Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Shimada, Masako; Chen, Xin; Cvrk, Tomas; Hilfiker, Helene; Parfenova, Maria; Segre, Gino V.

doi:10.1074/jbc.m204166200

Cited by 25 publications

(20 citation statements)

References 87 publications

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“…An epitope tag comprising codons encoding a 9 amino acid sequence of rhodopsin (rho-tag) was introduced at the C-terminal end of the receptor sequence for the affinity purification of the receptor. The rho epitope was first used successfully in a purification scheme for the parathyroid hormone receptor [14] and subsequently has been used for purification of other GPCRs [5]. After sequence confirmation, the resulting plasmid was digested with Bam HI and EcoRI, ligated into p424GPD overexpression vector [15], and digested with the same restriction enzymes generating pBKY1.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…However, a direct comparison of affinity of the purified receptor between our study and that using a fluorescent ligand was not possible due to the difference in the ligands used and to the fact that the binding of the fluorescent analog was not measured in cell membranes from HEK293 cells or yeast. The difference in α-factor affinity for purified Ste2p-rho compared to its affinity for Ste2p-rho in cell membranes ( Figure 2A) may result from either the absence of G-proteins, which are known to be required for the high affinity state of Ste2p [29] and other GPCRs such as the parathyroid hormone receptor [14,30], in the purified Ste2p-rho sample, or differences in the environment between Ste2p solubilized in detergent as opposed to its phospholipid-embedded state in native membranes. Scatchard analysis of binding (data not shown) indicated that ~40 % of the purified receptor retained binding activity (Table 1), if it is assumed that each receptor has one ligand binding site.…”

Section: Ligand Binding Analysismentioning

confidence: 99%

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Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Lee

Jung

Son

et al. 2007

Protein Expression and Purification

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We present an example of expression and purification of a biologically active G protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR α-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 μg of purified α-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified α-factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the α-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

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Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Ligand Binding Analysismentioning

confidence: 99%

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Lee

Jung

Son

et al. 2007

Protein Expression and Purification

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“…The specific antibody can be directed either against the receptor [39,[68][69][70][71][72][73][74] or against a sequence fused to the recombinant receptor. Several tags are often used as epitopes for immunoaffinity purification, such as the rhodopsin tag, which consists of the C-terminal 9 amino acids of bovine rhodopsin (also known as the rho tag), [33,36,75,76], or the FLAG tag, which is the 8-9 amino acid leader peptide of the gene-10 product from bacteriophage T7 [26,43,48,61,62,77,78]. For this latter peptide, the affinity of the corresponding monoclonal antibody is Ca 2+ dependent, and elution of the bound receptor can either be performed with EDTA or the FLAG peptide.…”

Section: Immunoaffinity-based Chromatographymentioning

confidence: 99%

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

Sarramegna

Muller

Milon

et al. 2006

Cell. Mol. Life Sci.

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G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.

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“…10,11 Given its high specificity, it has been widely used in studies addressing structure and function of rhodopsin, 12 as well as a reliable fusion tag for purification of nonrelated receptors. 13,14 In mice, it has been used for the detection of rhodopsin 15 and its subcellular distribution 16 during retinal degeneration. This antibody is commercially available (ab5417; Abcam, Cambridge, UK; R5403; Sigma-Aldrich, St. Louis, MO; sc-57,432; SCBT, Santa Cruz, CA; MAB5356; Millipore, Billerica, MA) and according to the supplier is used to detect rhodopsin also in zebrafish.…”

Section: Resultsmentioning

confidence: 99%

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

Yin

Brocher

Linder

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration.METHODS. The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish.RESULTS. We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones.CONCLUSIONS. Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish. (Invest Ophthalmol Vis Sci. 2012;53:4943-4951)

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Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Cited by 25 publications

References 87 publications

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

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