A Transplantable Sorting Signal That Is Sufficient to Mediate Rapid Recycling of G Protein-coupled Receptors

Abstract

The ␤ 2 -adrenergic receptor and ␦ opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the ␤ 2 -adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed ␦ opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism.

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…This could be the consequence of increased channel density, augmented open probability, and/or unitary conductance. NHERF overexpression may increase the recycling efficiency of internalized CFTR, as described for the ␤ 2 -adrenergic and the ␦-and the -opioid receptors in nonpolarized cells (24,26,50) and suggested for GFP-CFTR (35). Whereas a role of NHERF in the recycling of CFTR cannot be completely discounted, we were unable to detect attenuated expression, metabolic destabilization, or any functional defect of the truncated CFTR in pancreatic duct and BHK cells (11).…”

“…Deletion of the last 26 amino acid residues had no impact on the whole cell current densities in NIH 3T3 cells as reported earlier (36). Since defective recycling of the Gprotein-coupled receptor in the absence of NHERF binding is invariably associated with the accelerated degradation of the receptors (24,26,50), our observations, collectively, argue against the role of NHERF in CFTR recycling. Although the functional studies imply that the endogenous level of NHERF is insufficient to influence CFTR activity in BHK and PANC-1 cells, variations in the concentration and/or the compartmentalization of NHERF may ensure a regulatory role for NHERF in CFTR activation in other cells (64), including the sweat duct and Calu-3 epithelia (29,65).…”

“…4B). Considering that the complex-glycosylated form stability reflects the overall characteristic of post-Golgi, cell surface, and endosomal CFTR pools, and NHERF may facilitate recycling of CFTR as observed for G-protein-coupled receptors (24,26,35,50), next we evaluated the effect of C-terminal truncation on the apical localization and channel function of CFTR.…”

“…The D1 and D2 domains bind with nanomolar affinity to the PDZbinding motifs of CFTR ( 1477 DTRL) and to the C terminus of other transport proteins, signaling molecules, and receptors (18,(22)(23)(24)(25)(26). The ERM-binding domain tethers the complex via ezrin to cytoskeletal elements in a phosphorylation-dependent manner and to the catalytic and regulatory subunit of PKA (19,(27)(28)(29).…”

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

“…In addition, recycling of the GPCR is intimately involved in maintaining receptor density in cells and tissues because prolonged incubation of cells with isoproterenol did not cause significant degradation of ␤ 2 -AR even after several hours of continuous exposure to high levels of the ␤-agonist (50). However, mutations that interfered with the recycling of the ␤ 2 -AR resulted in degradation of mutant ␤ 2 -AR in cells chronically exposed to ␤-agonists (50). Therefore, recycling of the GPCR was associated with maintenance of GPCR density and signaling intensity.…”