Two barcodes encoded by the type-1 PDZ and by phospho-Ser³¹² regulate retromer/WASH-mediated sorting of the...

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 (FAM21_C). 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 FAM21_C-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.