Local Confinement within Plasma Membrane Nanodomains Promotes Constitutive Activity of GPCRs

Abstract

Many G protein-coupled receptors (GPCRs) exhibit constitutive activity through spontaneous conformational transitions that facilitate G protein coupling and downstream signaling in the absence of ligand binding. This baseline activity, which is critical for cellular homeostasis, can be influenced by receptor conformational ensembles and plasma membrane organization. We used live-cell signaling assays, single-particle tracking (SPT) and fluorescence cross-correlation spectroscopy (FCCS) to investigate how membrane organization impacts the basal activity of the M1 muscarinic receptor (M1R) and the adenosine A2A receptor (A2AR). M1R showed minimal basal Ca2+ responses, whereas A2AR exhibited basal cAMP production that was eliminated by an inverse agonist. FCCS showed that in the absence of ligands only a small portion of M1R co-diffused with its cognate Ga11 protein without ligand, whereas a larger fraction of A2AR co-diffused with Gas. Interestingly, SPT revealed that A2AR was enriched in slowly diffusing, confined states with spatial scales around 150-200 nm. The sensitivity to cholesterol- and membrane-order-altering agents suggests they are associated with raft-related membrane organization. Dual-color tracking and diffusion mapping demonstrated that A2AR and Gas shared confinement domains under basal conditions, whereas M1R and Ga11 only showed such co-confinement in the active state. We propose a model in which the co-confinement of GPCRs and G proteins within plasma membrane nanodomains promotes constitutive activity by increasing opportunities for transient productive encounters. This model links receptor conformational dynamics and nanoscale membrane organization to basal signaling output.