Two barcodes encoded by the type-1 PDZ and by phospho-Ser 312 regulate retromer/WASH-mediated sorting of the ß 1 -adrenergic receptor from endosomes to the plasma membrane.

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß1 -AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß1 -AR is inhibited when PKA or its substrate serine at position 312 (Ser312 ) are inactivated. We tested the hypothesis that phospho-Ser312 provided a second barcode for ß1 -AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß1 -AR trafficking. Recycling of WT ß1 -AR or WT ß2 -AR was dependent on targeting the retromer to endosomal membranes via SNX3 and rab7a, and on complexing the retromer to the WASH pentamer via the C-tail of FAM21 (FAM21C ). These maneuvers however, did not inhibit the recycling of a phospho-Ser312 ß1 -AR mimic ((S312D) ß1 -AR). Knockdown of the trans-acting PDZ protein sorting nexin27 (SNX27) inhibited the recycling of WT ß1 -AR and WT ß2 -AR, but had no effect on (S312D) ß1 -AR∆PDZ or on phosphorylation of WT ß1 -AR by PKA at Ser312 . However, depletion of FKBP15, a FAM21C -binding endosomal protein, selectively inhibited WT ß1 -AR but not ß2 -AR recycling, suggesting divergence might exist in GPCR trafficking roadmaps. These results indicate that two barcodes are involved in sorting WT ß1 -AR out of early endosomes. The first and antecedent "barcode" was the "type-1 PDZ", followed by a second reversible "phospho-Ser312 " verification "barcode". This organization allows tight regulation of ß1 -AR density to signaling intensity in conditions associated with aberrant ß1 -AR signaling such as in hypertension and heart failure.