Endocytosis sculpts distinct cAMP signal transduction by endogenously coexpressed GPCRs

Many G protein-coupled receptors (GPCRs) trigger a second phase of G protein-dependent signaling from internal membranes after agonist-induced endocytosis. However, individual GPCRs differ significantly in their ability to internalize, and it remains unclear if this confers selectivity on cellular signaling through natively coexpressed GPCRs. We addressed this question by examining the activation of the cyclic AMP (cAMP) / cAMP-dependent protein kinase (PKA) pathway by three ligands that stimulate three distinct, endogenously coexpressed GPCRs in HEK293 cells: isoproterenol (Iso) which primarily activates the β2-adrenergic receptor (β2AR), vasoactive intestinal peptide (VIP) which primarily activates the VIP receptor 1 (VIPR1/VPAC1), and 5’-N-ethylcarboxamidoadenosine (NECA) which primarily activates the adenosine 2B receptor (A _2B R). Using location-targeted biosensors and a transcriptional reporter, we demonstrate that each ligand triggers a unique cellular signaling profile and that these responses are differentially sensitive to endocytic inhibition. VIP elicited a response that was endocytosis-dependent at every level in the pathway, from upstream global cAMP elevation to downstream activation of nuclear PKA, while Iso elicited a response that was dependent on endocytosis selectively at downstream steps. In contrast, NECA robustly activated the entire cAMP signaling cascade independently of endocytosis, consistent with our observation that human A _2B R does not robustly internalize after activation. We conclude that endocytosis indeed sculpts downstream cAMP signaling by GPCRs in a receptor-specific manner. Our results add to the evolving view of compartmentalized signaling in the cAMP / PKA pathway and suggest that differences in GPCR trafficking can encode receptor-specific signaling profiles through a shared signal transduction pathway.