Shift in the relation between flash‐induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes

Emeis, D.; Hofmann, Klaus Peter

doi:10.1016/0014-5793(81)80618-7

Cited by 65 publications

(42 citation statements)

References 14 publications

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“…The G protein transducin (Gt) stabilizes the light-activated rhodopsin metarhodopsin II (MII), which has an absorption maximum of 380-390 nm, whereas metarhodopsin I has an absorption maximum at 480 nm (27)(28)(29)(30). A synthetic peptide specific to 11 amino acids at the C terminus of the a subunit of Gt, at, has been shown to stabilize MIT (5,6).…”

Section: Resultsmentioning

confidence: 99%

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Kisselev

Pronin

Ермолаева

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

101

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Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G protein y subunit, which is masked in the fy complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the fty complex in regulation of effector function.Coupling of cell surface receptors with intracellular heterotrimeric (afry) guanine nucleotide-binding proteins (G proteins) is a primary level of control over specificity in transmembrane signaling (1-4). The molecular mechanisms underlying specific interaction of various G proteins with diverse receptors are not fully understood. A role for the a subunit in receptor coupling is indicated by evidence that shows direct association of a peptide specific to the C-terminal domain of an a subunit with a receptor, rhodopsin, and additionally, from the demonstration that switching the C-terminal tail of one a subunit type with a corresponding portion of another a subunit type changes the receptor target of a G protein (5-8). However, even before the subunits of G proteins were clearly identified there were indications that the 13,y complex is a prerequisite for effective interaction of a G protein with a receptor (9, 10). Although purification of the subunits has confirmed this obligatory requirement in a variety of receptor systems (11-13), the role of the g3y complex has remained unidentified. The function of this complex in receptor-G protein interaction is especially enigmatic because (i) both the /B and fy subunits are families of diverse proteins (2) and (ii) comparison of the crystal structures of the GDP-and GTPbound a subunit reveals little change on the surface of the a subunit that is thought to interact with the receptor (3,14,15). This is surprising, since conformational rearrangements at this surface of a would be required to allow a GDP-bound G protein to associate with a receptor and, after exchanging GTP for GDP, to dissociate from the same receptor.Based on our recent results that indicate interaction between a peptide specific to a G-protein 'y subunit and a receptor, rhodopsin, we predicted that in the fry complex the C-terminal tail of the y subunit provides high-affinity contact with a receptor (16). However, since the f3-y complex by itself does not interact with rhodopsin (11, 17, 18), we proposed that the C-terminal tail of the y subunit is masked in the free Py complex but becomes available for interaction with rhodopsin during the course of receptor-afry complex association. Results presented here support these predictions.

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Section: Resultsmentioning

confidence: 99%

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Kisselev

Pronin

Ермолаева

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

101

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“…In analogy to G protein-coupled receptors with diffusible ligands in which G protein binding stabilizes a receptor conformation with a high affinity ligand-binding site, MII is stabilized at the expense of its tautomeric forms by the binding of G t or G tderived peptides. This stabilization of MII is the basis of the "extra-MII" assay (34,35). This assay, however, can only be applied under conditions of a dynamic equilibrium between metarhodopsin I and MII, which is exquisitely sensitive to membrane environment, pH, temperature, ionic strength, etc.…”

Section: Discussionmentioning

confidence: 99%

Mutation of the Fourth Cytoplasmic Loop of Rhodopsin Affects Binding of Transducin and Peptides Derived from the Carboxyl-terminal Sequences of Transducin α and γ Subunits

Ernst¹,

Meyer²,

Marin³

et al. 2000

Journal of Biological Chemistry

156

102

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The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G t ), is not well defined. We developed a novel assay to measure the ability of G t , or G t -derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340 -350 of the ␣ subunit of G t , or a cysteinylthioetherfarnesyl peptide corresponding to residues 50 -71 of the ␥ subunit of G t , are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the ␥-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G t (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930Chem. 275, -1936. Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the ␣ subunit of G t . We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the ␣ subunit of G t and plays a role in the regulation of ␤␥ subunit binding.G protein-coupled receptors transmit extracellular signals to the cell's interior via heterotrimeric G proteins and effector enzymes or ion channels (1, 2). Rhodopsin is one of the archetypes of the G protein-coupled receptor superfamily. It triggers the biochemical amplification machinery of the visual cascade in the rod photoreceptor cell, which comprises the G protein transducin (G t ) 1 and the effector, a cyclic GMP-specific phosphodiesterase (3, 4). The transduction of a light signal begins with the photochemical cis-trans isomerization of the chromophore, 11-cis-retinal. Protein conformational changes are transmitted from the ligand-binding site to the cytoplasmic surface of the receptor where catalytic activation of G t occurs. This intramolecular conversion from inactive (rhodopsin) to active (R*) states mediated by chromophore isomerization has been termed "signal transmission" (5). Key structural correlates of the transition to the active state include deprotonation of the retinylidene Schiff base (6) with concomitant protonation of the Glu 113 counterion (7,8) and the protonation of the cytoplasmic surface of rhodopsin (9, 10) mediated by the highly conserved Glu 134 residue at the cytoplasmic border of transmembrane (TM) helix 3. Movements of TM helices have been proposed to accompany the signal transmission process, with a change in the orientation of TM helices 3 and 6 relative to each other as the most prominent feature (11-13).The cytoplasmic surface of rhodopsin comprises four loops and a carboxyl-terminal tail. The first (C1), second (C2), and third (C3) cytoplasmic loops connect adjacent TM helices. The fourth cytoplasmic loop (C4) is bounded by TM helix 7 at its ...

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“…The use of stepwise photolysis with a series of flashes, "exhaustion curves," has been described in detail by Emeis and Hofmann (1981). Briefly each flash photoexcites a fixed mole fraction p of rhodopsin.…”

Section: Nir-scattering and Absorption Measurementsmentioning

confidence: 99%

Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein

Schleicher

Hofmann

1987

J. Membrain Biol.

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Formation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (RM) leads to a change in the light scattering of the disk membranes (binding signal or signal P). The signal measured on isolated disks (so-called PD signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than the RMG binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed the PD-signal level and kinetics as a function of flash intensity and compared it to the RMG-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of the PD signals decrease with flash intensity when the signals are normalized to the same final level. This finding prevents an explanation of the scattering signal by a slow postponed reaction of the RMG complex. We propose to interpret the scattering change as a redistribution of G-protein between a membrane-bound and a solved state. The process is driven by the complexation of membrane-bound G to flash-activated rhodopsin (RM). The experimental evidence for this two-state model is the following: The intensity dependence of the initial rate of the PD signal is explained by the model. Under the assumption of a bimolecular reaction of free G with sites at the membrane, equal to rhodopsin in their concentration, the measured rates yield a KD of 10(-5) M. Evaluation of the extra MII kinetics yields a biphasic rise at saturating flashes. The measured rates fit to the supply of free and membrane-bound G-protein for the reaction with RM. Quantitative estimation of the expected scattering intensity changes gives a comprehensive description of binding signal and dissociation signal by the gain and loss of G-protein scattering mass. The temperature dependence of the PD-signal rate leads to an activation energy of the membrane-association process of E alpha = 44 kJ/mol.

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Shift in the relation between flash‐induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes

Cited by 65 publications

References 14 publications

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Mutation of the Fourth Cytoplasmic Loop of Rhodopsin Affects Binding of Transducin and Peptides Derived from the Carboxyl-terminal Sequences of Transducin α and γ Subunits

Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein

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