Two barcodes encoded by the type-1 PDZ and by phospho-Ser312regulate retromer/WASH-mediated sorting of the ß1-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-Ser312provided 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-Ser312ß1-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.
Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA; Department of Biochemistry, Faculty of Pharmacy Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
Mohammed M
MM
Nooh
Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA. Electronic address: sbahouth@uthsc.edu.
Suleiman W
SW
Bahouth
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D054731:PDZ Domains; D028044:Phosphate-Binding Proteins; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000378:metabolism*; D058305:Sorting Nexins / Q000378:metabolism; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
D007545:Isoproterenol; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D028044:Phosphate-Binding Proteins; D018129:Phosphatidylinositol Phosphates; D010768:Phosphoserine; D011506:Proteins; D034741:RNA, Small Interfering; D018342:Receptors, Adrenergic, beta-1; D058305:Sorting Nexins; D022021:Tacrolimus Binding Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C540489:FKBP15 protein, human; C522885:SNX27 protein, human; C513980:SNX3 protein, human; C000605267:WASHC2C protein, human; C055525:phosphatidylinositol 3-phosphate
Confocal microscopy; GPCR trafficking; Retromer; Type-1 PDZ; WASH complex; β-adrenergic receptors