The Long and the Short Cycle: ALTERNATIVE INTRACELLULAR ROUTES FOR TRAFFICKING OF G-PROTEIN-COUPLED RECEPTORS

Giulio Innamorati,Christian Le Gouill,Michael Balamotis,Mariel Birnbaumer

doi:10.1074/jbc.m009780200

Giulio Innamorati, Christian Le Gouill + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m009780200

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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 beta-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.

Highlights

Vasopressin [1] is a small peptide hormone recognized by three different G protein-coupled receptors (GPCRs)1 of which
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
Substituting the C terminus with the homologous portion of the V1a receptor (V1aR) resulted in a chimeric V2/V1a receptor that was still coupled to Gs [15]

Summary

The Long and the Short Cycle

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. 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. Phosphorylation sites between positions 345 and 361 (of the 371 V2R amino acids; see Fig. 2) appear to regulate the interaction with the internalization machinery, since their removal modifies the extent of receptor internalization [9] These phosphorylation sites together with others further downstream, serine and threonine from positions 345– 364, create a signal regulating the traffic of the receptor back to the plasma membrane [10].2. The pathway followed by the V2R was compared using confocal microscopy to that of other receptors known to recycle in HEK 293 cells, such as the V1a and the ␤2-adrenergic receptors [12, 13] with the purpose to identify possible differences in their intracellular localization and to determine at which point the recycling and nonrecycling pathways physically diverged

EXPERIMENTAL PROCEDURES

RESULTS

It has been suggested that a strong and long term interaction

DISCUSSION