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            <sup>19</sup>
            F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

Loewen, Michèle C.; Klein‐Seetharaman, Judith; Getmanova, E. V.; Reeves, Philip J.; Schwalbe, Harald; Khorana, H G

doi:10.1073/pnas.051633098

Cited by 86 publications

(87 citation statements)

References 28 publications

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“…Photoaffinity labeling of receptor regions and residues adds still more direct information, and the most detailed, yet least common, insights are to be found in the application of biophysical techniques. Although biophysical studies like NMR and crystallography can only be applied to molecules that can be purified to homogeneity in large amounts, like rhodopsin (2,25), fluorescence in principle can be applied much more generally across the G protein-coupled receptor superfamily.…”

Section: Lifetime Measurements For Fluorescent Ligandsmentioning

confidence: 99%

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Harikumar

Pinon

Wessels

et al. 2002

Journal of Biological Chemistry

103

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Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa 488 , nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.Cell surface receptors present on essentially every excitable cell of the body are important targets for pharmacotherapy. A detailed understanding of the structure of these molecules and the molecular basis of their activation should contribute to the rational design and refinement of ligands for these receptors. Receptor-bound, environmentally sensitive fluorescent reporters can provide information regarding ligand-binding domains (1). In this work, we utilize this approach to gain insight into agonist-and antagonist-binding domains of the type A cholecystokinin (CCK) 1 receptor, a physiologically important member of the rhodopsin/␤-adrenergic receptor family of guanine nucleotide-binding protein (G protein)-coupled receptors.The superfamily of G protein-coupled receptors represents the largest group of membrane receptors. They are remarkable for the diversity in structure of the natural agonist ligands that can activate them and initiate intracellular signaling cascades. These range in size from small photons and odorants to peptides, proteins, and even large viral particles. Tertiary structure determination by x-ray crystallography has provided the most incisive insight into the structure of superfamily members, which bind small ligands in the intramembranous helical bundle domain ...

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Section: Lifetime Measurements For Fluorescent Ligandsmentioning

confidence: 99%

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Harikumar

Pinon

Wessels

et al. 2002

Journal of Biological Chemistry

103

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“…To meet these needs, stable mammalian cell lines for high-level expression of the opsin gene and its mutants were developed (1,2). The application of NMR spectroscopy to structure-function studies of rhodopsin thus became possible (3)(4)(5)(6). Recently, evidence has been accumulating that certain gene products either fail to be expressed in adequate amounts in the constitutive expression systems above or that viable stable cell lines cannot be generated for certain mutant opsin genes.…”

mentioning

confidence: 99%

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

Reeves

Kim

Khorana

2002

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

Self Cite

192

196

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Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg͞liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H 2L d promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q͞E134Q͞M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.G protein-coupled receptors ͉ HEK293S cells ͉ cytomegalovirus promoter ͉ glycosylation ͉ sodium butyrate C ertain studies on rhodopsin and its mutants require relatively large amounts of purified proteins. To meet these needs, stable mammalian cell lines for high-level expression of the opsin gene and its mutants were developed (1, 2). The application of NMR spectroscopy to structure-function studies of rhodopsin thus became possible (3-6). Recently, evidence has been accumulating that certain gene products either fail to be expressed in adequate amounts in the constitutive expression systems above or that viable stable cell lines cannot be generated for certain mutant opsin genes. For example, stable cell lines carrying the rhodopsin kinase gene expressed the kinase at levels much lower than those predicted from the transient transfection experiments (7). Further, we failed to isolate a viable stable cell line for expression of the strongly constitutively active opsin mutant, E113Q͞E134Q͞M257Y. It seems likely that the proteins produced by unregulated expression of the genes above are cytotoxic and, indeed, in certain cases may be lethal. To overcome these difficulties, we wanted to develop a strategy frequently used in bacterial expression systems (8, 9) in which membrane proteins toxic to the bacterial cells are expressed by induction of the desired gene only after achieving growth of cells to nearmaximum density. Previously, this strategy enabled large-scale production in Escherichia coli of numerous toxic bacterio-opsin mutants (10). Therefore, our present aim was to develop an inducible expression system in stable mammalian cell lines.A tetracycline (tet)-regulated gene expression system in mammalian cells was first described by Gossen and Bujard (11). By fusing the bacterial tet repressor (TetR) with the activating domain of the viral protein 16 (VP-16), a tetracycline-controlled transactivator was generated that stimulated transcription of a desired gene from a minimal cytomegalovirus (CMV) promo...

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“…For example, a detergent-solubilized rhodopsin protein that is suitable for solution NMR spectroscopy has been reported (38)(39). The differential dynamics of the rhodopsin protein in solution were revealed by solution NMR (39).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the NMR spectroscopic technique has been recognized as one of the most powerful tools for characterizing the structural and functional relationship of GPCRs in solution (35)(36)(37)(38)(39). For example, a detergent-solubilized rhodopsin protein that is suitable for solution NMR spectroscopy has been reported (38)(39).…”

Section: Discussionmentioning

confidence: 99%

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies

Ruan

Cervantes

2008

Biochemistry

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Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by several different detergents for comparison, and was finally purified to a single band at a nearly perfect ratio of 1:0.9 ± 0.05 (ligand:receptor molecule) in ligand binding using a Ni-column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNAdigestion, DM detergent-extraction, centrifugation and FPLC-purification. The purity and quantity of the purified TP, using the high-yield approach, was suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution 1D and 2D 1 H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.

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Solution ¹⁹ F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

Cited by 86 publications

References 28 publications

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies

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Solution 19 F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

Cited by 86 publications

References 28 publications

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Environment and Mobility of a Series of Fluorescent Reporters at the Amino Terminus of Structurally Related Peptide Agonists and Antagonists Bound to the Cholecystokinin Receptor

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A2 Receptor in Full Size for Structural Studies

Contact Info

Product

Resources

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Solution ¹⁹ F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies