E. S. (2002) Annu. Rev. Biophys. Biomol. Struct. 31, 443-484[CrossRef][Medline]
[Order article via Infotrieve]
- Arshavsky, V. Y., Lamb, T. D., and Pugh, E. N., Jr. (2001) Annu. Rev. Physiol. 64, 153-187[CrossRef]
- Burns, M. E., and Baylor, D. A. (2001) Annu. Rev. Neurosci. 24, 779-805[CrossRef][Medline]
[Order article via Infotrieve]
- Bockaert, J., and Pin, J. P. (1999) EMBO J. 18, 1723-1729[Abstract/Free Full Text]
- Wess, J. (1997) FASEB J. 11, 346-354[Abstract/Free Full Text]
- Marinissen, M. J., and Gutkind, J. S. (2001) Trends Pharmacol. Sci. 22, 368-376[CrossRef][Medline]
[Order article via Infotrieve]
- Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745[Abstract/Free Full Text]
- Teller, D. C., Okada, T., Behnke, C. A., Palczewski, K., and Stenkamp, R. E. (2001) Biochemistry 40, 7761-7772[CrossRef][Medline]
[Order article via Infotrieve]
- Okada, T., Fujiyoshi, Y., Silow, M., Navarro J., Landau, E. M., and Shichida, Y. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5982-5987[Abstract/Free Full Text]
- Okada, T., Ernst, O. P., Palczewski, K., and Hofmann, K. P. (2001) Trends Biochem. Sci. 26, 318-324[CrossRef][Medline]
[Order article via Infotrieve]
- Lu, Z. L., Saldanha, J. W., and Hulme, E. C. (2002) Trends Pharmacol. Sci. 23, 140-146[CrossRef][Medline]
[Order article via Infotrieve]
- Meng, E. C., and Bourne, H. R. (2001) Trends Pharmacol. Sci. 22, 587-593[CrossRef][Medline]
[Order article via Infotrieve]
- Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128[CrossRef][Medline]
[Order article via Infotrieve]
- Gomes, I., Jordan, B. A., Gupta, A., Rios, C., Trapaidze, N., and Devi, L. A. (2001) J. Mol. Med. 79, 226-242[CrossRef][Medline]
[Order article via Infotrieve]
- Berson, E. L. (1993) Investig. Ophthalmol. Vis. Sci. 34, 1659-1676[Medline]
[Order article via Infotrieve]
- Farrar, G. J., Kenna, P. F., and Humphries, P. (2002) EMBO J. 21, 857-864[Abstract/Free Full Text]
- Garriga, P., and Manyosa, J. (2002) FEBS Lett. 528, 17-22[CrossRef][Medline]
[Order article via Infotrieve]
- Stojanovic, A., and Hwa, J. (2002) Receptors Channels 8, 3-18[Medline]
[Order article via Infotrieve]
- Liu, X., Garriga, P., and Khorana, H. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4554-4559[Abstract/Free Full Text]
- Garriga, P., Liu, X., and Khorana, H. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4560-4564[Abstract/Free Full Text]
- Hwa, J., Garriga, P., Liu, X., and Khorana, H. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10571-10576[Abstract/Free Full Text]
- Andrés, A., Kosoy, A., Garriga, P., and Manyosa, J. (2001) Eur. J. Biochem. 268, 5696-5704[Abstract/Free Full Text]
- Dryja, T. P., Hahn, L. B., Cowley, G. S., McGee, T. L., and Berson, E. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9370-9374[Abstract]
- Sung, C.-H., Davenport, C. M., Hennessey, J. C., Maumenee, I. H., Jacobson, S. G., Heckenlively, J. R., Nowakowski, R., Fishman, G., Gouras, P., and Nathans, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6481-6485[Abstract]
- Macke, J. P., Davenport, C. M., Javcobson, S. G., Hennessey, J. C., Gonzalez-Fernandez, F., Conway, B. P., Keckenlively, J., Palmer, R., Maumenee, I. H., Sieving, P., Gouras, P., Good, W., and Nathans, J. (1993) Am. J. Hum. Genet. 53, 80-89[Medline]
[Order article via Infotrieve]
- Sung, C.-H., Davenport, C., and Nathans, J. (1993) J. Biol. Chem. 268, 26645-26649[Abstract/Free Full Text]
- Sung, C.-H., Schneider, B., Agawal, N., Papermaster, D. S., and Nathans, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8840-8844[Abstract]
- Kaushal, S., and Khorana, H. G. (1994) Biochemistry 33, 6121-6128[Medline]
[Order article via Infotrieve]
- Molday, R. S., and MacKenzie, D. (1983) Biochemistry 22, 653-660[Medline]
[Order article via Infotrieve]
- Hicks, D., and Molday, R. S. (1986) Exp. Eye Res. 42, 55-71[Medline]
[Order article via Infotrieve]
- Ferretti, L., Karnik, S. S., Khorana, H. G., Nassal, M., and Oprian, D. D. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 599-603[Abstract]
- Franke, R. R., Sakmar, T. P., Oprian, D. D., and Khorana, H. G. (1988) J. Biol. Chem. 263, 2119-2122[Abstract/Free Full Text]
- Andersson, S., Davis, D. N., Dhloback, H., Jornvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229[Abstract/Free Full Text]
- Oprian, D. D., Molday, R. S., Kaufman, R. J., and Khorana, H. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8874-8878[Abstract]
- Ridge, K. D., Lu, Z., Liu, X., and Khorana, H. G. (1995) Biochemistry 34, 3261-3267[Medline]
[Order article via Infotrieve]
- Sakmar, T. P., Franke, R. R., and Khorana, H. G. (1989) Proc. Natl. Acad. Sci. U. S. A. 88, 3079-3083
- Farrens, D. L., and Khorana, H. G. (1995) J. Biol. Chem. 270, 5073-5076[Abstract/Free Full Text]
- Ting, T. D., Goldin, S. B., and Ho, Y. K. (1994) in Methods in Neurosciences (Hargrave, P. A. (ed)) Vol. 15, pp. 180-195, Academic Press, Inc., San Diego, CA
- Fahmy, K., and Sakmar, T. P. (1993) Biochemistry 32, 7229-7236[Medline]
[Order article via Infotrieve]
- Jager, F., Jager, S., Krautle, O., Friedman, N., Sheves, M, Hoffman, K. P., and Siebert, F. (1994) Biochemistry 33, 7389-7397[Medline]
[Order article via Infotrieve]
- Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768-770[Abstract/Free Full Text]
- Doi, T., Molday, R. S., and Khorana, H. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4991-4995[Abstract]
- Ramon, E., del Valle, L. J., and Garriga, P. (2003) J. Biol. Chem. 278, 6427-6432[Abstract/Free Full Text]
- del Valle, L. J., Ramon, E., Cañavate, X., Dias, P., and Garriga, P. (2003) J. Biol. Chem. 278, 4719-4724[Abstract/Free Full Text]
- Janz, J. M., Fay, J. F., and Farrens, D. L. (2003) J. Biol. Chem. 278, 16982-16991[Abstract/Free Full Text]
- Gales, C, Kowalski-Chauvel, A., Dufour, M. N., Seva, C., Moroder, L., Pradayrol, L., Vaysse, N., Fourmy, D., and Silvente-Poirot, S. (2002) J. Biol. Chem. 275, 17321-17327[Abstract/Free Full Text]
- Gripentrog, J. M., Jesaitis, A. J., and Miettinen, H. M. (2000) Biochem. J. 352, 399-407[CrossRef][Medline]
[Order article via Infotrieve]
- Govaerts, C., Lefort, A., Costagliola, S., Wodak, S. J., Ballesteros, J. A., Van Sande, J., Pardo, L., and Vassart, G. (2001) J. Biol. Chem. 276, 22991-22999[Abstract/Free Full Text]
- Prioleau, C., Visiers, I., Ebersole, B. J., Weinstein, H., and Sealfon, S. C. (2002) J. Biol. Chem. 277, 36577-36584[Abstract/Free Full Text]
- Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., Hofmann, K. P., and Ernst, O. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2290-2295[Abstract/Free Full Text]
- Han, M., Smith, S. O., and Sakmar, T. P. (1998) Biochemistry 37, 8253-8261[CrossRef][Medline]
[Order article via Infotrieve]
- Davidson, F. F., Loewen, P. C., and Khorana, H. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4029-4033[Abstract]
- Heck, M., Schädel, S. A., Maretzki, D., Bartl, F. J., Ritter, E., Palczewski, K., and Hofmann, K. P. (2003) J. Biol. Chem. 278, 3162-3169[Abstract/Free Full Text]
- Rao, V. K., Cohen, G. B., and Oprian, D. D. (1994) Nature 367, 639-642[CrossRef][Medline]
[Order article via Infotrieve]
- Fryxell, K. J., and Meyerowitz, E. M. (1987) EMBO J. 6, 443-451[Abstract]
- Alkorta, I., and Du, P. (1994) Protein Eng. 7, 1231-1238[Abstract]
- Gartner, W., and Towner, P. (1995) Photochem. Photobiol. 62, 1-16[Medline]
[Order article via Infotrieve]
- Miura, S., and Karnik, S. S. (2002) J. Biol. Chem. 277, 24299-24305[Abstract/Free Full Text]
- Miura, S. I., Zhang, J., Boros, J., and Karnik, S. S. (2003) J. Biol. Chem. 278, 3720-3725[Abstract/Free Full Text]
- Berson, E. L., Rosner, B., Weigel-DiFranco, C., Dryja, T. P., and Sandberg, M. A. (2002) Investig. Ophthalmol. Vis. Sci. 43, 3027-3036[Abstract/Free Full Text]
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