Local Confinement within Plasma Membrane Nanodomains Drives Constitutive Activity of GPCRs

ABSTRACT 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 receptor’s 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 (M 1 R) and the adenosine A 2A receptor (A 2A R). In live-cell signalling assays, M 1 R showed minimal agonist-independent Ca²⁺ responses, while A 2A R exhibited significant basal cAMP production that was eliminated by an inverse agonist. FCCS showed that, without ligand, only a small portion of M 1 R co-diffuses with its cognate Gα 11 protein, whereas a much larger fraction of A 2A R co-diffuses with the Gα S protein. SPT revealed that A 2A R, but not M 1 R, 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 A 2A R and Gα S share confinement domains under basal conditions, while M 1 R and Gα 11 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.