Quantification of The Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.

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High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710000500202 ◽

2000 ◽

Vol 5 (2) ◽

pp. 63-69 ◽

Cited By ~ 75

Author(s):

Michael Allen ◽

Julian Reeves ◽

Geoffrey Mellor

Keyword(s):

Cell Surface ◽

High Throughput ◽

Fluorescence Polarization ◽

Radioligand Binding ◽

Low Cost ◽

G Protein Coupled Receptors ◽

Cell Surface Receptors ◽

Binding Assays ◽

Surface Receptors ◽

G Protein Coupled

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and δ-opioid) and nonpeptide (β-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at β1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.

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Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103257555 ◽

2003 ◽

Vol 8 (5) ◽

pp. 500-510 ◽

Cited By ~ 22

Author(s):

Anke Niedernberg ◽

Sorin Tunaru ◽

Andree Blaukat ◽

Bruce Harris ◽

Evi Kostenis

Keyword(s):

G Protein ◽

Partial Agonist ◽

A Priori ◽

G Protein Coupled Receptors ◽

Binding Assays ◽

Functional Assays ◽

Gtpγs Binding ◽

Sphingosine 1 Phosphate ◽

Intracellular Ca ◽

G Protein Coupled

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P5 receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPγS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca2+ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC50 values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPγS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPγS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. ( Journal of Biomolecular Screening 2003:500-510)

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