Distinct Signaling Patterns of Allosteric Antagonism at the P2Y₁ Receptor

Abstract

Traditionally, G protein-coupled receptor antagonists are classified as competitive or noncompetitive and surmountable or insurmountable based on functional antagonism. P2Y 1 receptor (P2Y 1 R) 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 P2Y 1 R 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 59-diphosphate trisodium salt and MRS2365 (59-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 59-3-O-(thio)triphosphate binding and b-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 P2Y 1 R 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.

Update of P2Y receptor pharmacology: IUPHAR Review 27

“…The principal G protein coupling of each of the P2Y receptor subtypes are indicated in Table 1. However, each of the P2Y receptors can be associated with multiple G proteins and other signalling pathways, as was shown for P2Y 1 receptors, including both G protein‐dependent and G protein‐independent, for example, β‐arrestin, pathways (Gao & Jacobson, 2017). Some of the P2Y receptors additionally couple to G 12/13 (P2Y 2 and P2Y 6 ) or G s (P2Y 11 ).The P2Y receptors regulate MAPK pathways and consequently affect cell survival and proliferation (Miras Portugal et al, 2019).…”

“…The non‐nucleotide compound MRS2950 ( 11 ) was identified by virtual screening of the modelled receptor orthosteric site (Costanzi, Kumar, Balasubramanian, Harden, & Jacobson, 2012). Another non‐nucleotide compound, BPTU ( N ‐[2‐[2‐(1,1‐dimethylethyl)phenoxy]‐3‐pyridinyl]‐ N ′‐[4‐(trifluoromethoxy)phenyl]urea, 12 ) and its analogues act as an allosteric inhibitors (Gao & Jacobson, 2017; Peng et al, 2018; Wang, Qiao, et al, 2013; Yuan et al, 2016; Zhang, Gao, et al, 2015).…”

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells

“…Our results indicate that I-191 is a negative allosteric modulator that may bind at a site on PAR2 that is different from that occupied by 2f-LIGRL-NH 2 or tethered agonist. Third, allosteric behavior is context dependent, and these ligands may behave differently under different conditions, in other cell types and signaling pathways (Gao and Jacobson, 2017). The description here of allosteric modulation may be context and system dependent.…”

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y2 antagonist

“…This is relevant because it is now clear that the apparent mode of antagonism of NAMs can vary depending upon the agonist used, the signalling pathway studied, and the extent of signalling amplification. For example, BPTU, a P2Y 1 NAM, caused a progressive rightwards parallel shift of the 2‐methylthioADP CRC, with no decrease in maximum, when inositol phosphate production or ERK1/2 stimulation was measured, but suppressed the maximum when β‐arrestin2‐mediated P2Y 1 receptor internalisation was studied (Gao & Jacobson, ). Furthermore, BPTU had a different activity profile against another P2Y 1 agonist.…”