title: Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs.
abstract: Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the beta2-adrenergic receptor (beta2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the delta-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.
authors.affiliation: Program in Pharmaceutical Sciences and Pharmacogenomics, Department of Psychiatry, University of California, San Francisco, California 94158-2140, USA.
authors.forename: Benjamin E L
authors.initials: BE
authors.lastname: Lauffer
authors.forename: Stanford
authors.initials: S
authors.lastname: Chen
authors.forename: Cristina
authors.initials: C
authors.lastname: Melero
authors.forename: Tanja
authors.initials: T
authors.lastname: Kortemme
authors.forename: Mark
authors.initials: M
authors.lastname: von Zastrow
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Vargas
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D040761:Biomimetic Materials / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D054731:PDZ Domains; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D000199:Actins; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; D043562:Receptors, G-Protein-Coupled; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate