The Long and the Short Cycle

Abstract

The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in ␤-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the "long cycle." The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane.Vasopressin (1) is a small peptide hormone recognized by three different G protein-coupled receptors (GPCRs) 1 of which the V1a and V1b subtypes are coupled by G q/11 and the V2 is coupled to G s . Interaction with the agonist instantly leads to GPCR activation, phosphorylation, desensitization, and sequestration, followed by the return of the dephosphorylated receptors to the cell surface. In a few exceptions, the internalized receptor is degraded in the lysosomal compartment. The pathway by which GPCRs are recycled is under intense study, and an increasing number of elements specific for the receptor or the cell type have been identified, although little is known about how the different pathways and organelles are involved. The majority of GPCRs internalize via clathrin-coated pits, although some, such as the somatostatin 2 receptor, have been found mostly in uncoated vesicles (1). Furthermore, in the case of the ␤ 2 adrenergic receptor (␤ 2 -AR), the endothelin receptor, and the cholecystokinin receptor, an additional internalization mechanism has been described involving ...

Resistance of the Human β1-Adrenergic Receptor to Agonist-mediated Down-regulation

“…There is increasing evidence the C-tails of GPCRs are important determinants in regulating their endosomal trafficking (33)(34)(35)(36). For the C-tail of ␤ 2 AR, tyrosine 350 and 354 are required for receptor down-regulation (9), and the terminal four amino acid residues (DSLL) represent a PDZ domain binding motif that is involved in the sorting of ␤ 2 AR between recycling endosomes and lysosomes (34,36).…”

“…Role of the C-tail of ␤AR in Agonist-mediated Regulation-As the C-tails of a number of GPCRs have been identified as structural determinants in targeting the receptors to lysosomes (33)(34)(35), we explored the effect of replacing the C-tail of one subtype with that of the other on agonist-mediated regulation. As the first step in the regulatory process is receptor phosphorylation, we 32 P-labeled BHK cells transiently expressing ␤ 1 AR, ␤ 2 AR, or the chimeras, ␤ 1 /␤ 2 ct-AR and ␤ 2 /␤ 1 ct-AR, stimulated the cells with agonist, and purified and analyzed the receptors for 32 P incorporation (Fig.…”

“…In addition, we investigated whether the two subtypes differed in basal or agonist-mediated turnover and whether basal and agonist-mediated turnover involved different pathways. Finally, as the C-tails of GPCRs including the ␤ 2 AR have been identified as important determinates of sorting between recycling and degradation (33)(34)(35)(36), we examined the effects of exchanging the C-tails of the two subtypes on receptor regulation. Our results indicate that in BHK cells, ␤ 1 AR is resistant to agonist-mediated down-regulation and instead undergoes up-regulation as does its mRNA; increased ␤ 1 AR internalization does not result in down-regulation; basal turnover is similar for both subtypes and appears to be non-lysosomal whereas agonist-mediated turnover of ␤ 2 AR is lysosomal; and finally the C-tails are key determinants of down-regulation, the ␤ 1 AR C-tail conferring resistance and the ␤ 2 AR conferring C-tail susceptibility.…”

Ezrin-Radixin-Moesin-binding Phosphoprotein-50/Na+/H+ Exchanger Regulatory Factor (EBP50/NHERF) Blocks U50,488H-induced Down-regulation of the Human κ Opioid Receptor by Enhancing Its Recycling Rate

“…The recycling pathway appears to be the default pathway for membrane proteins that are not trafficked to the lysosomal pathway (11). Although this view is generally true for the constitutively internalized and recycled molecules, such as transferrin, recent studies have indicated that recycling of internalized GPCRs is regulated (12)(13)(14)(15).…”

Phosphorylation Analysis of G Protein-Coupled Receptor by Mass Spectrometry: Identification of a Phosphorylation Site in V2 Vasopressin Receptor

“…Mutagenesis of single serines or threonines in the C-terminal tail reduced phosphorylation to different degrees and had variable effects on V2R recycling 8,19 . Ser 255 represents the first phosphorylation site identified in the 3 rd intracellular loop of V2R (Figure 3).…”