Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

Abstract

The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)(2)-Suc-TMR(8)]VP or (VP(TMR)). This ligand is fully functional as revealed by its high binding affinity toward V2R [(K(d)) =157 ± 52 nM] and ability to increase intracellular cAMP 32-fold. VP(TMR) induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VP(TMR) and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VP(TMR) and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VP(TMR)-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VP(TMR), and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VP(TMR) ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism.

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

“…Consistent with our recent study done with the identical HEK293/HA-V2R cell model, cAMP generation in response to one "pulse" of VP is maintained at time points after internalization of V2R, which begins as soon as 5 min after challenge with a saturating concentration of VP (14). These data raised the hypothesis that sustained cAMP signaling mediated by VP is associated with internalization of V2R in early endosomes.…”

“…These data raised the hypothesis that sustained cAMP signaling mediated by VP is associated with internalization of V2R in early endosomes. Although residual V2R is often observed on the cell surface after challenge and ligand washout, most to all of VP TMR internalizes (14), indicating that the majority of VP-V2R complexes are within the cytoplasmic compartment and may continue to signal.…”

“…In brief, cell imaging was performed at room temperature in Hepes buffer containing 0.1% BSA using a Nikon A1s confocal microscope attached to a Ti-E inverted base using a ϫ60 1.45 NA plan-apo objective. Live cell time-lapse confocal imaging of transfected cells treated with tetramethylrhodamine (TMR)-labeled vasopressin (VP TMR ) (14) was performed at 37°C using a Nikon A1-Rs confocal system attached to a Ti-E inverted base and an apo ϫ60 1.49 NA TIRF objective. The microscope was encased in a microscope cage incubator to maintain the temperature at 37°C.…”

“…To address this hypothesis, we used a series of biochemical, optical, and physiological assays to study V2R signaling in response to its two native ligands: vasopressin and oxytocin (13,14). These experiments employed cultured human embryonic kidney (HEK293) cells stably expressing recombinant V2R and a line derived from renal principal cells of the mouse collecting duct, mpkCCD C14 , which expresses V2R and the epithelial sodium channel (ENaC) (19,20).…”

Congestive heart failure in rats is associated with increased collecting duct vasopressin sensitivity and vasopressin type 2 receptor reexternalization

“…Upon ligand-induced receptor internalization, G-coupled receptors are most commonly recycled to the plasma membrane. However, previous studies have shown that this may not be the case for the V 2 receptor (2,5,11,12). Consequently, it could be of interest to examine whether experimentally induced CHF is associated with changes in receptor internalization and recycling abilities that could contribute to the apparent increased AVP sensitivity in CDs.…”