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 β₁ adrenergic receptor (β₁AR) 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_s subunit (mini G_s) and show preferential coupling to β₁AR with an intact ICL3 (β₁AR_ICL3) compared to the truncated β₁AR. Next, using hydrogen-deuterium exchange (HDX)-MS, we show how helix 5 of mini G_s 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_s helix 5 presents a different binding conformation to the surface of β₁AR_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_s 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_s.