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. Author manuscript; available in PMC: 2007 Dec 1.
Published in final edited form as: Vision Res. 2006 Sep 20;46(27):4493–4501. doi: 10.1016/j.visres.2006.08.003

PALMITYLATION OF CONE OPSINS

Zsolt Ablonczy 1,*, Masahiro Kono 1, Daniel R Knapp 2, Rosalie K Crouch 1
PMCID: PMC2025682  NIHMSID: NIHMS14180  PMID: 16989884

Abstract

Palmitylation is a widespread modification in G-protein-coupled receptors and often a dynamic process. In rhodopsins, palmitylation is static on C322/C323. Red/green (M/LWS) cone opsins have no cysteines at corresponding positions and no palmitylation. SWS2 cone opsins have a single corresponding cysteine and mass spectrometric analysis showed partial palmitylation of salamander SWS2 cone opsin. Ultraviolet (SWS1) cone opsins have one corresponding cysteine, but only unpalmitylated opsin was observed for mouse and salamander. The results show that the static palmitylation found on rhodopsin is not found on cone opsins and suggest the possibility of an unidentified role for opsin palmitylation in cones.

Keywords: cone, opsin, palmitylation, mass spectrometry, G-protein coupled receptor

Palmitylation, glycosylation, and phosphorylation are the three post-translational modifications characteristic of the G-protein-coupled receptors (GPCRs), one of the largest protein families in the human genome (Qanbar & Bouvier, 2003). Nearly all GPCRs are palmitylated (Tobin & Wheatley, 2004), but whereas glycosylation and phosphorylation are well-characterized for many GPCRs and their functional significance is clear, the role of palmitylation has proven to be more elusive (Qanbar & Bouvier, 2003). Sequence alignments of the GPCRs show the common one to three cysteine residues at the carboxyl terminus of the protein. The fatty acid palmitate is normally linked to these cysteines through a thioester linkage, although there is some evidence that suggests yet unidentified additional palmitylation sites exist outside this area for some GPCRs (Chen, Shahabi, Xu & Liu-Chen, 1998, Hawtin, Tobin, Patel & Wheatley, 2001). Although palmityl transferase enzymes have been identified (Fukata, Fukata, Adesnik, Nicoll & Bredt, 2004), these enzymes have not yet been shown to be present in GPCRs. Because the palmitylation sites on proteins are so diverse, it has not been possible to identify a consensus sequence or other structural feature, which could serve for enzyme recognition. Autopalmitylation has been suggested as a possible acylation mechanism (Bano, Jackson & Magee, 1998, O’Brien, St Jules, Reddy, Bazan & Zatz, 1987, Ovchinnikov Yu, Abdulaev & Bogachuk, 1988, Veit, Sachs, Heckelmann, Maretzki, Hofmann & Schmidt, 1998). Other fatty acid acylations have also been found on GPCRs but they are far less common.

The palmitylation of GPCRs might serve several purposes. Constitutive, static palmitylation has been implicated in different structural roles: it affects membrane localization and interaction with other proteins (Dunphy & Linder, 1998, Karnik, Ridge, Bhattacharya & Khorana, 1993), and also has been proposed to stabilize the protein or the GPCR ligand (Heck, Schadel, Maretzki & Hofmann, 2003, Sachs, Maretzki, Meyer & Hofmann, 2000, Traxler & Dewey, 1994). Other studies showed that in addition to structural roles, palmitylation might also be dynamic and essential for GPCR signaling, as palmitylation might regulate receptor turnover (Carman & Benovic, 1998, Ferguson, Zhang, Barak & Caron, 1998, Kraft, Olbrich, Majoul, Mack, Proudfoot & Oppermann, 2001) (as the internalized receptor is inactive), as well as regulate the interaction between the receptor and the G-protein or arrestin (Groarke, Drmota, Bahia, Evans, Wilson & Milligan, 2001, Moffett, Adam, Bonin, Loisel, Bouvier & Mouillac, 1996, Soskic, Nyakatura, Roos, Muller-Esterl & Godovac-Zimmermann, 1999) (thus modulating the signaling time). The modulation of the receptor-G-protein interaction often takes place through another dynamic GPCR modification, phosphorylation. The absence of palmitylation allows better solvent exposure of nearby phosphorylation sites, thus promoting phosphorylation and receptor deactivation (Ponimaskin, Dumuis, Gaven, Barthet, Heine, Glebov, Richter & Oppermann, 2005, Wang, Wen, Ablonczy, Crouch, Makino & Lem, 2005).

In contrast to most GPCR systems, which are dynamically palmitylated, the palmitylation of rhodopsin is constitutive. Rhodopsin, the dim light photoreceptor of the eye, attained the status of a “prototypical” GPCR receptor, because it is easily available in large quantities and is the only GPCR to date for which the crystal structure has been determined (Palczewski, Kumasaka, Hori, Behnke, Motoshima, Fox, Le Trong, Teller, Okada, Stenkamp, Yamamoto & Miyano, 2000). Rhodopsin was the first GPCR where the presence of palmitylation was reported (O’Brien & Zatz, 1984, Papac, Thornburg, Bullesbach, Crouch & Knapp, 1992) is reported to be independent of light conditions, age, chromophore ligands, and the localization of rhodopsin within the outer segment (Young & Albert, 2001). Other fatty acid acylations on rhodopsin are also present, representing about 10% of the total (Young & Albert, 2001).

There are five distinct opsin subfamilies, which are comprised of photopigments with similar absorption and signaling properties: rhodopsin (RH1), rhodopsin-like (RH2), short wavelength sensitive cones class 1 (SWS1 or ultraviolet cone opsins), short wavelength sensitive cones class II (SWS2 or blue cone opsins), and mid/long wavelength sensitive cone opsins (M/LWS or red/green cone opsins) (Ebrey & Koutalos, 2001). Although these subfamilies potentially differ by sequence comparisons, little is known about the actual structure and post-translational modifications of the opsin subfamilies outside rhodopsins. Interestingly, the number and relative position of potential palmitylation sites are characteristic of the various opsin families, but their significance, although potentially and functionally important, is not known. As the cones are the photoreceptors used for our everyday human vision this heightens the importance of gaining knowledge on the structural features of these opsins. Moreover, recent studies are showing that our considerable knowledge about rhodopsin structure may not translate to the structural and functional properties of cones, and indeed that cone opsins themselves may have quite diverse properties (Birge & Knox, 2003, Corson, Kefalov, Cornwall & Crouch, 2000, Das, Crouch, Ma, Oprian & Kono, 2004).

Until recently, it has not been possible to characterize cone opsin structural features, because these opsins could only be obtained in low quantities, and no analytical methodologies and instruments existed for their in-depth study. Our laboratory, which has extensive experience in the mass spectrometric (MS) analysis of rhodopsins (Ablonczy, Crouch & Knapp, 2005, Ablonczy, Kono, Crouch & Knapp, 2001, Ball, Oatis, Dharmasiri, Busman, Wang, Cowden, Galijatovic, Chen, Crouch & Knapp, 1998), has now developed novel methodologies that make it possible to analyze and characterize cone opsin proteins. We report here that the SWS1 salamander and mouse opsins, which are closely related to the human blue cone opsin, and the gecko and mouse M/LWS opsins, which align with the human red and green cone opsins, lack palmitylation. The SWS2 salamander opsin is found in a partially palmitylated state.

EXPERIMENTAL PROCEDURES

Pigments from photoreceptor outer segments

Frozen bovine retina was obtained from WL Lawson Co. (Lincoln, NE) and stored at −80°C until used. Geckos (Tokay gecko) were obtained from Diamond Reptile Breeders (Bushnell, FL). Retinae were dissected under dim red light and the tissue processed immediately. Bovine and gecko rod outer segments (ROS) were purified as published previously (McDowell & Kuhn, 1977, Yuan, Chen, Anderson, Kuwata & Ebrey, 1998). The final ROS membranes were suspended in 100 mM sodium phosphate buffer (pH: 7.4; Sigma Chem. Co., St. Louis, MO) at a concentration of 1 mg/mL and stored in the dark at −80°C for later use. The pigment quantities were determined by absorption (Cary 300, Varian Inc., Palo Alto, CA) at 495 nm (bovine rhodopsin) or 521 nm (gecko M/LWS opsin). For MS analysis, 10 μg pigment containing membranes were centrifuged at 100,000g and the pellet was incubated in 70 μL 0.1% dodecyl-maltoside (DM, Calbiochem-Novabiochem, La Jolla, CA) in water at room temperature for 15 min. This sample was then centrifuged again at the same speed and the solubilized fraction used for the analysis.

Photopigments from whole retinae

Nrl−/− mice were a kind gift from Dr. Anand Swaroop (University of Michigan, W.K. Kellogg Eye Center, Ann Arbor, MI). Retinae were dissected under dim red light, two retinae were pooled, and the tissue immediately saturated with 6 M urea to stop enzymatic processes, and frozen until used. After thawing the retinae, the urea was removed and the retinae were washed with water two-times, then the water was removed and the pellet processed immediately. Care was taken to minimize pain or discomfort in the animals (both geckos and mice). All experiments were carried out in accordance with the European Communities Council directive of 24 November 1986 (86/609/EEC) and with the Guidelines laid down by the NIH on the care and use of animals for experimental procedures.

Photopigments expressed in COS cells

Expression, harvest and purification of the synthetic salamander genes for the SWS1 and SWS2 cone pigments (Das et al., 2004) and the bovine rhodopsin gene (Ablonczy et al., 2001) were described previously. All expressed salamander opsins were tagged with the 1D4 epitope at their carboxy termini (Das et al., 2004). Pigments were immunopurified with the anti-rhodopsin 1D4 antibody, which recognizes the last eight amino acids of bovine rhodopsin and the recombinant salamander pigments (Molday & MacKenzie, 1983). The pigments were kept in the dark, and their concentration determined by their absorbance at 432 nm (recombinant salamander SWS2 cone opsin), 356 nm (recombinant salamander SWS1 cone opsin), and 495 nm (recombinant bovine rhodopsin). For MS analysis, 5 μg detergent-solubilized pigments (~70 μL final volume) were used.

Generation of peptides for sequence analysis

The bovine, gecko, and recombinant salamander pigment samples were reduced by the addition of 2 μL of 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP; ABI, Foster City, CA) and alkylated by the addition of 1 μL 10 mM methyl-methanethiosulfonate (MMTS; ABI) or 0.5 μL straight 4-vinylpyridine (Sigma). Both reactions were performed at room temperature for 1 hour under argon with constant shaking on a rotator. Afterwards, 200 μL trifluoroacetic acid (TFA) (Acros Organics, Fair Lawn, NJ) was added to acidify the samples. Without the previous steps, the mouse retinas were directly dissolved in 400 μL TFA then diluted with 180 μL water to adjust the concentration to 70% TFA. All the samples were cleaved with cyanogen bromide (CNBr) by adding 10 μL, 5 mol/L CNBr solution in acetonitrile (Sigma). The cleavage was carried out with shaking in the dark under argon overnight at room temperature. The reaction was quenched by the addition of 1 mL water and the solvents evaporated under vacuum (SpeedVac SC110, Savant Instruments Inc., Farmingdale, NY).

Mass spectrometry

The dried fragment mixture was redissolved in 5 μL TFA, 42 μL acetonitrile (Fisher), and 84 μL isopropanol (J. T. Baker, Phillipsburg, NJ) and brought up to 5 mL with water. Three mL of the resulting sample was loaded onto a 2.1 mm × 100 mm, OD-300 PE Brownlee Aquapore ODS cartridge column (Bodman Industries, Aston, PA) at a 400 μL/min flow of 0.05% TFA in 2.5% organic mobile phase (2:1 isopropanol/acetonitrile) with an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA). The peptides were eluted at a flow rate of 200 mL/min with an 80 min gradient from 2.5% to 97.5% organic phase followed by a 45 min wash with 97.5% organic phase. The column effluent was split 10:1 and then directed into the IonMax source of a Finnigan LTQ ion-trap mass spectrometer (Thermo-Finnigan Instrument Systems Inc., San Jose, CA). In each cycle, one MS and one MS/MS spectra of the three most abundant molecular ions were automatically acquired using Xcalibur software (version 1.4 SR2) with repeatless dynamic exclusion. The collected data were further analyzed with the TurboSequest unit of the Bioworks 3.2 software. The mouse and salamander data was searched against the NCBI nonredundant database limited to mouse and salamander headings. Scores higher than Xcorr=1 were considered as valid matches, but the obtained sequences have also been hand-checked.

RESULTS

Native and recombinant rhodopsins are doubly palmitylated

Rhodopsins have two C-terminal cysteine palmitylation sites. The palmitylation of bovine rhodopsin was first shown with the incorporation of [3H]palmitic acid into rod outer segment preparations (O’Brien & Zatz, 1984). The two adjacent C-terminal cysteines (C322 and C323) were inferred to be palmitylated by indirect evidence (Ovchinnikov Yu et al., 1988), and then later shown to be palmitylated by MS (Papac et al., 1992). The methodology applied in these experiments was further developed for the analysis of the entire bovine rhodopsin sequence in a single experiment (Ball et al., 1998) that allowed the routine examination of the rhodopsin sequences and post-translational modifications in several species, rat (Ablonczy, Knapp, Darrow, Organisciak & Crouch, 2000), mouse (Ablonczy, Crouch, Goletz, Redmond, Knapp, Ma & Rohrer, 2002), pig (Ablonczy, Goletz, Knapp & Crouch, 2002), and salamander (unpublished data). The C322 and C323 sites were found to be palmitylated independent of age and light condition, but recently, trace amounts of monopalmitylated rhodopsin C-termini have also been detected in some samples (unpublished data). The comparison of the selected ion chromatograms (SIC) of the doublypalmitylated versus the calculated unpalmitylated molecular ions shows that the cysteines are both palmitylated in the bovine rhodopsin (Figure 1A) with no indication of the missing palmitates in this sample. As during sample workup, the free cysteines are pyridylethylated, the molecular weight for the calculated un- or monopalmitylated ions reflects 4-vinylpyridine incorporations. Prior to blocking the free cysteines with pyridylethylation, the disulfide bond of rhodopsin was cleaved with TCEP, which does not affect the palmitate linkage. Expressed bovine rhodopsin has also been shown to have two palmitates, originally by [3H]palmitic acid labeling (Karnik et al., 1993) and later confirmed by MS (Ablonczy et al., 2001). Figure 1B shows the tandem mass spectrum (MS/MS) of the palmitylated molecular ion of the C-terminal peptide fragment of bovine rhodopsin expressed in COS cells, which does not differ from that of the native rhodopsin and confirms the incorporation of two palmitylated cysteines.

Figure 1.

Figure 1

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Bovine rhodopsin palmitylation. A, Selected ion chromatograms corresponding to possible C-terminal rhodopsin CNBr fragments. The top trace shows the presence of the native palmitylated molecular ion. The bottom trace shows that the calculated nonpalmitylated molecular ion of the same rhodopsin fragment is not present in quantities above the baseline noise. B, MS/MS sequence of the palmitylated molecular ion of the C-terminal peptide fragment of recombinant bovine rhodopsin expressed in COS cells. The letter “J” stands for S-palmityl cysteine. The sequence confirms the presence of palmitylation.

The recombinant salamander SWS2 opsin is partially palmitylated

Unlike rhodopsin, SWS2 opsins have only a single C-terminal cysteine. We have mapped the recombinant salamander SWS2 cone opsin with our CNBr cleavage-based MS method and analyzed the palmitylation state of the pigment. Figure 2A shows SICs of C-terminal CNBr fragments showing a mixture of palmitylated (bottom trace) and nonpalmitylated (top trace) molecular ions from the same linear HPLC gradient. Characteristic of the hydrophobic nature of palmitylation, there is a large difference in the retention times (15.2 min for the unpalmitylated versus 34.9 min for the palmitylated). As the sample was pyridylethylated, the molecular weight for the unpalmitylated peak reflects this addition. The palmitylated (m/z=1338.3) and the nonpalmitylated (m/z=1294.1) molecular ions both reflect +3 charge states. The ratio of the palmitylated to the unpalmitylated opsin is estimated to be 1:1 (assuming equal MS ion current yield for the two forms).

Figure 2.

Figure 2

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Palmitylation of the recombinant salamander SWS2 pigment. A, Selected ion chromatograms of the same C-terminal CNBr fragment calculated with and without palmitylation showing a mixture of nonpalmitylated (top trace), palmitylated (bottom trace) and molecular ions. B, Base peak chromatograms of two SWS2 cone opsin samples with two different cysteine labels. In the top trace, the cysteines were labeled with 4-vinylpyridyne, in the bottom trace they were labeled with MMTS. Note that the molecular weight of the C-terminal CNBr fragment (#16) shifts, indicating the presence of labeled cysteine - unpalmitylated opsin. C, MS/MS sequence showing the nonpalmitylated form of SWS2 cone opsin C-terminus. “O” stands for pyridylethyl cysteine. D, MS/MS sequence showing the palmitylated form of SWS2 cone opsin C-terminus. “J” stands for S-palmityl cysteine. Panels C and D indicates only the N- and C- terminal regions of the sequences crucial for the identification of the cysteine sites.

Figure 2B demonstrates the presence of nonpalmitylated C-terminal cysteine, as the molecular weight of the C-terminal CNBr fragment (#16) shifts consistently with the molecular weights calculated with the two different substituents (m/z=1294 for the pyridylethylated and m/z=1274 for the MMTS-treated, as the MMTS reaction produces a methyl disulfide derivative on the cysteine side chain). The CNBr fragment 16 molecular weights reflect +3 charge states. In addition to the change in the molecular weight, the methyl sulfide derviative also causes a slight increase (~2 min) in the retention time of all the free cysteine containing CNBr peptides (#8, #12, #15-16, #16), while the molecular weights of the peptides without a cysteine do not change (#1-3, #1-4, #3-4, #4, #6, #13, #13-14).

Figures 2C and 2D compare the MS/MS sequences of the molecular ions for the nonpalmitylated SWS2 cone opsin C-terminus (Figure 2C) and the palmitylated form (Figure 2D) from the same experiment (chromatographic gradient). The figure indicates the N- and C- terminal regions of the sequences crucial for the identification of the cysteine site, showing ions consistent with their respective cysteine molecular weights confirming the validity of the results and peak identifications shown in Figure 2A.

SWS1 opsins are not palmitylated

All the mammalian short wavelength cone opsins, including the human blue cone opsin, belong to the SWS1 family; therefore, they are highly significant for human vision. These opsins also have a single potential palmitylation site. By MS, we have analyzed both the recombinant salamander SWS1 cone opsin and the mouse SWS1 cone opsin from the Nrl−/− mouse line.

Figure 3A shows the MS/MS spectrum of the nonpalmitylated recombinant salamander SWS1 opsin C-terminus. A complete sequence ladder can be observed, which confirms the presence of a pyridylethyl cysteine, indicating that the C-terminal cysteine was free before cysteine blocking with 4-vinylpyridyne. Unlike the rest of the opsins, the SWS1 opsins have a methionine after the C-terminal cysteine; therefore the fragment shown in Figure 3A is not the C-terminal CNBr fragment but an incomplete cleavage product. The palmitylated cysteines in rhodopsin align with the last cysteine in the salamander SWS1 opsin fragment. Additional digestion of the C-terminus with trypsin has confirmed the presence of unpalmitylated cysteine (data not shown).

Figure 3.

Figure 3

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SWS1 cone opsin palmitylation. A, MS/MS sequence of an incomplete CNBr cleavage fragment from recombinant salamander SWS1 cone pigment. The sequence shows the presence of nonpalmitylated opsin C-terminus. “O” stands for pyridylethyl cysteine, “X” for homoserine lactone. B, MS/MS sequence of an incomplete CNBr cleavage fragment from mouse SWS1 cone pigment. The sequence shows the presence of nonpalmitylated opsin C-terminus. “B” stands for a homoserine that lost a water molecule, “X” for homoserine lactone.

As the salamander SWS1 opsin is recombinant, it was important to check the palmitylation state of a native SWS1 pigment as well. Figure 3B presents the MS/MS sequence of an incomplete CNBr cleavage fragment from the SWS1 cone pigment of the Nrl−/− mouse. The sequence also shows the presence of an opsin C-terminus that has a nonpalmitylated cysteine. In this particular experiment the cysteines were not blocked with an extra label; therefore, the sequence shows free cysteines, the second of which aligns with the palmitylated cysteines in rhodopsin. In other experiments, when the sample was treated with alkylating agents, the cysteine was found to be labeled correspondingly (data not shown). The methionine within the sequence where no CNBr cleavage took place converted into a homoserine with a water molecule lost. Exhaustive searches with the TurboSequest engine against the mouse database did not reveal any evidence for a palmitylated C-terminal cysteine in the mouse SWS1 pigment.

M/LWS opsins are not palmitylated

The M/LWS cone opsins do not have a cysteine aligning with either of the palmitylated cysteines in rhodopsin. Moreover, previous [3H]palmitic acid incorporation experiments with recombinant M/LWS opsin pigments confirmed that they were not palmitylated (Ostrer, Pullarkat & Kazmi, 1998). Theoretically, fatty acid derivatization could occur at another cysteine, and in some GPCRs such an incorporation has been identified (Chen et al., 1998, Hawtin et al., 2001); therefore, it was important to examine native M/LWS pigments as well, using MS analysis procedures. These studies were conducted on both the abundant native gecko M/LWS opsin pigment and on the mouse M/LWS cone opsin from the Nrl−/− mouse.

Figure 4A shows the MS/MS sequence of the CNBr fragment 9 of gecko M/LWS opsin containing the C-terminal cysteine. This cysteine, however, lines-up with C316 in rhodopsin, which is nonpalmitylated. The presence of the pyridylethyl label on the cysteine indicates cysteine that was not palmitylated. Extensive searches did not show the presence of a palmitylated version of the peptide. Similar analysis was performed for all the CNBr fragments that contain cysteines, with no indication for palmitate incorporation at any of the other sites either.

Figure 4.

Figure 4

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Red/green cone opsin palmitylation. A, MS/MS sequence of CNBr fragment #9 from gecko red opsin. The sequence shows the presence of nonpalmitylated pigment. “O” stands for pyridylethyl cysteine, “X” for homoserine lactone. B, MS/MS sequence of the C-terminal CNBr cleavage fragment from mouse green cone pigment. The sequence shows the presence of nonpalmitylated opsin C-terminus. Only the N- and C- terminal regions of the sequence are indicated, which are crucial for the identification of the cysteine site.

Although the gecko M/LWS opsin in its native membranes is abundantly available from outer segment preparations, and its genetic, spectroscopic and biochemical properties match other M/LWS opsins, the gecko photoreceptors with this pigment have rod-like features (Kojima, Okano, Fukada, Shichida, Yoshizawa & Ebrey, 1992, Pedler & Tilly, 1964). Therefore, it was important to check the palmitylation of an M/LWS cone opsin originating from native cones. Figure 4B presents the MS/MS sequence of the C-terminal CNBr cleavage fragment from the mouse M/LWS cone pigment. This fragment is considerably longer than that of the gecko shown in Figure 4A, because it lacks a methionine where CNBr cleavage could occur. In this particular preparation the cysteines were not blocked and the peptide fragment thus contains a free cysteine which aligns with C316 in rhodopsin, indicating that it is not palmitylated. This figure also indicates only the N- and C- terminal regions of the sequences crucial for the identification of the cysteine site, confirming the validity of the peak identification. Similar to the gecko M/LWS opsin, database searches with TurboSequest did not show the presence of a palmitylated version of the peptide, and palmitylation was not found on any cysteine in the opsin.

DISCUSSION

Rhodopsins and cone opsins are not palmitylated the same

Different GPCRs show a wide range of palmitylation levels from unpalmitylated to triply-palmitylated states (Qanbar & Bouvier, 2003). However, it is expected that receptors within a GPCR family will have similar palmitylation patterns. Rhodopsin palmitylation is constitutive on two adjacent sites, C322 and C323 (Ovchinnikov Yu et al., 1988, Papac et al., 1992). Therefore, it was assumed that constitutive palmitylation is a conserved post-translational modification of the opsins, although, the emerging gene sequences for cone opsins showed that the number and localization of palmitylation sites are characteristic of the opsin families (Ebrey & Koutalos, 2001). Figure 5 shows the C-terminal part of a multiple CLUSTALW sequence alignment of the different opsins analyzed in this study. The alignment was performed at the Network Protein Sequence Analysis (NPS@) site of the Pole BioInformatique Lyonnais, in Lyon, France in Lyon, France (Combet, Blanchet, Geourjon & Deleage, 2000).

Figure 5.

Figure 5

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Multiple C-terminal alignment of opsin sequences. The CLUSTALW sequence alignment of was performed at the Network Protein Sequence Analysis (NPS@) site of the Pole BioInformatique Lyonnais, in Lyon, France in Lyon, France (Combet et al., 2000). The following settings were used: endgaps=1, gapdist=8, gapext=0.2, gapopen=10.0, hgapresidues=GPSNDQERK, ktuple=1, matrix=gonnet, maxdiv=30, outorder=aligned, pairgap=3, score=percent, topdiags=5, type=PROTEIN, window=5.

Our mapping experiments of cone opsins demonstrate that the cone opsin palmitylation is fundamentally different from that of rhodopsins. The M/LWS opsins (red/green cone opsins) are not palmitylated. However, this result is not surprising because they do not have a cysteine aligning with the bovine rhodopsin palmitylation sites. The palmitylation of the SWS1 and SWS2 was more surprising. While SWS2 opsins have a single, partially palmitylated site which aligns with C323 in bovine rhodopsin in our alignment in Figure 2, SWS1 opsins have a single cysteine aligning with C322, but it is not palmitylated.

These results are in contrast with previous findings for the primary palmitylation site of recombinant rhodopsins (Karnik et al., 1993). Analysis of the C322S and the C323S mutant opsins expressed in COS cells, showed that the C322S mutation prevents palmitylation of C323, but the C323S mutation allows partial palmitylation of C322 (Karnik et al., 1993). Therefore, it was concluded that C322 is the primary palmitate acceptor, followed in sequence by the palmitylation of C323. However, the analysis of the two short-wavelength-sensitive salamander cone opsins seems to be just the opposite, as the primary palmitylation site should be the one aligning with C323 in Figure 5. This implies the possibility that the local structure of cone opsins and rhodopsins is different around the C-terminal cysteines, resulting in a change in the cysteine solvent exposures that might affect accessibility for palmitylation.

The selection of native and recombinant pigments

Salamanders have four visual pigments. Being the most abundant pigment, to date, it has only been possible to analyze the salamander rhodopsin by mass spectrometry (data not shown), however, all the salamander pigments have been expressed in a COS-cell based system (Das et al., 2004, Ma, Kono, Xu, Das, Ryan, Hazard, Oprian & Crouch, 2001b, Xu, Hazard, Lockman, Crouch & Ma, 1998), and are abundantly available. Therefore, we have studied the least abundant two salamander pigments, SWS1 and SWS2 in their recombinant form. The C-terminus of each of these pigments has been altered from the native sequence for purification purposes (see Figure 5) (Ma et al., 2001b, Xu et al., 1998). The obtained pigments have undergone substantial biochemical characterization (Das et al., 2004, Ma et al., 2001b, Xu et al., 1998) with results comparable to single cell physiological experiments, and are routinely used in our laboratory.

The concern could arise as to whether the structural characteristics of the recombinant pigments faithfully reflect those of the native ones. Bovine rhodopsin is expressed in the same cellular expression system. Earlier experiments of the Khorana laboratory (Kaushal, Ridge & Khorana, 1994), and our mass spectrometric characterization of rhodopsin expressed in the same system (Ablonczy et al., 2001) shows that the only rhodopsin post-translational modification altered by the expression system is glycosylation. Therefore, it is expected that if recombinant rhodopsin is natively palmitylated in COS cells (see Figure 1B) then the recombinant cone opsins would be natively palmitylated as well. An additional question arises because of the C-terminal sequence insertions into the recombinant salamander opsins. However, the chain length and amino acid composition of the opsin C-termini vary; even within the same opsin family (see Figure 5). Moreover the insertion site is relatively far from the potentially palmitylated cysteines. Therefore, it is not expected that these sequence insertions alter the palmitylation levels. However, in order to make sure that this assumption is correct, we compared the palmitylation of the recombinant salamander SWS1 pigment and the native mouse SWS1 pigment, and obtained the same results.

The difficulties with the purification of native M/LWS cone opsins from the eye are similar to those of the short wavelength sensitive opsins. However, the gecko M/LWS opsin is an exception, because it is abundantly available from the major photoreceptor of the gecko retina. But these photoreceptors are rod like, containing a real M/LWS cone opsin (Kojima et al., 1992). Thus, the question might arise if the palmitylation of the red/green opsins depends on the type of photoreceptor cell in which they are expressed. Therefore, we compared the palmitylation of the gecko M/LWS pigment and the mouse green pigment, and observed the same results.

Due to the low number of cone cells vs. the rod cells, it is not normally possible to extract and analyze cone pigments from mammalian eyes. The high rhodopsin background and the low dynamic range of the mass spectrometers make mass spectrometric analysis prohibitive. However, the native mouse SWS1 and M/LWS pigments were extracted from the Nrl−/− mouse. In this model, the lack of the NRL gene prevents the development of the rod photoreceptor cells, and therefore the eyes of these mice contain only cone cells, and in higher numbers than normal (Nikonov, Daniele, Zhu, Craft, Swaroop & Pugh, 2005, Nikonov, Kholodenko, Lem & Pugh, 2006). The cones of this model contain a mixture of SWS1 and M/LWS pigments, coexpressed in the same cell. The resulting lack of rhodopsin makes it possible to extract sufficient cone opsins for the mass spectrometric analysis.

The palmitylation pattern is characteristic of opsin subfamilies

Our data demonstrate the presence or absence of palmitylation for four out of the five opsin subfamilies. Rhodopsins are constitutively doubly palmitylated, SWS1 opsins are not palmitylated, SWS2 opsins are partially palmitylated, and M/LWS opsins are not palmitylated. Rhodopsin-like (RH2) pigments were not analyzed, but as a consequence of their high homology to rhodopsins, they are expected to be also doubly palmitylated.

The mammalian cone opsins (all SWS1 and M/LWS pigments) seem to be not palmitylated, which is in sharp contrast to the RH1 pigments; however, the functional significance of this is not yet understood. It is not certain whether the palmitylation state of the SWS1 pigments is constitutive or dynamic, but the capability of the salamander SWS2 opsin to be palmitylated or nonpalmitylated suggests dynamic palmitylation. Dynamic palmitylation might indicate a yet unidentified signaling process, which is involved in the palmitylation of short-wavelength-sensitive cone opsins. It is also remarkable, that the salamander SWS2 pigment is found to be present in both rod and cone cells of the salamander (Ma, Znoiko, Othersen, Ryan, Das, Isayama, Kono, Oprian, Corson, Cornwall, Cameron, Harosi, Makino & Crouch, 2001a), with the potential possibility that it is palmitylated differently in the rod and cone cells. This warrants further investigation, as we have only examined expressed salamander cone opsins.

When nonpalmitylated rhodopsin was first expressed in COS cells, no effects on the light-induced transducin binding were observed; therefore, it was concluded that the functional efficiency of the proximal part of the C-terminus is not dependent on the presence of palmitates (Karnik et al., 1993). However, the Palm−/− mouse showed a small, but definite physiological difference (Wang et al., 2005). The lack of palmitylation enhances phosphorylation and slightly speeds-up the shutoff of signal transduction. This in vivo result was in good agreement with other GPCRs, where dynamic palmitylation promoted phosphorylation of nearby sites and receptor inactivation (Ponimaskin et al., 2005, Qanbar & Bouvier, 2003), but it was different from the in vitro rhodopsin studies, which showed no changes in activation and a slight reduction in phosphorylation (Karnik et al., 1993). Therefore, the results for the Palm−/− mouse and other GPCR systems taken together with our results for cone opsin palmitylation, suggest that the significance of the differential palmitylation of opsin subfamilies may be in the fine tuning of opsin deactivation, and this fine tuning might be characteristic of the opsin subfamily.

In summary, the results show that it is not possible to generalize the post-translational modifications of rhodopsin to cone opsins. Therefore, it will be necessary to obtain detailed knowledge about the structural features of all opsin families to achieve a detailed understanding of the structural features underlying cone opsin physiology.

Acknowledgments

The authors thank Patrice Goletz, Greg Beall and Dr. Jie Fan for technical assistance. Supported by NIH grants EY-04939 (RKC), EY-08239 (DRK), EY-013748 (MK), EY-14793, and C06 RR015455, and an unrestricted grant to the Department of Ophthalmology at Medical University of South Carolina from Research to Prevent Blindness, Inc. (RPB, New York, NY). R.K.C. is an RPB Senior Scientific Investigator.

Footnotes

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