B2AR phosphorylation at S345 and S346 restricts B2AR to ASRT endosomal microdomains. (A) Images of iso-induced B2AR endocytosis and PKA inhibition. Before iso, B2ARs localize to the cell surface. 5 min after iso, B2AR redistributes to endosomes and is sorted into tubules (arrowheads). KT was added 5 min after iso. Bars: (main images) 5 µm; (insets) 2 µm. (B) Example images of B2AR and coronin (ASRT marker) with iso and KT. KT causes B2AR to enter non-ASRT domains. (C) Quantitation of percent B2AR in ASRT and non-ASRT tubules. n = 31 (−KT, iso) and 14 (KT) cells. (D) Example images of B2AR and SNX1 (ASRT marker) with iso and KT. (B and D) Arrowheads show B2AR in ASRT domains, and arrows show B2AR in non-ASRT domains. Bars, 2 µm. (E) Quantitation of the percent B2AR in ASRT and non-ASRT tubules. n = 11 (−KT, iso only) and 18 (+KT, PKA inhibited) cells. (F) 5 min after iso, SS>AA also localized to endosomes and tubules. Bars, 5 µm. (G) Example images of B2AR and SS>AA with coronin. SS>AA localizes to both ASRT domains (arrowheads) and constitutive tubules (arrows). Bars, 2 µm. (H) Quantitation of percent B2AR and SS>AA ASRT tubules. SS>AA localizes to both ASRT and non-ASRT tubules. n = 13 (B2AR) and 14 (SS>AA) cells. Error bars in all graphs are SEM across cells.