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.

Effect of carboxylic acid side chains on the absorption maximum of visual pigments.

The proposal that the absorption maximum of the visual pigments is governed by interaction of the 11-cis-retinal chromophore with charged carboxylic acid side chains in the membrane-embedded regions of the proteins has been tested by mutating five Asp and Glu residues thought to be buried in rhodopsin. Changing Glu113 to Gln causes a dramatic shift in the absorption maximum from 500 nanometers to 380 nanometers, a decrease in the pKa (acidity constant) of the protonated Schiff base of the chromophore to about 6, and a greatly increased reactivity with hydroxylamine. Thus Glu113 appears to be the counterion to the protonated Schiff base. Wavelength modulation in visual pigments apparently is not governed by electrostatic interaction with carboxylate residues, other than the counterion.

Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore.

Rhodopsin and the visual pigments are a distinct group within the family of G-protein-linked receptors in that they have a covalently bound ligand, the 11-cis-retinal chromophore, whereas all of the other receptors bind their agonists through noncovalent interactions. The retinal chromophore in rhodopsin is bound by means of a protonated Schiff base linkage to the epsilon-amino group of Lys-296. Two rhodopsin mutants have been constructed, K296G and K296A, in which the covalent linkage to the chromophore is removed. Both mutants form a pigment with an absorption spectrum close to that of the wild type when reconstituted with the Schiff base of an n-alkylamine and 11-cis-retinal. In addition, the pigment formed from K296G and the n-propylamine Schiff base of 11-cis-retinal was found to activate transducin in a light-dependent manner, with 30 to 40% of the specific activity measured for the wild-type protein. It appears that the covalent bond is not essential for binding of the chromophore or for catalytic activation of transducin.

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.

Constitutively active mutants of rhodopsin.

Two critical amino acids in the visual pigment rhodopsin are Lys-296, the site of attachment of retinal to the protein through a protonated Schiff base linkage, and Glu-113, the Schiff base counterion. Mutation of Lys-296 or Glu-113 results in constitutive activation of opsin, as assayed by its ability to activate transducin in the absence of added chromophore. We conclude that opsin is constrained to an inactive conformation by a salt bridge between Lys-296 and Glu-113. Recently, one of the mutants, K296E, was found in a family with retinitis pigmentosa, suggesting that degeneration of the photoreceptor cells in individuals with this mutation may result from persistent stimulation of the phototransduction pathway.

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.

Molecular determinants of spectral properties and signal transduction in the visual pigments.

Site-directed mutagenesis of the visual pigment rhodopsin has provided a wealth of information regarding amino acid residues responsible for the determination of the spectral properties of the chromophore, the amino acids involved in activation and inactivation of the protein, and the effect of amino acid substitutions found in patients with retinitis pigmentosa. In addition, cell culture systems have now been established for expression of the three human color vision pigments, opening the way for a similar attack on the structure and function of these important proteins.

The ligand-binding domain of rhodopsin and other G protein-linked receptors.

Rhodopsin is a member of the very large family of G protein-linked receptors. The members of this family show clear signs of evolutionary relatedness, primarily in amino acid sequence homology, topographical structure of the proteins in the membrane, and the fact that all of the receptors function through the intermediary action of a GTP-binding regulatory protein or G protein. Recently, it has become clear that the structural similarity of these receptors extends well beyond the rather crude comparison of membrane topography. Reviewed here are several studies in which site-directed mutagenesis and active-site-directed reagents were used to show that the ligand-binding pockets of these receptors are highly similar. They are similar despite the fact that the structures of their various ligands are very different.

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.

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.

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.

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.

Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH.

NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.

Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296.

In previous studies, mutation of either Lys296 or Glu113 in bovine rhodopsin has been shown to result in constitutive activation of the apoprotein form, opsin [Robinson et al. (1992) Neuron 9, 719-725]. In this report, pH-rate profiles for the rhodopsin-catalyzed exchange of GTPgS for GDP on transducin are established for the constitutively active opsin mutants. All of the mutants, including the double-mutant E113Q,K296G, show a bell-shaped pH-rate profile. Therefore, it is evident that at least two ionizable groups in addition to Lys296 and Glu113 control the formation of the active opsin state. The sole effect of mutation at position 113 or 296 is to alter the ionization constant of the group with the higher pKa, called pka2. pKa2 decreases in the following order: rhodopsin/light (9.0) > K296E = K296G = E113Q,K296G (8.0) > E113Q (6.8) > K296H (6.6) >> wild-type opsin (< 5.0). These results are consistent with a model where activation of opsin involves (i) breaking of the salt bridge between Lys296 and Glu113, (ii) deprotonation of Lys296, and (iii) the net uptake of a proton from the solvent. Furthermore, exogenous addition of the chromophore all-trans-retinal shifts the wild-type and E113Q opsin equilibrium to favor the active state. In all these respects, the light-independent activation of the opsin mutants appears to proceed by a mechanism similar to that of light-activated rhodopsin.

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.

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.

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.

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.

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.