Activation of rhodopsin kinase.
Ontology highlight
ABSTRACT:
SUBMITTER: McCarthy NE
PROVIDER: S-EPMC1222487 | biostudies-other | 2002 Apr
REPOSITORIES: biostudies-other
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Project description:Light-induced extrasynaptic dopamine release in the retina reduces adenosine 3',5'-cyclic monophosphate (cAMP) in rod photoreceptor cells, which is thought to mediate light-dependent desensitization. However, the fine time course of the cAMP dynamics in rods remains elusive due to technical difficulty. Here, we visualized the spatiotemporal regulation of cAMP-dependent protein kinase (PKA) in mouse rods by two-photon live imaging of retinal explants of PKAchu mice, which express a fluorescent biosensor for PKA. Unexpectedly, in addition to the light-on-induced suppression, we observed prominent light-off-induced PKA activation. This activation required photopic light intensity and was confined to the illuminated rods. The estimated maximum spectral sensitivity of 489 nm and loss of the light-off-induced PKA activation in rod-transducin-knockout retinas strongly suggest the involvement of rhodopsin. In support of this notion, rhodopsin-deficient retinal explants showed only the light-on-induced PKA suppression. Taken together, these results suggest that, upon photopic light stimulation, rhodopsin and dopamine signals are integrated to shape the light-off-induced cAMP production and following PKA activation. This may support the dark adaptation of rods.
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Project description:Activation of class-A G-protein-coupled receptors (GPCRs) involves large-scale reorganization of the H3/H6 interhelical network. In rhodopsin (Rh), this process is coupled to a change in the protonation state of a key residue, E134, whose exact role in activation is not well understood. Capturing this millisecond pH-dependent process is a well-appreciated challenge. We have developed a scheme combining the harmonic Fourier beads (HFB) method and constant-pH molecular dynamics with pH-based replica exchange (pH-REX) to gain insight into the structural changes that occur along the activation pathway as a function of the protonation state of E134. Our results indicate that E134 is protonated as a consequence of tilting of H6 by ca. 4.0° with respect to its initial position and simultaneous rotation by ca. 23° along its principal axis. The movement of H6 is associated with breakage of the E247-R135 and R135-E134 salt bridges and concomitant release of the E134 side chain, which results in an increase in its pKa value above physiological pH. An increase in the hydrophobicity of the environment surrounding E134 leads to further tilting and rotation of H6 and upshift of the E134 pKa. Such atomic-level information, which is not accessible through experiments, refines the earlier proposed sequential model of Rh activation (see: Zaitseva, E.; et al. Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: The Meta IIa Conformational Substate . J. Am. Chem. Soc. 2010, 132, 4815) and argues that the E134 protonation switch is both a cause and a consequence of the H6 motion.
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Project description:Rhodopsin is the first G-protein-coupled receptor (GPCR) with its three-dimensional structure solved by X-ray crystallography. The crystal structure of rhodopsin has revealed the molecular mechanism of photoreception and signal transduction in the visual system. Although several other GPCR crystal structures have been reported over the past few years, the rhodopsin structure remains an important model for understanding the structural and functional characteristics of other GPCRs. This review summarizes the structural features, the photoactivation, and the G protein signal transduction of rhodopsin.
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Project description: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.
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Project description:G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.
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Project description:Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to approximately 50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.
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