The characteristic wavelength at which a visual pigment absorbs light is regulated by interactions between protein (opsin) and retinylidene Schiff base chromophore. By using site-directed mutagenesis, charged amino acids in bovine rhodopsin transmembrane helix C were systematically replaced. Substitution of glutamic acid-134 or arginine-135 did not affect spectral properties. However, substitution of glutamic acid-122 by glutamine or by aspartic acid formed pigments that were blue-shifted in light absorption (Amax = 480 nm and 475 nm, respectively). While the substitution of glutamic acid-113 by aspartic acid gave a slightly red-shifted pigment (Amax = 505 nm), replacement by glutamine formed a pigment that was strikingly blue-shifted in light absorption (Amax = 380 nm). The 380-nm species existed in a pH-dependent equilibrium with a 490-nm species such that at acidic pH all of the pigment was converted to Amax = 490 nm. We conclude that glutamic acid-113 serves as the retinylidene Schiff base counterion in rhodopsin. We believe that this opsin-chromophore interaction is an example of a general mechanism of color regulation in the visual pigments.

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

SummaryImmunoglobulin (Ig) antigen receptors are composed of a noncovalently-associated complex of Ig and two other proteins, Igo~ and Ig3. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Igc~-IgB heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca 2+ and phosphorylation responses also uncouple IgM from Igoe-Ig/3. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca 2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Igct with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig~ to the inactive Ig reconstitutes only Ca z+ responses. Thus, Igor and IgB are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that IgoL and IgB can activate different signaling pathways.

Glu-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Purified mutant rhodopsin pigments were prepared in which Glu-113 was replaced individually by Gin (Ell3Q), Asp (E113D), Asn (E113N), or Ala (E113A). E113Q, E113N, and E113A existed as pH-dependent equilibrium mixtures of unprotonated and protonated Schiff base (PSB) forms. The Schiff base pK. values determined by spectrophotometric titration were 6.00 (E113Q), 6.71 (E113N), and 5.70 (E113A). Thus, mutation of Glu-113 markedly reduced the Schiff base pK,,. The addition of NaCI promoted the formation of a PSB in E113Q and E113A. An exogenously supplied solute anion replaced Glu-113 to compensate for the positive charge of the PSB in these mutants. The A.m values of the PSB forms of the mutants in NaCl were 496 nm (E113Q), 506 nm (E113A), 510 nm (E113D), and 520 mn (E113N). To evaluate the effect of different types of solute anions on A.values, mutants were prepared in sodium salts of halides, perchlorate, and a series ofcarboxylic acids ofvarious sizes and acidity. The A,,, values of E113Q and E113A depended on the solute anion present and ranged from 488 nm to 522 nm for E113Q and from 486 nm to 528 am for Ei13A. The solute anion affected the A.. values of E113N and E113D to lesser degrees.The reactivities of the mutants to hydroxylamine were also studied. Whereas rhodopsin was stable to hydroxylamine in the dark, E113N reacted slowly and E113Q reacted rapidly under these conditions, indicating structural differences in the Schiff base environments. The A,, values and solute anion dependencies of the Glu-113 mutants indicate that interactions between Schiff base and its counterion play a significant role in determining the A.,1 of rhodopsin.Rhodopsin belongs to the family of receptors that activates guanine nucleotide-binding regulatory proteins in signal transduction. The primary structure of rhodopsin has been determined (1-3), and models propose the existence of seven transmembrane segments, a common motif in this membrane receptor family (4). In rhodopsin, these transmembrane segments form a binding pocket for the 11-cis-retinal chromophore that is linked to the opsin through a protonated Schiffbase (PSB) at Lys-296. Rhodopsin has a Ama,, at 500 nm; human cone pigments, which also contain 11-cis-retinal, display Amas values ranging from 440 nm (blue) to 530 nm (green) to 560 nm (red) (5). A central problem has been to understand how the interactions between opsin proteins and retinal chromophores determine their spectral properties.Recently, we investigated the role of charged amino acids in transmembrane helix C on the structure and function of bovine rhodopsin by site-directed mutagenesis and concluded that Glu-113 served as the counterion of the retinylidene Schiff base (6). Zhukovsky and Oprian (7) and Nathans (8) came to the same conclusion. We further studied a set of four mutants, each of which contains a single amino acid substitution at position 113, (E113D, E113Q, E113N, and E113A). Mutant pigments E113Q, E113N, ...