Characterization of Rhodopsin Congenital Night Blindness Mutant T94I

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO T4R/T4R dog retina, we found that the mutation abolished glycosylation at Asn 2 , whereas glycosylation at Asn 15 was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho* lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G t ). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

Zhu

Jang

Jastrzębska

et al. 2004

“…In cone visual cells, the light-independent G protein activation by the retinal-free protein acts to desensitize the cells by lowering their overall photosensitivity (30,31). Similarly, in rod visual cells, the constitutive G protein activity accompanying some rhodopsin mutants that exhibit faster retinal-Schiff base hydrolysis can cause night blindness by desensitization of the cells (53,59,64). If retinal-free mammalian melanopsins also inherently possess some level of constitutive G protein activity, the spontaneous Schiff base hydrolysis and retinal release could similarly act to desensitize ipRGCs.…”

Section: Dog Opn4mentioning

confidence: 99%

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Tsukamoto

Kubo

Farrens

et al. 2015

Background: Melanopsin is involved in non-visual photoreception in mammals, but molecular characteristics underlying these non-visual functions are unclear. Results: Stability of the bond with the retinal chromophore is weakened and diversified in mammalian melanopsins. Conclusion: Mammalian melanopsins have acquired characteristics suited for their non-visual functions. Significance: The weaker retinal attachment in melanopsin may contribute to the functional tuning of non-visual photoreception in mammals.

“…Photolysis of rhodopsin was carried out using a continuous wave argon ion laser directed into the sample compartment from above by a concave mirror that dispersed the beam evenly over the cuvette, with beam gating carried out with a manual shutter. Complete photoisomerization of rhodopsin and all mutants was achieved without significant heating in less than 1 s. Transducin activation assays were carried out as described previously (39).…”

Section: Gpcr Family Alignments-blastmentioning

confidence: 99%

“…In summary, our results suggest that ET is a reliable and highly efficient method for Transducin activation by wild-type (WT) and mutant opsins is shown in the presence and absence of 11-cis-retinal and light. Transducin activity was assayed using membranes from transfected COS cells as previously described (39): OE, in the absence of retinal; q, in the presence of retinal; and E, time course for the reaction in the presence of retinal after exposure to light (h ) for each mutant listed. Membrane amounts were selected to give the same light-dependent transducin activation kinetics; experiments with detergent-purified rhodopsins confirmed that the specific activities in the light were not measurably different for the mutants and wild-type (data not shown).…”

Section: Et Residues Cluster At Functional Sites Inmentioning

confidence: 99%

Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions

Madabushi

Gross

Philippi

et al. 2004

Self Cite

185

171

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 subdivided 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.The profusion and diversity of G protein-coupled receptors (GPCRs) 1 give them a central role in health and disease. In humans, over 1000 genes encode these receptors (1), each of which responds to a single or few ligands by activating G proteins, which then modulate enzymes and channels to initiate highly amplified signaling cascades. Such cascades control sight, taste, smell, slow neurotransmission and the responses to most water-soluble hormones and chemokines. In fact, GPCRs are so ubiquitous that, although they are the targets of nearly 50% of current drugs (2), this is still a small fraction of their pharmacological potential (3).Some of the major questions relevant to GPCR pharmacology include the following: What residues are critical for ligand binding and G protein activation? What do different receptor families have in common with regard to their activation mechanism? From a structural perspective, it is known that all GPCRs form a seven transmembrane (TM) ␣-helical bundle, connected by three intracellular and three extracellular loops, with an extracellular N terminus and an intracellular C terminus (4, 5). However, low overall sequence identity of 25% even within class A GPCRs (6 -8) suggests that significant deviations can occur in ligand binding pockets and in interhelical contacts that stabilize or mediate the transition between a...