Biomedical subjects

Alecia K Gross

Publications and source records attributed to Alecia K Gross.

6 recordsLinked to original sources

PubMed2006

Defective development of photoreceptor membranes in a mouse model of recessive retinal degeneration.

Retinal neurodegeneration occurs in several inherited diseases. Some of the most severe disease alleles involve mutations at the C-terminus of rhodopsin, but in no case is the pathogenic mechanism leading to cell death well understood. We have examined a mouse model of recessive retinal degeneration caused by a knock-in of a human rhodopsin-EGFP fusion gene (hrhoG/hrhoG) at the rhodopsin locus. Whereas heterozygous mutant mice were indistinguishable from control mice, homozygous mutant mice had retinal degeneration. We hypothesized that degeneration might be due to aberrant rhodopsin signaling; however, inhibiting signaling by rearing mice in total darkness had no effect on the rate of degeneration. Using confocal and electron microscopy, we identified the fundamental defect as failed biogenesis of disk membranes, which is observed at the earliest stages of outer segment development. These results reveal that in addition to its role in transport and sorting of rhodopsin to disk membranes, rhodopsin is also essential for formation of disks.

Animals↗

PubMed2005

Rhodopsin-EGFP knock-ins for imaging quantal gene alterations.

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

Animals↗

PubMed2003

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions.

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.

Amino Acid Sequence↗

PubMed2003

An opsin mutant with increased thermal stability.

This report describes the biochemical characterization of a double mutant of rhodopsin (N2C,D282C) in which Cys residues engineered into the protein at positions 2 (in the amino-terminal extracellular domain) and 282 (in the extracellular loop between transmembrane helices 6 and 7) are shown to form a disulfide bond and increase the thermal stability of the unliganded or opsin form of the protein. Wild-type opsin does not survive detergent solubilization and purification at pH 7.5 and 25 degrees C. In contrast, the N2C,D282C mutant opsin survives the purification protocol and loses less than 50% activity after incubation for 20 days under the same conditions. Less than 5% is lost after 20 days at 4 degrees C. While the disulfide bond clearly has a dramatic effect on protein stability, it has a minor impact on the activity of the pigment. The MII lifetime of the mutant (6.6 min) is similar to that of the wild type (7.9 min), and the specific activity of the light-activated mutant for activation of transducin is within 20% of the wild-type activity. Therefore, it seems likely that the disulfide bond does not perturb greatly the structure of the protein. For these reasons, we anticipate that the mutant may be of use in detailed kinetic and mechanistic investigations of the ligand binding reaction and for crystallization trials involving recombinant rhodopsin, especially the unliganded opsin form of the protein.

Amino Acid Sequence↗

PubMed2003

Slow binding of retinal to rhodopsin mutants G90D and T94D.

In an examination of the effect of three rhodopsin night blindness mutations on the rate of association of 11-cis-retinal with opsin, one of the mutations (G90D) was found to slow the rate of reaction by more than 80-fold. This effect does not appear to be general to night blindness mutations as the two other mutants (A292E and T94I) were not found to bind retinal with slowed kinetics. However, T94D was similar to G90D in that the rate of retinal binding was dramatically slowed. Gly90 and Thr94 are both located in the active site of the protein close to the Schiff base counterion Glu113. Thus, the slow kinetics of Schiff base formation appear to correlate with the introduction of a negative charge close to the Schiff base counterion, suggesting a possible role for Glu113 as a catalytic base in this reaction. Consistent with this model, the E113Q mutant was also found to bind retinal more slowly than the wild type.

Alanine↗

PubMed2003

Characterization of rhodopsin congenital night blindness mutant T94I.

The Thr94 --> Ile mutation in the second transmembrane segment of rhodopsin has been reported to be associated with a congenital night blindness phenotype in a large Irish pedigree. Previously, two other known rhodopsin mutants that cause congenital night blindness, A292E and G90D, have been shown in vitro to constitutively activate the G protein transducin in the absence of a chromophore. The proposed mechanism of constitutive activation of these two mutants is an electrostatic disruption of the active site salt bridge between Glu113 and Lys296 that contributes to stabilization of the protein in the inactive state. Here, the T94I rhodopsin mutant is characterized and compared to the two other known rhodopsin night blindness mutants. The T94I mutant opsin is shown also to constitutively activate transducin. The T94I mutant pigment (with a bound 11-cis-retinal chromophore), like the other known rhodopsin night blindness mutants, is not active in the dark and has wild-type activity upon exposure to light. Similar to the Gly90 --> Asp substitution, position 94 is close enough to the Schiff base nitrogen that an Asp at this position can functionally substitute for the Glu113 counterion. However, in contrast to the other night blindness mutants, the T94I MII intermediate decays with a half-life that is approximately 8-fold slower than in the wild-type MII intermediate. Thus, the one phenotype shared by all congenital night blindness mutants that is different from the wild-type protein is constitutive activation of the apoprotein.

Amino Acid Sequence↗