Measuring arrestin binding to G Protein Coupled Receptors: BRET and recruitment assays tell different stories.

Abstract

In mammals, visual (Arr1) and cone arrestin (Arr4) target activated photoreceptors, whereas ubiquitous arrestin2 and 3 (Arr2 and Arr3) regulate non‐visual GPCRs. We explored the interaction of Arr1, 2 and 3 with the M1–M2 muscarinic acetylcholine receptors (M1/M2AChR) and the β2‐adrenergic receptor (β2AR) by direct visualization of arrestin recruitment and bioluminescence resonance energy transfer (BRET). Venus‐tagged arrestins were expressed in HEK‐293 cells and imaged after agonist stimulation of co‐transfected M1AChR, M2AChR or β2AR. The formation of puncta corresponding to the receptor‐arrestin complexes recruited to coated pits was observed for M1AChR and β2AR with Arr3, but not Arr1. For BRET assays, the same receptors fused with Renilla luciferase 8 (RLuc8) were co‐expressed in COS‐7 cells with increasing amounts of Venus‐tagged arrestins. In contrast to recruitment assays, all three arrestins yielded saturable BRET signals with near‐identical profiles. Considering that measured affinity of Arr2 and Arr3 for M2AChR or β2AR is much higher than that of Arr1, our results suggest that BRET is less sensitive to differences in receptor preference of arrestins than recruitment assays.NIH grants GM077561, GM081756 (VVG), MH073676 (PJC), and F32 MH087039 (GJD)