The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609

Free backbone carbonyls mediate rhodopsin activation

Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration

Ligand-Dependent Conformations and Dynamics of the Serotonin 5-HT2A Receptor Determine Its Activation and Membrane-Driven Oligomerization Properties

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization

Microglial activation and chronic neurodegeneration

Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurode-generative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including tumor necrosis factor-α, nitric oxide, interleukin-1β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (e.g., lipopolysaccharide or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss with time. Although the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s disease. We review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe