A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Salamander UV cone pigment: sequence, expression, and spectral properties.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

G protein-coupled receptor activation: analysis of a highly constrained, "straitjacketed" rhodopsin.

G protein-coupled receptor (GPCR) activation is generally assumed to result in a significant structural rearrangement of the receptor, presumably involving the rigid body movement of transmembrane helices. We have investigated the activation of the GPCR rhodopsin by the construction and analysis of a mutant which contains a total of four disulfide bonds connecting the cytoplasmic ends of helices 1 and 7, and 3 and 5, and the extracellular ends of helices 3 and 4, and 5 and 6. Despite the constraints imposed by four disulfides, this "straitjacketed" receptor retains the ability to activate the G protein transducin and, therefore, provides insight into the molecular mechanism of the initial step in signal transduction of this important class of receptors.

Tertiary interactions between transmembrane segments 3 and 5 near the cytoplasmic side of rhodopsin.

Previous studies [Yu, H., Kono, M., and Oprian, D. D. (1999) Biochemistry 38, xxxx-xxxx] using split receptors and disulfide cross-linking have shown that native cysteines 140 and 222 on the cytoplasmic side of transmembrane segments (TM) 3 and 5 of rhodopsin, respectively, can cross-link to each other upon treatment with the oxidant Cu(phen)3(2+). In this paper we show that although the 140-222 cross-link does not affect the spectral properties of rhodopsin, it completely and reversibly inactivates the ability of the receptor to activate transducin. Following on this lead we further investigate the cytoplasmic region of TM3 and TM5 and identify three additional pairs of residues that when changed to Cys are capable of forming disulfide cross-links in the protein: 140/225, 136/222, and 136/225. These disulfides are able to form without addition of the Cu(phen)3(2+) oxidant. Similar to the 140-222 cross-link, none of the additional disulfides affect the spectral properties of rhodopsin. Also like the 140-222 bond, the 136-222 disulfide completely and reversibly inactivates the light-dependent activation of transducin by the receptor. In contrast, the 140-225 and 136-225 disulfides have no effect on the ability of rhodopsin to activate transducin. The pattern of cross-linking observed in Cys and disulfide scans of the protein is consistent with helical secondary structure in TM3 from 130 to 142 and in TM5 from 218 to 225.

State-dependent disulfide cross-linking in rhodopsin.

In previous studies, we developed a new method for detecting tertiary interactions in rhodopsin using split receptors and disulfide cross-linking. Cysteines are engineered into separate fragments of the split opsin, the disulfide bond can be formed between the juxtaposed residues by treatment with Cu(phen)3(2+), and then disulfide cross-links can be detected on the gel by an electrophoretic mobility shift. In this study, we utilized this method to examine the cross-linking reactions between native cysteines in the ground state and after photoexcitation of rhodopsin. In the dark, Cys140 on transmembrane segment (TM) 3 cross-links to Cys222 on TM5. After photobleaching, Cys140 cross-links to Cys316 and Cys222, and the rate of the cross-linking reaction between Cys140 and Cys222 significantly increases.

Spectral tuning in the human blue cone pigment.

The first determination of the absolute absorption maximum of the human blue cone visual pigment is presented. After expression in COS cells, reconstitution with 11-cis-retinal, and purification, the blue pigment exhibits an absolute absorption maximum of 414 nm. The pigment reacts rapidly with hydroxylamine in the dark and is capable of activating bovine rod transducin in a light-dependent manner. Products of mutations of proposed spectral tuning residues in the blue pigment do not behave as predicted when using rhodopsin mutants as a model. Mutations of amino acids in the ring portion of the chromophore binding pocket of rhodopsin serve well as a predictive model for mutations in the blue pigment, but mutations near the Schiff base do not.

Tertiary interactions between the fifth and sixth transmembrane segments of rhodopsin.

We have used cysteine scanning mutagenesis and disulfide cross-linking in a split rhodopsin construct to investigate the secondary structure and tertiary contacts of the fifth (TM5) and sixth (TM6) transmembrane segments of rhodopsin. Using a simple increase in pH to promote disulfide bond formation, three cross-links between residues on the extracellular side of TM5 (at positions 198, 200, and 204) and TM6 (at position 276) have been identified and characterized. The helical pattern of cross-linking observed indicates that the fifth transmembrane helix extends through residue 200 but does not include residue 198. Rhodopsin mutants containing these disulfides demonstrate nativelike absorption spectra and light-dependent activation of transducin, suggesting that large movements on the extracellular side of TM5 with respect to TM6 are not required for receptor activation.

Disulfide bond exchange in rhodopsin.

Rhodopsin contains two cysteines (Cys110 and Cys187) that are highly conserved among members of the G protein coupled receptor family and that form a disulfide bond connecting helixes 3 and 4 on the extracellular side of the protein. However, recent work on a rhodopsin mutant split in the cytoplasmic loop connecting helixes 3 and 4 has shown that the amino- and carboxy-terminal fragments of this split protein do not comigrate on nonreducing SDS-PAGE gels, suggesting that the native Cys110-Cys187 disulfide bond is not present in this mutant [Ridge et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3204-3208; Yu et al. (1995) Biochemistry 34, 14963-14969]. We show here that the inability to observe the disulfide bond on SDS gels is the result of a disulfide bond exchange reaction which occurs when this split rhodopsin is denatured in preparation for SDS-PAGE. Cys185 reacts with the native disulfide, displacing Cys110 and forming a new disulfide with Cys187. If the sulfhydryl-specific reagent N-ethylmaleimide is included in the sample during preparation for electrophoresis or if Cys185 is changed to Ser, the two fragments do comigrate with full-length rhodopsin on SDS gels and, therefore, are connected by the native Cys110-Cys187 disulfide bond. In related experiments, we find no evidence that the Cys110-Cys187 disulfide bond is broken upon formation of the active intermediate metarhodopsin II.

In vitro assay for trans-phosphorylation of rhodopsin by rhodopsin kinase.

Trans-phosphorylation of rhodopsin refers to a reaction in which a rhodopsin kinase molecule that has been activated by a light-activated rhodopsin molecule collides with and phosphorylates a second molecule of rhodopsin that has not been activated by light. It has been invoked as a mechanism for high-gain phosphorylation, a phenomenon that is observed at low bleaching levels where up to several hundred moles of phosphate are added to the rhodopsin pool per mole of photolyzed rhodopsin. Trans-phosphorylation is an appealing mechanism to propose for high-gain phosphorylation, but it has not been tested directly because of the difficulty inherent in unambiguous identification of light-activated and dark forms of rhodopsin present in the same reaction mixture. We report here a direct assay for trans-phosphorylation of rhodopsin. The assay is based on the use of a split receptor mutant of rhodopsin, SR(1-4/5-7), in which the fully functional protein is assembled from two separately expressed fragments. Because of different electrophoretic mobilities, SR(1-4/5-7) and wild-type rhodopsin can be monitored independently for phosphorylation while in the same reaction mixture. Thus, if wild-type rhodopsin is exposed to light and then incubated in the dark with SR(1-4/5-7), ATP, and rhodopsin kinase, phosphorylation of SR(1-4/5-7) would be a clear demonstration that trans-phosphorylation has occurred. Despite numerous attempts using several different experimental configurations, we have been unable to detect trans-phosphorylation of dark rhodopsin with this system.

Resonance Raman examination of the wavelength regulation mechanism in human visual pigments.

Resonance Raman spectra of recombinant human green and red cone pigments have been obtained to examine the molecular mechanism of color recognition by visual pigments. Spectra were acquired using a 77 K resonance Raman microprobe or preresonance Raman spectroscopy. The vibrational bands were assigned by comparison to the spectra of bovine rhodopsin and model compounds. The C=NH stretching frequencies of rhodopsin, the green cone pigment, and the red cone pigment in H2O (D2O) are found at 1656 (1623), 1640 (1618), and 1644 cm(-1), respectively. Together with previous resonance Raman studies on iodopsin [Lin, S. W., Imamoto, Y., Fukada, Y., Shichida, Y., Yoshizawa, T., & Mathies, R. A. (1994) Biochemistry 33, 2151-2160], these values suggest that red and green pigments have very similar Schiff base environments, while the Schiff base group in rhodopsin is more strongly hydrogen-bonded to its protein environment. The absence of significant frequency and intensity differences of modes in the fingerprint and the hydrogen out-of-plane wagging regions for all these pigments does not support the hypothesis that local chromophore interactions with charged protein residues and/or chromophore planarization are crucial for the absorption differences among these pigments. However, our data are consistent with the idea that the Schiff base group in blue visual pigments is stabilized by protein and water dipoles and that the removal of this dipolar field shifts the absorption maximum from blue to green. A further red shift of the lambda(max) from the green to the red pigment is successfully modeled by the addition of hydroxyl-bearing amino acids (Ser164, Tyr261, and Thr269) close to the ionone ring that lower the transition energy by interacting with the change of dipole moment of the chromophore upon excitation. The increased hydrogen bonding of the protonated Schiff base group in rhodopsin is predicted to account for the 30 nm blue shift of its absorption maximum compared to that of the green pigment.

Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa.

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

Activating mutations of rhodopsin and other G protein-coupled receptors.

Rhodopsin, the visual pigment of rod photoreceptors cells, is a member of the large family of G protein-coupled receptors. Rhodopsin is composed of two parts: a polypeptide chain called opsin and an 11-cis-retinal chromophore covalently bound to the protein by means of a protonated Schiff base linkage to Lys296 located in the seventh transmembrane segment of the protein. Several mutations have been described that constitutively activate the apoprotein opsin. These mutations appear to activate the protein by a common mechanism of action. They disrupt a salt-bridge between Lys296 and the couterion Glu113 that helps constrain the protein to an inactive conformation. Four of the mutations have been shown to cause two different diseases of the retina, retinitis pigmentosa and congenital night blindness. Recently, several other human diseases have been shown to be caused by constitutively activating mutations of G protein-coupled receptors.

A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.

A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive activation of opsin: interaction of mutants with rhodopsin kinase and arrestin.

Mutation of Gly90, Glu113, Ala292, and Lys296 in the visual pigment rhodopsin constitutively activates the protein for activation of the G protein transducin. Three of these mutations have been shown to cause two different human diseases. Mutation of Gly90 and Ala292 results in complete night blindness, and mutation of Lys296 results in the degenerative disease retinitis pigmentosa. We show here that the mutants not only constitutively activate transducin but are also constitutively activated for phosphorylation by rhodopsin kinase. In addition, the phosphorylated mutants are shown to bind tightly to the inhibitory protein arrestin in a reaction that quenches the activity toward transducin. Thus the same mutations that result in constitutive activation of transducin also result in constitutive phosphorylation by rhodopsin kinase and binding of arrestin to inhibit the activity. This implies that the same conformational change may be responsible for activation of transducin and rhodopsin kinase. It also suggests that degeneration of photoreceptor cells in retinitis pigmentosa results indirectly from the activated state of the receptor, perhaps as a consequence of phosphorylation and persistent binding of arrestin.

Molecular determinants of human red/green color discrimination.

The human red and green color vision pigments are identical at all but 15 of their 364 amino acids, and yet their absorption maxima differ by 31 nm. In an extensive mutagenesis study, including a set of 28 chimeric proteins modeled after pigments in the color-deficient human population and an additional 30 single and multiple point mutants, the spectral difference between these 2 pigments is shown to be determined by 7 and only 7 amino acid residues. In going from the red pigment to the green pigment, the 7 residues are Ser116-->Tyr, Ser180-->Ala, Ile230-->Thr, Ala233-->Ser, Tyr277-->Phe, Thr285-->Ala, and Tyr309-->Phe.

Active site-directed inactivation of constitutively active mutants of rhodopsin.

Recently, mutations of the active site Lys296 residue in rhodopsin (Lys296-->Glu and Lys296-->Met) have been found as the cause of disease in some patients with autosomal dominant retinitis pigmentosa. In vitro, these mutations result in constitutive activation of the protein. In an effort to develop a potential therapeutic agent for treatment of the disease, we have examined various amine derivatives of 11-cis- and 9-cis-retinal for ability to irreversibly inactivate a related constitutively active mutant, K296G. Three amines were prepared by reductive amination of retinal: 11-cis-retinylpropylamine, 11-cis-retinylamine, and 9-cis-retinylamine. All three compounds inactivated K296G, and the inactivation could not be reversed upon exposure to light. None of the compounds inactivated the wild-type protein. Although the amines were not effective on the naturally occurring retinitis pigmentosa mutants, presumably because of unfavorable steric interactions with the bulky Glu and Met side chains at position 296, the success with K296G makes it highly encouraging that this approach will evolve related compounds that are capable of inactivating the naturally occurring mutants as well.

Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness.

Mutations in the gene for the visual pigment rhodopsin cause retinitis pigmentosa (RP) and congenital night blindness. Inheritance of the diseases is generally autosomal dominant and about 40 different rhodopsin mutations have been documented. Although the cell death and retinal degeneration associated with RP have been suggested to result from improper folding and accumulation of the mutant proteins in rod photoreceptor cells, this may not account for the disease in all cases. For example, RP mutations at Lys 296, site of Schiff base linkage to the retinal chromophore, result in constitutive activation of the protein in vitro; that is, the mutants can catalytically activate the G protein transducin in the absence of chromophore and in the absence of light. Similarly, mutation of Ala 292-->Glu activates opsin in vitro and causes night blindness. We show here that the mutation Gly 90-->Asp (G90D) in the second transmembrane segment of rhodopsin, which causes congenital night blindness, also constitutively activates opsin. Furthermore, we show that Asp 90 can substitute for the Schiff base counterion, Glu 113, which is located in the third transmembrane segment of the protein. This demonstrates the proximity of Asp 90 and Lys 296 in the three-dimensional structure of rhodopsin and suggests that the constitutively activating mutations operate by a common molecular mechanism, disrupting a salt bridge between Lys 296 and the Schiff base counterion, Glu 113.

Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness.

A number of mutations in the rhodopsin gene have been shown to cause both dominant and recessive retinitis pigmentosa. Here we describe another phenotype associated with a defect in this gene. We discovered a patient with congenital stationary night blindness who carries the missense mutation Ala292Glu. When coupled with 11-cis-retinal in vitro, Ala292Glu rhodopsin is able to activate transducin in a light-dependent manner like wild-type rhodopsin. However, without a chromophore, Ala292Glu opsin anomalously activates transducin. We speculate that the rod dysfunction in this patient is due to an abnormal, continuous activation of transducin by mutant opsin molecules in photoreceptor outer segments.

Constitutive activation of opsin: influence of charge at position 134 and size at position 296.

In previous studies, mutation of Lys296 or Glu113 in opsin has been shown to result in constitutive activation of the protein--that is, these mutants can activate the G protein transducin in the absence of chromophore and in the absence of light. These and other data have led to the suggestion that a salt bridge between Lys296 and Glu113 helps to constrain opsin to an inactive conformation. It is shown here that of 12 different amino acids substituted at position 296, all, except Arg and the wild-type Lys, are constitutively active at neutral pH, lending further support to this suggestion. However, activation of opsin appears also to be influenced significantly by the size of amino acid side chain at position 296. Thus, there are multiple effects of the mutations. Wild-type opsin is also shown to be weakly active at pH 6.1. Five other charged amino acids in the membrane-embedded region of the protein (Asp83, Glu122, Glu134, Arg135, and Glu201) were mutated to see if they affect constitutive activity. Of these amino acids, only mutation of Glu134 results in an increase in the activity of opsin. Changing Glu134 to Gln increases the activity of opsin, while changing Glu134 to Asp inhibits activity. These results suggest that a negative charge on Glu134 is important in stabilizing the inactive state of opsin. Glu134 is highly conserved in all visual pigments and most of the other G protein-linked receptors.