Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGF_xxxa or VEGF_xxxb isoforms. Alternative splicing events at exons 5–7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF₁₆₅a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

Bioluminescence resonance energy transfer, from the substrate of a luciferase fused with a G protein–coupled receptor to a fluorescent dye covalently linked to a receptor ligand, allows the profiling of ligand affinity and binding kinetics. Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

G protein-coupled receptors modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This ‘biased signaling’ paradigm requires that we now characterize physiological signaling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signaling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models.