G protein–coupled receptors (GPCRs) are recycled via a sequence-dependent pathway that is spatially and biochemically distinct from bulk recycling. Why there are two distinct recycling pathways from the endosome is a fundamental question in cell biology. In this study, we show that the separation of these two pathways is essential for normal spatial encoding of GPCR signaling. The prototypical β-2 adrenergic receptor (B2AR) activates Gα stimulatory protein (Gαs) on the endosome exclusively in sequence-dependent recycling tubules marked by actin/sorting nexin/retromer tubular (ASRT) microdomains. B2AR was detected in an active conformation in bulk recycling tubules, but was unable to activate Gαs. Protein kinase A phosphorylation of B2AR increases the fraction of receptors localized to ASRT domains and biases the downstream transcriptional effects of B2AR to genes controlled by endosomal signals. Our results identify the physiological relevance of separating GPCR recycling from bulk recycling and suggest a mechanism to tune downstream responses of GPCR signaling by manipulating the spatial origin of G protein signaling.

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