State-Dependent Disulfide Cross-Linking in Rhodopsin

Yu, Hwa‐Lung; Kono, Masahiro; Oprian, Daniel D.

doi:10.1021/bi990948+

Cited by 45 publications

(45 citation statements)

References 22 publications

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“…Disulfide cross-linking approaches have been used previously to investigate light-induced structural changes in the photoreceptor, rhodopsin (9,(25)(26)(27)(28)(29). However, this is the first study demonstrating the usefulness of a disulfide mapping strategy to investigate activity-dependent structural changes in a hormone-activated GPCR.…”

Section: Discussionmentioning

confidence: 98%

“…To address this issue, we decided to employ Cys substitution mutagenesis followed by disulfide cross-linking of Cys residues that are adjacent to each other in the three-dimensional structure of the receptor. This approach has been used successfully in the past to monitor dynamic changes in a number of different membrane proteins including various bacterial chemoreceptors (21-24) and, more recently, bovine rhodopsin (9,(25)(26)(27)(28)(29).To examine the potential proximity of the cytoplasmic ends of TM III and TM VI, we recently described a cross-linking protocol involving the use of a series of Cys-substituted mutant M 3 muscarinic receptors (30). The observed disulfide crosslinking patterns suggested that the cytoplasmic surface of the M 3 receptor protein is highly dynamic.…”

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confidence: 99%

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confidence: 99%

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Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Ward

Hamdan

Bloodworth

et al. 2002

Journal of Biological Chemistry

135

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The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M 3 muscarinic receptor, a prototypical G q -coupled receptor, as a model system. It is known that a tyrosine residue (Tyr 254 ) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M 3 receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M 3 receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys 484-Ser 493 at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M 3 receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing aninsitudisulfidecross-linkingstrategytoexamineagonistdependent dynamic structural changes in a G proteincoupled receptor. G protein-coupled receptors (GPCRs)1 constitute the largest class of signaling molecules in the mammalian genome (1-3).All GPCRs are predicted to share a conserved molecular architecture consisting of a bundle of seven ␣-helically arranged transmembrane domains (TM I-VII) linked by alternating intracellular and extracellular loops (Fig. 1). Despite the remarkable structural diversity of the ligands that exert their physiological functions via interaction with specific classes of GPCRs, all GPCRs are thought to share a conserved mechanism of activation. Several lines of evidence indicate that the binding of ligands to the extracellular side of the receptor leads to changes in the arrangement of distinct TM helices, which are then propagated to the intracellular surface of the receptor, thus enabling the receptor to recognize and activate specific classes of heterotrimeric G proteins (4 -7).Accumulating evidence suggests that GPCR activation may involve a change in the relative disposition of TM III and VI (4 -11). Elegant site-directed spin labeling studies (9) carried out with t...

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Section: Discussionmentioning

confidence: 98%

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confidence: 99%

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confidence: 99%

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Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Ward

Hamdan

Bloodworth

et al. 2002

Journal of Biological Chemistry

135

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“…Disulfides have previously been successfully used to restrict intramolecular mobility and probe conformational change in bacterial chemoreceptors (13,14) and rhodopsin (15), as well as to increase the stability of proteins (16). However, efforts to lock in active conformations with disulfides have been challenging.…”

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confidence: 99%

“…However, efforts to lock in active conformations with disulfides have been challenging. Rhodopsin has thus far been locked only in the inactive form (15); the bacterial aspartate receptor has been locked in forms with up to 4-fold higher enzymatic activity and 8-fold higher affinity for aspartate (17).…”

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Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activityin vivo

Shimaoka

Palframan

et al. 2001

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

210

258

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Toward the active conformations of rhodopsin and the β₂‐adrenergic receptor

Gouldson¹,

et al. 2004

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Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.

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State-Dependent Disulfide Cross-Linking in Rhodopsin

Cited by 45 publications

References 22 publications

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activityin vivo

Toward the active conformations of rhodopsin and the β₂‐adrenergic receptor

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State-Dependent Disulfide Cross-Linking in Rhodopsin

Cited by 45 publications

References 22 publications

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activityin vivo

Toward the active conformations of rhodopsin and the β2‐adrenergic receptor

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Toward the active conformations of rhodopsin and the β₂‐adrenergic receptor