Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin α-Subunit upon Binding a Soluble Mimic of Light-Activated Rhodopsin

Brabazon, Danielle M.; Abdulaev, N.G.; Marino, John P.; Ridge, Kevin D.

doi:10.1021/bi0268899

Cited by 20 publications

(18 citation statements)

References 68 publications

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“…Specifically, HPTRX/CDEF displayed virtually identical kinetics of G t activation when compared with light-activated ROS rhodopsin (38), suggesting that the CD and EF loops in the fusion protein can adopt a conformation that is a close approximation to that present in R*. It was also shown that HPTRX/CDEF bound G t␣ (340 -350) peptides (52) inducing structural changes in these peptides analogous to those observed previously in studies with native, light-activated rhodopsin in ROS membranes (28 -30).…”

Section: Biochemical Analysis Of a Complex Between A Solublementioning

confidence: 95%

“…Biochemical and structural studies (38,52) have shown that some of these rhodopsin cytoplasmic fragment/thioredoxin fusion proteins exhibit properties similar to those of R*. In particular, the HPTRX/ CDEF fusion protein, which contains tandemly linked segments from the CD (amino acids 132-154) and EF (amino acids 231-252) loops of rhodopsin (Fig.…”

Section: Biochemical Analysis Of a Complex Between A Solublementioning

confidence: 99%

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Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit

Ridge

Abdulaev

Zhang

et al. 2006

Journal of Biological Chemistry

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Overall, these results demonstrated that R*-induced changes in G ␣ can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation. G-protein coupled receptors (GPCRs)3 represent a diverse group of seven transmembrane (TM) helix receptors that require agonist-dependent activation to initiate heterotrimeric (␣␤␥) G-protein-mediated intracellular signaling cascades. GPCR activation of cognate G-proteins are the first steps in cellular communication pathways responsible for signaling cascades that mediate vision, olfaction, taste, and the action of numerous hormones and neurotransmitters (1). Activation of a G-protein by its agonist-stimulated GPCR (R*) requires the propagation of structural signals from the receptor-binding interface to the guanine nucleotide-binding pocket. The structural basis for the interaction of a GPCR with its cognate G-protein and the subsequent activation of the G-protein by R* are not fully understood.Using signaling of the retinal G-protein transducin (G t ) by rhodopsin as a model system, we are applying solution NMR methods to track changes in the G-protein ␣-subunit (G ␣ ) associated with activated R* interactions. Rhodopsin, the rod cell photoreceptor involved in dimlight vision, represents one of the best studied GPCRs in terms of structure and function (2, 3). Photon capture triggers cis 3 trans isomerization of the retinal chromophore, which initiates structural changes in the TM helices resulting in the formation of the light-activated signaling state metarhodopsin II, R*. This is accompanied by functionally significant changes at the cytoplasmic surface that leads to the formation of binding and activation sites for several signaling proteins, including G t (4 -10). Crystal structures for the inactive (dark) state of rhodopsin have provided a detailed view of the retinal binding site and the cytoplasmic region (11-15). Although remarkably informative, the crystal structures provide few solid insights into the mechanism of signal transfer from R* to G t .Binding of heterotrimeric G-proteins to activated GPCRs requires the presence of both G ␣ and G-protein ␤␥-subunits (G ␤␥ ). The following three regions on the ␣-subunit of G t (G t␣ ) are known to be important for receptor interactions; the amino-terminal 23 residues, an internal sequence from amino acids 305-315, and the carboxyl-terminal 11 amino acids (16 -18). Upon binding to R*, G t␣ is thought to undergo structural changes in both the amino-and carboxyl-terminal regions. High resolution crystal structures of G ␣ subunits, including G t␣ (19 -24) 3 The abbreviations used are: GPCR, G-protein coupled receptor; TM, transmembrane; R*, the agonist activated form of a GPCR; GTP␥S, guanosine 5Ј-O-3-thiotriphosphate; CMC, critical micelle concentration; DM, n-dodecyl ␤-D-maltopyranoside; ChiT, prodomain released chimeric G ␣ ; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; HPTRX/CDEF, protein chimera containing segments from the CD and EF loops of bovine opsin g...

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Section: Biochemical Analysis Of a Complex Between A Solublementioning

confidence: 95%

Section: Biochemical Analysis Of a Complex Between A Solublementioning

confidence: 99%

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit

Ridge

Abdulaev

Zhang

et al. 2006

Journal of Biological Chemistry

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“…The hypothesis was that TM6 movement exposes a binding site for the transducin Gα subunit (GTα) C-terminal tail (GTα C-term). The GTα C-term was known to be required for rhodopsin-transducin interaction [119], and peptide analogues of this region bind rhodopsin with high affinity [120] and become structured in the process [121, 122]. …”

Section: From Molecular To Cellular Insight In Visual Rhodopsin Fumentioning

confidence: 99%

Fluorescence spectroscopy of rhodopsins: Insights and approaches

Alexiev

Farrens

2014

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy.

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“…Several lines of evidence show that the rhodopsin cytoplasmic face directly interacts with the G T␣ C terminus. For example, peptides and peptide analogues corresponding to the G T␣ C terminus can bind and stabilize MII rhodopsin (18 -20), and binding induces structure in these peptides (21)(22)(23)(24). Rhodopsin mutagenesis studies show that residues in intracellular loops i2 and i3 are important in this interaction (25)(26)(27)(28), and a photocross-linking probe on loop i3 at residue 240 on rhodopsin cross-links to this region of G T␣ (29).…”

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confidence: 99%

Rhodopsin Activation Exposes a Key Hydrophobic Binding Site for the Transducin α-Subunit C Terminus

Janz

Farrens

2004

Journal of Biological Chemistry

103

145

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Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin ␣-subunit (G T␣ ) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G T␣ C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G T␣ C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G T␣ C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/ bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.Visual signal transduction begins with light-induced structural changes in the photoreceptor rhodopsin. These changes convert this model G-protein-coupled receptor (GPCR) 1 into an active form (called MII), which couples with and activates the G-protein transducin, thus initiating the biochemical cascade that results in vision (1-4).A key step in MII formation involves an outward movement of transmembrane helix 6 in the rhodopsin cytoplasmic face (5). This movement increases the exposure of residues on the inner face of this helix (6 -8) ( Fig. 1) and is needed for transducin (G T ) activation (5, 9). A similar movement appears to occur in ligand-activated GPCRs (10 -14), suggesting that helix 6 movement represents a conserved event in GPCR signaling (15).How might this movement enable coupling with transducin? We and others have hypothesized that helix 6 movement opens a cleft that provides a binding site for the transducin ␣ subunit C-terminal tail (G T␣ C terminus) (7,16,17) (Fig. 1). Several lines of evidence show that the rhodopsin cytoplasmic face directly interacts with the G T␣ C terminus. For example, peptides and peptide analogues corresponding to the G T␣ C terminus can bind and stabilize MII rhodopsin (18 -20), and binding induces structure in these peptides (21-24). Rhodopsin mutagenesis studies show that residues in intracellular loops i2 and i3 are important in this interaction (25-28), and a photocross-linking probe on loop i3 at residue 240 on rhodopsin cross-links to this region of G T␣ (29). However, direct evidence for where and how the G T␣ C terminus and rhodopsin interact is lacking.In the present manuscript we set out to test whether the G T␣ C terminus binds to the cleft formed during rhodopsin activation through the use of a combination of site-directed labeling and fluorescence methods. Using these approaches, we were ab...

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Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin α-Subunit upon Binding a Soluble Mimic of Light-Activated Rhodopsin

Cited by 20 publications

References 68 publications

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit

Fluorescence spectroscopy of rhodopsins: Insights and approaches

Rhodopsin Activation Exposes a Key Hydrophobic Binding Site for the Transducin α-Subunit C Terminus

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