Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and ...identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic ...nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.
Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in ...humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic
, which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ
knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins.
This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology.
The C-terminal tail of the transducin α subunit, Gtα(340−350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of ...Gtα(340−350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin-label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin-label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC343-Gtα(340−350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtα(340−350) analogue. A doubly labeled Proxyl/TOAC348-Gtα(340−350) was examined by DEER spectrocopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin-labels in this GPCR−G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin−G-protein interface.
A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these ...receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα (340-350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα (340-350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αLtype. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325-346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor-G protein interface is demonstrated.
Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and ...identical posttranslational farnesylation of their Ggamma subunits, rods and cones rely on unique Ggamma.sub.1 (Gngt1) and Ggamma.sub.c (Gngt2) isoforms, respectively. The only other farnesylated G-protein gamma-subunit, Ggamma.sub.11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Ggamma.sub.1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Ggamma.sub.1, Ggamma.sub.c, or Ggamma.sub.11 in Ggamma.sub.1 -deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Ggamma in rod cells and restoration of Galpha.sub.t1 expression, which is greatly reduced in Ggamma.sub.1 -deficient rods. Electroretinography showed restoration of visual function in all three transgenic Ggamma.sub.1 -deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Ggamma.sub.1 -deficient rods was also fully restored. In all dark-adapted transgenic lines, Galpha.sub.t1 was targeted to the outer segments, reversing its diffuse localization found in Ggamma.sub.1 -deficient rods. Bright illumination triggered Galpha.sub.t1 translocation from the rod outer to inner segments in all three transgenic strains. However, Galpha.sub.t1 translocation in Ggamma.sub.11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Ggamma.sub.11 transgenic mice but not in Ggamma.sub.1 controls. Thus, while farnesylated Ggamma subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Ggamma isoforms by the ubiquitous Ggamma.sub.11 affects the ability of rods to adapt to background light.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To characterize the possible role of transducin Gtβγ-complex in modulating the signaling properties of photoactivated rhodopsin and its lifetime in rod disc membranes and intact rods.
Rhodopsin ...photolysis was studied using UV-visible spectroscopy and rapid scanning spectroscopy in the presence of hydroxylamine in highly purified wild-type and Gtγ-deficient mouse rod disc membranes. Complex formation between photoactivated rhodopsin and transducin was measured by extra-metarhodopsin (meta) II assay. Recovery of dark current and flash sensitivity in individual intact wild-type and Gtγ-deficient mouse rods was measured by single-cell suction recordings.
Photoconversion of rhodopsin to meta I/meta II equilibrium proceeds normally after elimination of the Gtβγ-complex. The meta I/meta II ratio, the rate of meta II decay, the reactivity of meta II toward hydroxylamine, and the rate of meta III formation in Gtγ-deficient rod disc membranes were identical with those observed in wild-type samples. Under low-intensity illumination, the amount of extra-meta II in Gtγ-deficient discs was significantly reduced. The initial rate of dark current recovery after 12% rhodopsin bleach was three times faster in Gtγ-deficient rods, whereas the rate of the late current recovery was largely unchanged. Mutant rods also exhibited faster postbleach recovery of flash sensitivity.
Photoactivation and thermal decay of rhodopsin proceed similarly in wild-type and Gtγ-deficient mouse rods, but the complex formation between photoactivated rhodopsin and transducin is severely compromised in the absence of Gtβγ. The resultant lower transduction activation contributes to faster photoresponse recovery after a moderate pigment bleach in Gtγ-deficient rods.
Modulation of interactions between activated GPCRs (G-protein coupled receptors) and the intracellular (IC) signal transducers, heterotrimeric G-proteins, is an attractive, yet essentially ...unexplored, paradigm for treatment of certain diseases. Regulating downstream signaling for treatment of congenital diseases due to constitutively active GPCRs, as well as tumors where GPCRs are often overexpressed, requires the development of new methodologies. Modeling, experimental data, docking, scoring, and experimental testing (MEDSET) was developed to discover inhibitors that target the IC loops of activated GPCRs. As proof-of-concept, MEDSET developed and utilized a model of the interface between photoactivated rhodopsin (R*) and transducin (Gt), its G-protein. A National Cancer Institute (NCI) compound library was screened to identify compounds that bound at the interface between R* and its G-protein. High-scoring compounds from this virtual screen were obtained and tested experimentally for their ability to stabilize R* and prevent Gt from binding to R*. Several compounds that modulate signal transduction have been identified.
Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The βγ subunit ...complex, especially the C-terminal domain of the transducin γ subunit, Gtγ(60–71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin α subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the γ subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the βγ complex in signal transfer to G proteins.
Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin ...(Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt γ -subunit, Gtγ (50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtβ γ complex in direct activation assays. Competition by Gtγ (50-71) farnesyl with Gt for the active receptor suggests a pivotal role for Gtβ γ in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt α -subunit, Gtα (340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R*by Gtα (340-350) and Gtγ (50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.
PDF
