Molecular Determinants of the Reversible Membrane Anchorage of the G-Protein Transducin

Seitz, Harald R.; Heck, M.; Hofmann, Klaus Peter; Alt, Thomas; Pellaud, Jérôme; Seelig, Anna

doi:10.1021/bi990298+

Cited by 48 publications

(52 citation statements)

References 40 publications

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“…The hydrophobic free energy of binding of myristoylated peptides was determined as ⌬G 0 ϭ Ϫ7.9 kcal/mol (49), in close agreement with that of myristoylated proteins such as hisactophilin I and II (22), and that of the ␣-subunit of transducin (50). The free energy of binding of farnesylated peptides was determined as ⌬G 0 ϭ Ϫ9.3 kcal/mol (51) again in good agreement with that of the farnesylated ␤␥-subunit of transducin (50).…”

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confidence: 58%

Phospholipid Binding of Synthetic Talin Peptides Provides Evidence for an Intrinsic Membrane Anchor of Talin

Seelig

Blatter

Frentzel

et al. 2000

Journal of Biological Chemistry

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Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/ m). S19 partitions into charged monolayers/bilayers with a penetration area A p ‫؍‬ 140 ؎ 30 Å 2 and a free energy of binding of ⌬G 0 ‫؍‬ ؊5.7 kcal/mol, thereby forming a partially ␣-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A p ‫؍‬ 148 ؎ 23 Å 2 and A p ‫؍‬ 160 ؎ 15 Å 2 , respectively, forming an ␣-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is ⌬G 0 ‫؍‬ ؊9.4 kcal/mol with a hydrophobic contribution of ⌬G h ‫؍‬ ؊7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of ⌬G h ‫؍‬ ؊1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.Talin is a widespread actin-binding protein present in focal cell adhesions and ruffling membranes of moving cells (1, 2). In fibroblasts, talin binding to lipid membranes is associated with the establishment of a signaling cascade, mediated either by integrins (3, 4) leading to the formation of focal adhesions or, alternatively, by layilin (5) leading to a nucleation of actin assembly in membrane ruffles. In platelets, talin redistributes from the cytoplasm to the membrane during activation (6) where it colocalizes with the GPIIb/IIIa complex (7). Talin analogues have been identified in lower organisms like Dictyostelium (8) and Caenorhabditis elegans (9), the N termini and C termini being most preserved.Reasons to assume that talin is involved in a polarized assembly of the actin cytoskeleton by nucleating actin filament growth at lipid interfaces (10, 11) were provided from the finding that Dictyostelium mutants, which lack the entire protein, are massively impaired in adhesion and motility (12), and HeLa cells, when down-regulated in talin expression by antisense RNA, exhibit a reduced rate in cell spreading (13). Antibodies directed against talin, when microinjected were shown to inhibit fibroblast migration (14).Some of talin's functions have been attributed to specific protein domains. Calpain or thrombin cleavage in vitro yields two parts with 190 and 47 kDa, respectively. The C-terminal 190-kDa portion was shown to carry the actin binding sites (15) in form of a conserved sequence, the (I/L)WEQ module (16), and to be responsible f...

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confidence: 58%

Phospholipid Binding of Synthetic Talin Peptides Provides Evidence for an Intrinsic Membrane Anchor of Talin

Seelig

Blatter

Frentzel

et al. 2000

Journal of Biological Chemistry

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“…Fast and immediate docking is enabled by the precise orientation of the G protein to the membrane surface. The membrane anchor of the G protein made up of the myristoyl/ farnesyl modifications at the ␣-and ␥-subunits, respectively (25,29,30), now can play a decisive role in the initial encounter between the proteins. Evidence for a sequential interaction between different binding sites on G t and R*, which includes such a docking process, recently was presented (30,31).…”

Section: Discussionmentioning

confidence: 99%

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Ernst

Gramse

Kolbe

et al. 2007

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

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G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (G␣␤␥).Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with G␣␤␥. To investigate whether a G protein-coupled receptor monomer can activate G␣␤␥, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G t) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchange in G t. Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G t was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G t interaction yielded a maximum of Ϸ50 Gt molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of Gt with an activated rhodopsin monomer is sufficient for fully functional Gt activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated. enzyme catalysis ͉ nucleotide exchange ͉ light scattering

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“…Importantly, the membrane affinity of the ␣␤␥ heterotrimer is much higher than the affinities of individual ␣ and ␤␥ subunits (32,33). Transducin activation by photoexcited rhodopsin causes dissociation of the subunits from one another, thus reducing their membrane affinity.…”

Section: Discussionmentioning

confidence: 99%

Phosducin Facilitates Light-driven Transducin Translocation in Rod Photoreceptors

Sokolov

Strissel

Leskov

et al. 2004

Journal of Biological Chemistry

108

168

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Phosducin is a photoreceptor-specific protein known to interact with the ␤␥ subunits of G proteins. In pursuit of the function of phosducin, we tested the hypothesis that it regulates the light-driven translocation of G protein transducin from the outer segments of rod photoreceptors to other compartments of the rod cell. Transducin translocation has been previously shown to contribute to rod adaptation to bright illumination, yet the molecular mechanisms underlying the translocation phenomenon remain unknown. In this study we provide two major lines of evidence in support of the role of phosducin in transducin translocation. First, we have demonstrated that transducin ␤␥ subunits interact with phosducin along their entire intracellular translocation route, as evident from their co-precipitation in serial tangential sections from light-adapted but not darkadapted retinas. Second, we generated a phosducin knockout mouse and found that the degree of lightdriven transducin translocation in the rods of these mice was significantly reduced as compared with that observed in the rods of wild type animals. In knockout animals the translocation of transducin ␤␥ subunits was affected to a larger degree than the translocation of the ␣ subunit. We also found that the amount of phosducin in rods is sufficient to interact with practically all of the transducin present in these cells and that the subcellular distribution of phosducin is consistent with that of a soluble protein evenly distributed throughout the entire rod cytoplasm. Together, these data indicate that phosducin binding to transducin ␤␥ subunits facilitates transducin translocation. We suggest that the mechanism of phosducin action is based on the reduction of transducin affinity to the membranes of rod outer segments, achieved by keeping the transducin ␤␥ subunits apart from the ␣ subunit. This increased solubility of transducin would make it more susceptible to translocation from the outer segments.Vertebrate photoreceptors are highly specialized neurons responsible for the reception and primary processing of visual information. The outer segment compartment of the photoreceptor contains large amounts of the proteins involved in light detection and in the generation of the visual signal (for review, see Refs. 1-4). Photons entering the outer segment are absorbed by rhodopsin, which triggers sequential activation of many molecules of the photoreceptor-specific heterotrimeric G protein, transducin. Transducin activation consists of GTP binding to its ␣ subunit followed by dissociation of the ␣ subunit from the transducin ␤␥ subunits and the subsequent stimulation of the downstream effector, cGMP phosphodiesterase. Signaling persists until GTP is hydrolyzed and transducin subunits re-associate into a heterotrimer. In dark-adapted rods most of the transducin is located in the outer segments. However, prolonged exposures to bright light cause massive transducin translocation from rod outer segments to other subcellular compartments of the rod (5-10). In a recent st...

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Molecular Determinants of the Reversible Membrane Anchorage of the G-Protein Transducin

Cited by 48 publications

References 40 publications

Phospholipid Binding of Synthetic Talin Peptides Provides Evidence for an Intrinsic Membrane Anchor of Talin

Phospholipid Binding of Synthetic Talin Peptides Provides Evidence for an Intrinsic Membrane Anchor of Talin

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Phosducin Facilitates Light-driven Transducin Translocation in Rod Photoreceptors

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