Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling

G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and β2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple “on/off” switch. [Display omitted] •Receptors signal via receptor-associated independent cAMP nanodomains (RAINs)•RAINs constitute entirely self-sufficient, independent cell signaling units•When receptors are strongly stimulated, RAINs may fuse, and bulk cAMP increases•Cells may use thousands of independent RAINs to orchestrate signaling specificity GPCRs signal via localized regions of cAMP that do not mix, leading to action of independent on/off switches rather than coupling via a shared pool of signaling molecules.