From theory to platelets: unraveling the history and complexities of biased signaling

Abstract

Biased signaling refers to a phenomenon where a ligand preferentially activates 1 signaling pathway over another at the same receptor. It is best described for ligands that selectively activate G protein-coupled receptors through G protein or β-arrestin pathways. The concept of biased signaling has a rich history that has been experimentally characterized in the past 40 years. As early as the 1970s, models of biased signaling suggested that ligand-bound receptors have a rigid structure, whereas free receptors are fluid proteins with multiple potential active states. Recent cell signaling studies demonstrate that ligands block select signaling pathways but amplify others. This suggests that each ligand can stabilize a unique active conformation supporting the proposed model. Additional studies expanded our understanding of biased signaling to include biased receptors and system bias, which consider the impact of genetic differences and cellular context in which the signal is being studied. This is exemplified in platelet biology. Platelets are nonnucleated cells that rely on membrane receptors such as protease-activated receptor 1 (PAR1), PAR4, and Toll-like receptor 4 (TLR4) to facilitate platelet activation. There is now evidence of biased signaling through PAR1, PAR4, and TLR4 in platelets, making them attractive therapeutic targets. Here, we describe the origins of biased signaling theory and explore the concepts of biased agonists and systems through the lens of platelet activation.

Figure 1.

PAR1 and PAR4 cleavage sites. Proteases cleave PAR1 and PAR4 at different sites on their N terminus. The residues in green denote the canonical tethered ligand generated by thrombin. (A) Model of PAR activation mechanism via proteolysis of the N terminus. (B) The amino acid sequence of the N terminus of PAR1 or PAR4. The cleavage sites for thrombin, MMP1, MMP2, APC, and cathepsin G are inidicated. CatG, cathepsin G.

Figure 2.

Biased signaling in PAR1 and PAR4 in platelets. Biased agonists and interactions with other receptors result in biased signaling and platelet responses based on which pathways are favored. MMP2 requires αIIbβ3 as a cofactor to sensitize PAR1 (red) for thrombin signaling. In low concentrations of thrombin, PAR1 is favored over PAR4 (blue) signaling through Gαq and Gα12 pathways. MMP1 biases PAR1 signaling through the Gα12 pathway. PAR4 heterodimerization with P2Y12 (green) induces arrestin pathway signaling. Cathepsin G cleaves PAR4 at an additional site, increases calcium mobilization, and platelet aggregation. CatG, cathepsin G.

Figure 3.

TLR4-biased signaling in platelets. Different chemotypes of LPS trigger either the MyD88-independent or MyD88-dependent pathways in platelets. IKK, NF-κB kinase; IRAK, interleukin-1 receptor–associated kinase; NEMO, NF-κB essential modulator; TAK1, transforming growth factor-β–activated kinase 1; TBK1, TRAF family member–associated NF-κB activator binding kinase-1; TRAF, tumor necrosis factor receptor–associated factor; TRAM, TRIF-related adapter molecule.