COUPLING OF A G‐PROTEIN COUPLED RECEPTOR TO THE AGS3‐Galphai SIGNALING COMPLEX

Abstract

AGS3 and related entities serve as alternative binding partners for Gαi independent of the classical heterotrimer Gαβγ and this interaction is mediated by a 20–25 amino acid motif termed the G‐protein regulatory (GPR). Such a signaling module may function in the context of signal processing through G‐protein coupled receptors or actually regulate intracellular events independent of the classical receptor ‐ heterotrimeric G‐protein – effector model. We report the regulation of the GPR‐Gαi signaling module by a cell surface receptor using bioluminescence resonance energy transfer (BRET). AGS3‐Renilla luciferase (Rluc), and Gαi1‐yellow fluorescent protein (YFP) exhibited robust, specific BRET. AGS3‐Rluc ‐ Gαil‐YFP BRET was inhibited by activation of the α2‐adrenergic receptor (α2 ‐AR), a typical G‐protein coupled receptor that couples to the Gαi/Gαo class of heterotrimeric G‐proteins. The AGS3‐Gαi complex is in close proximity to the receptor as Gαi‐dependent and agonist‐sensitive BRET was observed between AGS3‐Rluc and the α2‐AR‐Venus. AGS3‐Gαi appears to be anchored within a receptor signaling complex at the membrane and the biophysical organization of this complex is stabilized or destabilized by specific conformations of the receptor.