Molecular determinants of visual pigment function.

Mutagenesis studies and comparisons of natural variants of rhodopsin and related visual pigments have led to new insights concerning photoreceptor function. The studies identify domains important for receptor folding, the residues that set the wavelength of absorption for the ligand 11-cis retinal, and residues, that when mutated, trigger the cell death of photoreceptors.     

Purification of the bovine rod outer segment Na+/Ca2+ exchanger

Optimal conditions for solubilization and stabilization of the Na+/Ca2+ exchanger from rod outer segments were examined. The exchanger was found to be most stable at low detergent concentrations (7.5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), greater than or equal to 100 mM NaCl, pH 7.0-7.5, and with 0.1% added soybean asolectin. The sulfhydryl-modifying reagent, dithiothreitol, caused a loss of exchanger activity and was omitted throughout the purification procedure. These conditions were used to purify the Na+/Ca2+ exchanger from rod outer segments by a combination of selective solubilization, ion exchange, and wheat germ agglutinin chromatography. The procedure achieves a 336-fold increase in exchanger specific activity. The presence of exchanger activity most closely correlates with a polypeptide of molecular mass 215-kDa. Exchanger activity in both the crude rod outer segments and the purified exchanger is specifically dependent upon the presence of K+ in the assay medium; neither choline nor Li+ can substitute for K+.     

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.     

Spectral tuning in the mammalian short-wavelength sensitive cone pigments

The wild-type mouse ultraviolet (UV) and bovine blue cone visual pigments have absorption maxima of 358 and 438 nm, respectively, while sharing 87% amino acid identity. To determine the molecular basis underlying the 80 nm spectral shift between these pigments, we selected several amino acids in helices II and III for site-directed mutagenesis. These amino acids included: (1) those that differ between mouse UV and bovine blue; (2) the conserved counterion, Glu113; and (3) Ser90, which is involved in wavelength modulation in avian short-wavelength sensitive cone pigments. These studies resulted in the identification of a single amino acid substitution at position 86 responsible for the majority of the spectral shift between the mouse UV and bovine blue cone pigments. This is the first time that this amino acid by itself has been shown to play a major role in the spectral tuning of the SWS1 cone pigments. A single amino acid substitution appears to be the dominant factor by which the majority of mammalian short-wavelength sensitive cone pigments have shifted their absorption maxima from the UV to the visible regions of the spectrum. Studies investigating the role of the conserved counterion Glu113 suggest that the bovine and mouse SWS1 pigments result from a protonated and unprotonated Schiff base chromophore, respectively.     

Relationships within the family of GTP-binding proteins isolated from bovine central nervous system

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.     

Beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase. Comparison with the phosphodiesterase family

A group of cDNA clones encoding the beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase were isolated for structural analysis. The encoded polypeptide has 853 residues with a calculated molecular mass of 98 kDa. The beta-subunit is 72% identical to the rod cGMP phosphodiesterase alpha-subunit. Like the alpha-subunit and the cone alpha'-subunit, the beta-subunit belongs to the family of phosphodiesterase genes. The beta- and alpha-subunits are more similar to each other than either is to the cone alpha'-subunit, suggesting either that the beta- and alpha-subunits diverged more recently or that their divergence was restrained by the rod functional environment.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.