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 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 expression in the cornea

2018 ◽  
Vol 35 ◽  
Author(s):  
ANTON DELWIG ◽  
SHAWNTA Y. CHANEY ◽  
ANDREA S. BERTKE ◽  
JAN VERWEIJ ◽  
SUSANA QUIRCE ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Nerve Fibers ◽  
Visual Signals ◽  
Light Responses ◽  
Rods And Cones ◽  
Retinal Tissue ◽  
Antibody Staining ◽  
Electrophysiological Recordings ◽  
Pupil Constriction ◽  
Sensory Fibers

AbstractA unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.

scholarly journals Recurrent axon collaterals of intrinsically photosensitive retinal ganglion cells

2013 ◽  
Vol 30 (4) ◽  
pp. 175-182 ◽  
Author(s):  
HANNAH R. JOO ◽  
BETH B. PETERSON ◽  
DENNIS M. DACEY ◽  
SAMER HATTAR ◽  
SHIH-KUO CHEN
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Plexiform Layer ◽  
Small Subset ◽  
Inner Plexiform Layer ◽  
Retinal Ganglion ◽  
Light Responses ◽  
Genetic Method ◽  
Axon Collaterals ◽  
The Brain

AbstractRetinal ganglion cells (RGCs), the output neurons of the retina, have axons that project via the optic nerve to diverse targets in the brain. Typically, RGC axons do not branch before exiting the retina and thus do not provide it with synaptic feedback. Although a small subset of RGCs with intraretinal axon collaterals has been previously observed in human, monkey, cat, and turtle, their function remains unknown. A small, more recently identified population of RGCs expresses the photopigment melanopsin. These intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit an irradiance-coding signal to visual nuclei in the brain, contributing both to image-forming vision and to several nonimage-forming functions, including circadian photoentrainment and the pupillary light reflex. In this study, using melanopsin immunolabeling in monkey and a genetic method to sparsely label the melanopsin cells in mouse, we show that a subgroup of ipRGCs have axons that branch en route to the optic disc, forming intraretinal axon collaterals that terminate in the inner plexiform layer of the retina. The previously described collateral-bearing population identified by intracellular dye injection is anatomically indistinguishable from the collateral-bearing melanopsin cells identified here, suggesting they are a subset of the melanopsin-expressing RGC type and may therefore share its functional properties. Identification of an anatomically distinct subpopulation in mouse, monkey, and human suggests this pathway may be conserved in these and other species (turtle and cat) with intraretinal axon collaterals. We speculate that ipRGC axon collaterals constitute a likely synaptic pathway for feedback of an irradiance signal to modulate retinal light responses.

scholarly journals Melanopsin- and L-cone–induced pupil constriction is inhibited by S- and M-cones in humans

2018 ◽  
Vol 115 (4) ◽  
pp. 792-797 ◽  
Author(s):  
Tom Woelders ◽  
Thomas Leenheers ◽  
Marijke C. M. Gordijn ◽  
Roelof A. Hut ◽  
Domien G. M. Beersma ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Brightness Discrimination ◽  
Light Response ◽  
Inhibitory Input ◽  
Human Retina ◽  
Constant Intensity ◽  
Light Responses ◽  
Pupillary Light ◽  
Pupillary Constriction ◽  
Pupil Constriction

The human retina contains five photoreceptor types: rods; short (S)-, mid (M)-, and long (L)-wavelength–sensitive cones; and melanopsin-expressing ganglion cells. Recently, it has been shown that selective increments in M-cone activation are paradoxically perceived as brightness decrements, as opposed to L-cone increments. Here we show that similar effects are also observed in the pupillary light response, whereby M-cone or S-cone increments lead to pupil dilation whereas L-cone or melanopic illuminance increments resulted in pupil constriction. Additionally, intermittent photoreceptor activation increased pupil constriction over a 30-min interval. Modulation of L-cone or melanopic illuminance within the 0.25–4-Hz frequency range resulted in more sustained pupillary constriction than light of constant intensity. Opposite results were found for S-cone and M-cone modulations (2 Hz), mirroring the dichotomy observed in the transient responses. The transient and sustained pupillary light responses therefore suggest that S- and M-cones provide inhibitory input to the pupillary control system when selectively activated, whereas L-cones and melanopsin response fulfill an excitatory role. These findings provide insight into functional networks in the human retina and the effect of color-coding in nonvisual responses to light, and imply that nonvisual and visual brightness discrimination may share a common pathway that starts in the retina.

Amacrine and ganglion cell contributions to the electroretinogram in amphibian retina

2001 ◽  
Vol 18 (1) ◽  
pp. 147-156 ◽  
Author(s):  
GAUTAM AWATRAMANI ◽  
JUE WANG ◽  
MALCOLM M. SLAUGHTER
Keyword(s):  
Contrast Agents ◽  
Ganglion Cell ◽  
Neuronal Activity ◽  
Opposite Effect ◽  
Cell Function ◽  
Ganglion Cells ◽  
Field Potential ◽  
Tiger Salamander ◽  
Third Order ◽  
Light Responses

The neuronal generators of the b- and d-waves of the electroretinogram (ERG) were investigated in the tiger salamander retina to determine if amacrine and ganglion cells contribute to this field potential. Several agents were used that affect third-order neurons, such as tetrodotoxin, baclofen, and NMDA agonists and antagonists. Baclofen, an agent that enhances light responses in third-order neurons, increased the d-wave and reduced the b-wave. In contrast, agents that decrease light responses in third-order neurons had the opposite effect of enhancing the b-wave and depressing the d-wave. The effect on the d-wave was particularly pronounced. The results indicate that third-order neuronal activity influences b- and d-waves of the ERG. The opposing actions suggest that the b-wave to d-wave ratio might serve as an measure of ganglion cell function.

Evidence for glycine, GABAA, and GABAB receptors on rabbit OFF-alpha ganglion cells

2003 ◽  
Vol 20 (3) ◽  
pp. 285-296 ◽  
Author(s):  
THOMAS C. ROTOLO ◽  
RAMON F. DACHEUX
Keyword(s):  
Ganglion Cells ◽  
Gabab Receptors ◽  
Direct Inhibition ◽  
Light Responses ◽  
Retinal Surface ◽  
Excitatory Response ◽  
Sulforhodamine B ◽  
Intact Rabbit ◽  
Specific Receptors ◽  
Whole Cell Recordings

Inhibitory synaptic transmission via GABA and glycine receptors plays a crucial role in shaping the excitatory response of neurons in the retina. Whole-cell recordings were obtained from ganglion cells in the intact rabbit eyecup preparation to correlate GABA- and glycine-activated currents with the presence of their specific receptors on morphologically identified α ganglion cells. Alpha ganglion cells were chosen based upon their large somata when viewing the retinal surface, and responses to light and dark spots were used to identify OFF-alpha ganglion cells. Light responses were abolished by superfusion of Ringer's containing cobalt to synaptically isolate the cell by blocking all Ca2+-mediated transmitter release. Pressure pulses of GABA and glycine were delivered to an area that encompassed the dendritic field while receptor antagonists were applied through superfusion to characterize the direct inhibition onto the ganglion cell. Physiological results indicated that OFF-α cells did not have any GABAC receptor-activated currents, but did express currents mediated by ionotropic GABAA receptors and metabotropic GABAB receptors that were blocked by their specific antagonists bicuculline and CGP55845, respectively. The amplitudes of strychnine-sensitive glycine-activated currents were always larger than the currents elicited by GABA. Confocal optical sections of physiologically identified, sulforhodamine B-stained cells displayed the localization of glycine and GABAA receptor subunit labeling dispersed over the stained dendrites.

scholarly journals Intrinsic light responses of retinal ganglion cells projecting to the circadian system

2003 ◽  
Vol 17 (9) ◽  
pp. 1727-1735 ◽  
Author(s):  
Erin J. Warren ◽  
Charles N. Allen ◽  
R. Lane Brown ◽  
David W. Robinson
Keyword(s):  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Circadian System ◽  
Retinal Ganglion ◽  
Light Responses

scholarly journals Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

2000 ◽  
Vol 20 (10) ◽  
pp. 3558-3567 ◽  
Author(s):  
Isabelle Mothe-Satney ◽  
Daqing Yang ◽  
Patrick Fadden ◽  
Timothy A. J. Haystead ◽  
John C. Lawrence
Keyword(s):  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Translational Repression ◽  
Phosphorylation Sites ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Dependent Processes ◽  
Constitutive Phosphorylation

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

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