Type I PDZ Ligands Are Sufficient to Promote Rapid Recycling of G Protein-coupled Receptors Independent of Binding to N-Ethylmaleimide-sensitive Factor

Abstract

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the ␤ 2 adrenergic receptor (␤ 2 AR) mediates both PDZ binding to Na ؉ /H ؉ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the ␤ 2 AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the ␤ 2 AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the ␤ 1 -adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the ␤ 2 AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.

A Novel Sorting Sequence in the β2-Adrenergic Receptor Switches Recycling from Default to the Hrs-dependent Mechanism

“…Mutation of the novel sorting sequence produced a dramatically different phenotype (default recycling) than mutation of a previously defined recycling signal present in the distal ␤ 2 ADR C-tail, which greatly inhibits recycling when mutated (5)(6)(7)15). This suggests an epistatic relationship between these distinct sorting sequences, in which the acidic dileucine sequence functions upstream of both Hrs-and the PDZ-dependent recycling sequence, mediating the initial sorting of endocytosed receptors to the specialized recycling mechanism (Fig.…”

“…studies defined a minimal recycling signal, contained within the distal 4 -10 residues of the ␤ 2 ADR C-terminal tail (C-tail), which is necessary for efficient recycling of the ␤ 2 ADR and sufficient to re-route a distinct GPCR from lysosomal to recycling pathways (5)(6)(7)15). We recently showed that a sequence corresponding to residues 366 -413 of the ␤ 2 ADR C-tail, which includes these distal residues, is sufficient to confer Hrs-dependent recycling when fused to a truncated mutant V2 vasopressin receptor (V2R 362T) that otherwise recycles by default and in an Hrs-independent manner (16,20,24).…”

“…Mutation of the C-terminal PDZ ligand (5)(6)(7)15), as well as depletion of cellular Hrs (16), strongly inhibits recycling of internalized receptors. Remarkably, neither of these manipulations produce default recycling of receptors, which is the trafficking phenotype expected in the absence of endocytic sorting signals (12).…”

“…Mutation of this "recycling signal," or disruption of cytoplasmic protein interactions with this domain, profoundly impairs recycling of the ␤ 2 ADR (5)(6)(7)15).…”

Functions of the Cytoplasmic Tails of the Human Receptor Activity-modifying Protein Components of Calcitonin Gene-related Peptide and Adrenomedullin Receptors

“…In that case, NHERF-1 is thought to act by tethering surface AM 2 receptors to the actin cytoskeleton in a manner also seen with epidermal growth factor receptors (42). By contrast, NHERF-1 promotes the agonist-mediated recycling of ␤ 2 -ARs, which also have a PDZ motif (SLL) (30). The mechanism by which NHERF-1 exerts these differing effects on different GPCRs remains unknown.…”