Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.

Kahlert, M.; König, Bernd; Hofmann, Klaus

doi:10.1016/s0021-9258(17)30605-1

Cited by 40 publications

(9 citation statements)

References 30 publications

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“…In this context, Figure gives a macroscopic GDP off rate constant ( k - a ) of 2 × 10 -4 s -1 for transducin at 35 °C. Using this value, together with a macroscopic dissociation constant ( K D ) of 25 μM (at 25 °C) ( , ), we calculate an effective forward rate constant k a = k - a / K D = 8 M -1 s -1 as the association rate for binding of GDP to G α . This tiny value for GDP binding to G α is even smaller than for EF, tubulin, ras, myosin, kinesin, etc., indicating quite hindered nucleotide binding mechanics and implying a slow intramolecular isomerization step.…”

Section: Discussionmentioning

confidence: 99%

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange

et al. 2001

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Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, with very high affinity. Yet, it is common to find that substantial amounts (30-90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become "denatured" during formation of the obligatory nucleotide-free or empty (MT) Galpha-state that is intermediary to GDP/GTP exchange at a single binding site. We find Galpha native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Galpha is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucleotide binding at micromolar ambient GTPgammaS concentrations. Because of the weak preequilibrium, quantitative protection against Galpha aggregation is only achieved at free nucleotide concentrations 10-100 times higher than those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an intramolecular Galpha refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Galpha x GTP isomers described here, only the second can bind Mg2+, "locking" GTP in place with a large net rise in GTP binding affinity. A companion Galpha x GDP isomerization reaction is identified as the cause of the very slow spontaneous GDP dissociation that characterizes G protein nucleotide exchange and low spontaneous background activity in the absence of GPCR activation. Galpha x GDP and Galpha x GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.

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Section: Discussionmentioning

confidence: 99%

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange

et al. 2001

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“…The number of ligands which exert their activity through a membrane bound G-protein coupled receptor is steadily increasing. Although there are differences in binding sites with small ligands (e.g., catecholamines, norepinephrine, adenosine, epinephrine) binding within the transmembrane helices, while peptide ligands bind somewhere in the extracellular domain, the mode of action is similar; ligand binding leads to the activation of a G-protein. ,, Peptide fragments of several G-protein coupled receptors are reported to modulate receptor/G-protein coupling and/or to directly stimulate GDP-GTP exchange of G-proteins. − In many of these studies peptides derived from the third intracellular loop (I3) of the G-protein coupled receptors were identified as the major, but not the only, interacting domain with the G-proteins. An example is peptide Q a tetradecapeptide that corresponds to the carboxy-terminal region of I3 of the α 2a -adrenergic receptor. , Peptide Q lowers binding affinity for radiolabeled agonist and activates the GTPase activity of a purified G-protein.…”

Section: Discussionmentioning

confidence: 99%

“…19,34,35 Peptide fragments of several G-protein coupled receptors are reported to modulate receptor/ G-protein coupling and/or to directly stimulate GDP-GTP exchange of G-proteins. [36][37][38][39][40][41][42][43][44][45] In many of these studies peptides derived from the third intracellular loop (I3) of the G-protein coupled receptors were identified as the major, but not the only, interacting domain with the G-proteins. An example is peptide Q a tetradecapeptide that corresponds to the carboxy-terminal region of I3 of the R 2a -adrenergic receptor.…”

Section: Discussionmentioning

confidence: 99%

Peptide Mimetic of the Third Cytoplasmic Loop of the PTH/PTHrP Receptor

et al. 1996

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The third cytoplasmic loop of seven transmembrane helix receptors is important both for the coupling to and activation of the heterotrimeric G-protein. Here, the structural characterization of two peptides containing the sequence of the putative third cytoplasmic loop of the parathyroid hormone/parathyroid hormone related peptide receptor in aqueous solution and in the presence of micelles is presented. The 27-amino acid peptide has been examined both in a linear form and cyclized with a linker of 8 methylenes designed to maintain a distance of 12 Å between the N- and C-termini, modeling the distance observed between transmembrane α-helices in bacteriorhodopsin. In aqueous solution both peptides are relatively unstructured. In contrast, in the presence of sodium dodecylsulfate micelles the linear peptide adopts a well-defined extended conformation with approximately 30% α-helix. The cyclic peptide possesses an N-terminal, ten amino acid α-helical domain followed by seven amino acids which protrude out in a well-defined loop. These results indicate the importance of the membrane environment and the role of the structural constraint achieved by cyclization; the octamethylene linker restricts the peptide to conformations possible while attached to transmembrane helices V and VI thus mimicking the conformation of the loop in the native receptor.

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“…Preparation of Rho and Rho Mutants. Disk membranes were isolated from bovine rod outer segments, and Rho was solubilized and purified on a Con A-Sepharose column in n-octyl β-D-glucoside as described (29,30). Purified Rho was dialyzed against 1 mM sodium phosphate buffer (pH 6.5).…”

Section: Methodsmentioning

confidence: 99%

Spectroscopic Evidence for Interaction between Transmembrane Helices 3 and 5 in Rhodopsin

1998

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Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.

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Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.

Cited by 40 publications

References 30 publications

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange

Peptide Mimetic of the Third Cytoplasmic Loop of the PTH/PTHrP Receptor

Spectroscopic Evidence for Interaction between Transmembrane Helices 3 and 5 in Rhodopsin

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Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.

Cited by 40 publications

References 30 publications

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg2+ “Lock” after GTP/GDP Exchange

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg2+ “Lock” after GTP/GDP Exchange

Peptide Mimetic of the Third Cytoplasmic Loop of the PTH/PTHrP Receptor

Spectroscopic Evidence for Interaction between Transmembrane Helices 3 and 5 in Rhodopsin

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Product

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Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange

Rapid Irreversible G Protein Alpha Subunit Misfolding Due to Intramolecular Kinetic Bottleneck that Precedes Mg²⁺ “Lock” after GTP/GDP Exchange