Local Confinement within Plasma Membrane Nanodomains Drives Constitutive Activity of GPCRs

Many G protein coupled receptors (GPCRs) exhibit constitutive (basal) activity, where they can signal in the absence of ligand binding through spontaneous conformational transitions that facilitate G protein coupling and downstream signaling. This intrinsic baseline activity is critical for cellular homeostasis and can be modulated by the receptors conformational ensemble, membrane organization, and interactions with intracellular effectors. In this study, we use live-cell signaling assays, fluorescence cross-correlation spectroscopy (FCCS), and single-particle tracking (SPT) to investigate how membrane organization influences the basal activity of two class A GPCRs: the M1 muscarinic receptor (M1R) and the adenosine A2A receptor (A2AR). In live-cell signalling assays, M1R showed minimal agonist-independent Ca{superscript 2} responses, while A2AR exhibited significant basal cAMP production that was eliminated by an inverse agonist. FCCS showed that, without ligand, only a small portion of M1R co-diffuses with its cognate G11 protein, whereas a much larger fraction of A2AR co-diffuses with the GS protein. SPT revealed that A2AR, but not M1R, is enriched in slowly diffusing, confined states with spatial scales around 150-200 nm and sensitivity to cholesterol- and raft-modulating agents, consistent with localization in lipid-raft nanodomains. Dual-color tracking and diffusion mapping demonstrated that a significant portion of A2AR and GS share confinement domains under basal conditions, while M1R and G11 only show such co-confinement in the active state. These findings support a model where the co-confinement of GPCRs and G proteins within plasma membrane nanodomains--rather than stable pre-coupled RG complexes-- determines the level of constitutive GPCR activity.