Ligand-Free Signaling of G Protein Coupled Receptors: Addressing Unresolved questions with Antagonist Probes and Genomics

G protein coupled receptors (GPCRs) exist each in multiple forms and aggregates distributed across various cell compartments, signaling along multiple pathways upon activation by agonists. In addition, pervasive ligand-free GPCR signaling also occurs with multiple functional states. I propose three distinct ligand-free receptor categories: first, low level slippage into active forms (‘spontaneous basal signaling’); second, ‘acutely activated ligand-free signaling’ which sustains signaling after the agonist has dissociated from the receptor; and third, ‘sustained ligand-free signaling’ that is regulated to sustain cellular responses. Studies of the µ opioid receptor differentiate these three receptor forms and suggest that continued agonist stimulation can lead to sustained ligand-free signaling with a role in opioid dependence. The serotonin 5HT_2A receptor also appears to support ligand-free signaling of physiological and pharmacological relevance. Yet, systematic studies of distinct ligand-free receptor forms are scarce. Sustained ligand-free signaling can arise from various mechanisms, such as tethered extracellular peptide regions of GPCRs stabilizing an active receptor-G protein state, demonstrated with the glucagon-like peptide 1 receptor GLP1R. Accurate assays are needed to measure ligand-free signaling along specific pathways, localized to cellular sub-compartments. Neutral antagonists (no effect on ligand-free signaling) and inverse agonists (block ligand-free signaling) distinguish between the various forms of ligand-free signaling and can lead to distinct therapeutic applications, highlighted with opioid, serotonin, and peptide hormone receptors, targeting pain, depression and schizophrenia, and metabolic disorders. GPCR mutations that activate or suppress ligand-free signaling reveal (patho)physiological functions, but genetic effects on distinct ligand-free receptor signaling pathways are largely unresolved. Clarifying ligand-free signaling pathways of GPCRs has the potential to uncover hidden disease risk factors and novel targets for therapeutic interventions.