GPCR-selective effects of endocytosis on cellular signaling through the cAMP/PKA cascade

Many G-protein-coupled receptors (GPCRs) use endocytosis to promote gene transcription by prolonging signaling through the Gs-coupled cAMP/cAMP-dependent PKA cascade. However, not all GPCRs efficiently internalize after agonist-induced activation, and, among those that do, considerable differences have been observed in the ability of different GPCRs to stimulate endosomal cAMP production in different cell types. We asked if endocytosis distinguishes the signaling profiles of GPCRs that are naturally coexpressed in the same cells, focusing on three Gs-coupled GPCRs endogenous to human embryonic kidney-derived (293) cells: the vasoactive intestinal peptide receptor-1 (VIPR1/VPAC1) and β2-adrenergic receptor (β₂AR/ADRB2), which both rapidly internalize after activation, and the adenosine-2B receptor (A_2BR/ADORA2B) which we show here does not. For VIPR1, endocytosis significantly prolongs both the global cAMP elevation and cytoplasmic PKA activity increase. For β2AR, endocytosis has little effect on the global cAMP elevation but, nonetheless, it significantly prolongs the cytoplasmic PKA activity increase. A_2BR differs still further, with endocytosis having little effect on signal duration measured at either intermediate step. We then show that further downstream steps in the cascade, nuclear PKA activation and transcriptional induction, are significantly endocytosis dependent when stimulated through VIPR1 and β2AR but endocytosis independent through A_2BR. We conclude that endocytosis indeed distinguishes the signaling profiles of endogenously coexpressed GPCRs. We propose that quantitative differences in GPCR internalization and activation in endosomes program in cells a GPCR-selective, spatiotemporal "cAMP code" that is spatially "decoded" by proximity to local cytoplasmic PKA stores and then temporally interpreted by the nucleus.