Coupling and Activation of the β1 Adrenergic Receptor - The Role of the Third Intracellular Loop.

G protein-coupled receptors (GPCRs) belong to the most diverse group of membrane receptors with a conserved structure of seven transmembrane (TM) α-helices connected by intracellular and extracellular loops. Intracellular loop 3 (ICL3) connects TM5 and TM6, the two helices shown to play significant roles in receptor activation. Herein, we investigate the activation and signaling of the β adrenergic receptor (βAR) using mass spectrometry (MS) with a particular focus on the ICL3 loop. First, using native MS, we measure the extent of receptor coupling to an engineered Gα subunit (mini G) and show preferential coupling to βAR with an intact ICL3 (βAR_ICL3) compared to the truncated βAR. Next, using hydrogen-deuterium exchange (HDX)-MS, we show how helix 5 of mini G reports on the extent of receptor activation in the presence of a range of agonists. Then, exploring a range of solution conditions and using comparative HDX, we note additional HDX protection when ICL3 is present, implying that mini G helix 5 presents a different binding conformation to the surface of βAR_ICL3, a conclusion supported by MD simulation. Considering when this conformatonal change occurs we used time-resolved HDX and employed two functional assays to measure GDP release and cAMP production, with and without ICL3. We found that ICL3 exerts its effect on G through enhanced cAMP production but does not affect GDP release. Together, our study uncovers potential roles of ICL3 in fine-tuning GPCR activation through subtle changes in the binding pose of helix 5, only after nucleotide release from G.