Autoregulation of GPCR signalling through the third intracellular loop

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation(1–3). Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling(4). Previous studies focusing on the β(2) adrenergic receptor (β(2)AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling(5–7). Here we derive mechanistic insights into the role of ICL3 in β(2)AR signalling, observing that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor’s G protein-binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, showing that G protein-mimetic effectors bias the exposed states of ICL3 to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signalling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein-selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding the range of known mechanisms by which receptors mediate G protein subtype selective signalling. Furthermore, our collective findings suggest ICL3 as an allosteric site for receptor- and signalling pathway-specific ligands.