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. 2013 Mar 28;56(6):2406-14.
doi: 10.1021/jm301749y. Epub 2013 Mar 12.

Colloidal aggregation causes inhibition of G protein-coupled receptors

Maria F Sassano  1 , Allison K Doak, Bryan L Roth, Brian K Shoichet
Affiliations
Free PMC article

Colloidal aggregation causes inhibition of G protein-coupled receptors

Maria F Sassano et al. J Med Chem. .
Free PMC article

Abstract

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field.

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Figures

Figure 1
Figure 1
Aggregating small molecules used in this study.
Figure 2
Figure 2
Centrifugation treatment. Concentration–response curves are shown for CCR4, CX3CR1, and V2R in the β-arrestin recruitment assay. Circles represent data points for aggregators without treatment; squares represent data points after centrifugation (±SEM). Calculated pIC50 and IC50 values are displayed in Table 1.
Figure 3
Figure 3
Particle formation by colloid-forming molecules measured by DLS in the (A) absence of Tween-80 and (B) presence of Tween-80.
Figure 4
Figure 4
Detergent treatment. Concentration–response curves are shown for CCR4, CX3CR1, and V2R in the β-arrestin recruitment assay. Circles represent data points for aggregators without treatment; squares represent data points after addition of detergent (±SEM). Calculated pIC50 and IC50 values are displayed in Table 1.
Figure 5
Figure 5
Concentration–response curves for CCR4 in the β-arrestin recruitment assay in the absence of the CCR4 agonist, CCL22 (±SEM). The depression of basal activity by the colloid-formers in the absence of the agonist suggests inverse agonism.
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