scholarly journals The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

2020 ◽  
Author(s):  
J.C. Valdez-Lopez ◽  
S.T. Petr ◽  
M.P. Donohue ◽  
R.J. Bailey ◽  
M. Gebreeziabher ◽  
...  
Keyword(s):  
G Protein ◽  
Visual Pigment ◽  
Image Formation ◽  
Phosphorylation Sites ◽  
Tyrosine Residue ◽  
Ionic Interaction ◽  
Cytoplasmic Loop ◽  
Light Responses ◽  
C Terminus ◽  
Activation Rate

ABSTRACTMelanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses including circadian photoentrainment and pupillary light reflexes, and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin’s deactivation have been characterized (i.e. C-terminal phosphorylation and β-arrestin binding), but a detailed analysis of melanopsin’s activation is lacking. We propose that an extended 3rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation which is stabilized by ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the 3rd cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin’s activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin—which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine—indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin’s C-terminus is proximal to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.STATEMENT OF SIGNIFICANCEMelanopsin is an important visual pigment in the mammalian retina that mediates non-image forming responses such as circadian photoentrainment and pupil constriction, and supports contrast detection for image formation. In this study, we detail two critical structural features of mouse melanopsin—its 3rd cytoplasmic loop and C-terminus—that are important in the activation of melanopsin’s light responses. Furthermore, we propose that these two regions directly participate in coupling mouse melanopsin to its G-protein. These findings contribute to further understanding of GPCR-G-protein coupling, and given recent findings suggesting flexibility of melanopsin signal transduction in the retina (possibly by coupling more than one G-protein type), these findings provide insight into the molecular basis of melanopsin function in the retina.

scholarly journals Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation

2020 ◽  
Author(s):  
Juan C. Valdez-Lopez ◽  
Sahil Gulati ◽  
Elelbin A. Ortiz ◽  
Krzysztof Palczewski ◽  
Phyllis R. Robinson
Keyword(s):  
Ganglion Cells ◽  
Proximal Region ◽  
Hek293 Cells ◽  
Small Subset ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Light Responses ◽  
Protein Receptor ◽  
C Terminus ◽  
Pupil Constriction

ABSTRACTMelanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsin’s Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and β-arrestin binding are centered around these critical phosphorylation sites (S388-S395). This degree of functional versatility could help explain the diverse ipRGC light responses as well as non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.

scholarly journals Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.

1996 ◽  
Vol 16 (1) ◽  
pp. 247-257 ◽  
Author(s):  
Q Chen ◽  
J B Konopka
Keyword(s):  
G Protein ◽  
Yeast Cells ◽  
Special Role ◽  
Phosphorylation Sites ◽  
Pheromone Receptor ◽  
Protein Activation ◽  
C Terminus ◽  
G Protein Activation ◽  
Alpha Factor ◽  
Increased Sensitivity

The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.

scholarly journals The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus. Vol. 279 (2004) 34240–34249

2005 ◽  
Vol 280 (7) ◽  
pp. 6252
Author(s):  
Sergej Milovic ◽  
Bibiane Steinecker-Frohnwieser ◽  
Wolfgang Schreibmayer ◽  
Lukas G. Weigl
Keyword(s):  
G Protein ◽  
K Channels ◽  
C Terminus

scholarly journals Differential Interactions of the C terminus and the Cytoplasmic I-II Loop of Neuronal Ca2+Channels with G-protein α and βγ Subunits

1998 ◽  
Vol 273 (28) ◽  
pp. 17595-17603 ◽  
Author(s):  
Taiji Furukawa ◽  
Reiko Miura ◽  
Yasuo Mori ◽  
Mark Strobeck ◽  
Kazuyuki Suzuki ◽  
...  
Keyword(s):  
G Protein ◽  
C Terminus ◽  
Ca2 Channels

scholarly journals Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

2018 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Pradhipa Karuna M ◽  
Leonie Witte ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Long Range ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Multivesicular Bodies ◽  
Phosphorylation Sites ◽  
Wnt Proteins ◽  
C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

scholarly journals Yeast-Based Directed-Evolution For High-Throughput Structural Stabilization of G Protein-Coupled Receptors (GPCRs)

2021 ◽  
Author(s):  
May Meltzer ◽  
Zvagelsky Tatiana ◽  
Niv Papo ◽  
Stanislav Engel
Keyword(s):  
G Protein ◽  
Resistant Cell ◽  
Single Step ◽  
G Protein Coupled Receptors ◽  
Structural Basis ◽  
Diffusion Method ◽  
Structural Stabilization ◽  
C Terminus ◽  
Screening Platform ◽  
G Protein Coupled

Abstract The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we presented a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The detergent-resistant cell wall of yeast provides a unique compartmentalization opportunity to physically link the receptor phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method offers important insights into the structural basis of GPCR stability, questioning the inherent instability of the GPCR scaffold, and revealing the potential role of the C-terminus in receptor stabilization.

scholarly journals Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

2009 ◽  
Vol 83 (20) ◽  
pp. 10710-10718 ◽  
Author(s):  
Steven Kwilas ◽  
Rachael M. Liesman ◽  
Liqun Zhang ◽  
Edward Walsh ◽  
Raymond J. Pickles ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Cell Cultures ◽  
Vero Cells ◽  
Target Cells ◽  
Apical Surface ◽  
Virus Attachment ◽  
C Terminus ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

scholarly journals A Reevaluation of the Role of the Heterotrimeric G Protein in Coupling Light Responses in Arabidopsis

2003 ◽  
Vol 131 (4) ◽  
pp. 1623-1627 ◽  
Author(s):  
Alan M. Jones ◽  
Joseph R. Ecker ◽  
Jin-Gui Chen
Keyword(s):  
G Protein ◽  
Light Responses ◽  
Heterotrimeric G Protein

scholarly journals Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors.

1986 ◽  
Vol 88 (5) ◽  
pp. 675-694 ◽  
Author(s):  
N J Mangini ◽  
D R Pepperberg ◽  
W Baehr
Keyword(s):  
G Protein ◽  
Visual Pigment ◽  
Outer Segment ◽  
Tight Binding ◽  
Beta Subunits ◽  
Hypotonic Medium ◽  
Intense Light ◽  
Low Levels ◽  
Toad Bufo ◽  
Peak Value

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.

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