Abstract

Traditionally, G protein–coupled receptor antagonists are classified as competitive or noncompetitive and surmountable or insurmountable based on functional antagonism. P2Y₁ receptor (P2Y₁R) structures showed two antagonists binding to two spatially distinct sites: nucleotide MRS2500 (orthosteric, contacting the helical bundle) and urea BPTU (allosteric, on the external receptor surface). However, the nature of their P2Y₁R antagonism has not been characterized. Here we characterized BPTU antagonism at various signaling pathways activated by structurally diverse agonists. BPTU rightward shifted the concentration-response curves of both 2-methylthioadenosine 5′-diphosphate trisodium salt and MRS2365 (5′-diphosphates) in some signaling events, such as extracellular signal-regulated kinase 1/2 and label free, in a parallel manner without affecting the maximum agonist effect (E_max) but antagonized insurmountably (suppressed agonist E_max) in signaling events such as guanosine 5′-3-O-(thio)triphosphate binding and β-arrestin2 recruitment. However, with dinucleotide Ap4A as an agonist, BPTU suppressed the E_max insurmountably in all signaling pathways. By comparison, MRS2500 behaved as surmountable antagonist rightward-shifting concentration-response curves of all three agonists in a parallel manner for all signaling pathways measured. Thus, we demonstrated a previously undocumented phenomenon that P2Y₁R antagonism patterns could vary in different signaling pathways, which could be related to conformational selection, signaling amplification, and probe dependence. This phenomenon may apply generally to other receptors considering that antagonism by a specific ligand is often not compared at multiple signaling pathways. Thus, antagonism can be surmountable or insurmountable depending on the signaling pathways measured and the agonists used, which should be of broad relevance to drug discovery and disease treatment.