Integrin Signaling Modulates AQP2 Trafficking via Arg-Gly-Asp (RGD) Motif

Abstract

Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively.

Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

“…Biocytin hydrazide was used to label apical membrane proteins following the method of Tamma et al (13) with modifications. Briefly, the apical plasma membrane of the cells were treated with sodium periodate, which oxidizes the glycan of the apical membrane proteins before biocytin hydrazide labeling.…”

“…However, several technical barriers prohibited deeper quantitative analysis: (i) labeling of the apical plasma membrane of native collecting ducts required technically difficult perfusion of the biotinylation reagent into the collecting duct lumens; (ii) AQP2, the major vasopressin target, was not efficiently biotinylated because it has only one extracellular NHS-reactive lysine that is likely occluded by an adjacent N-glycosylation site in the second extracellular loop; and (iii) investigation of native collecting ducts precluded the use of in vivo metabolic labeling for accurate quantification of protein abundance changes. In this study, we overcame these limitations by using stable-isotope labeling with amino acids in cell culture (SILAC) methodology in cultured mouse cortical collecting duct (mpkCCD-clone 11) cells (4) that had undergone apical surface biotinylation with a reagent that targets extracellular N-glycosyl groups (13). Biotinylated proteins were identified…”

Aquaporin 2

“…They showed that AQP2 interacts with integrins through the integrin-binding site in AQP2, Arg-Gly-Asp (RGD), which is the same motif identified by Tamma et al 10 They then observed that AQP2 promotes epithelial cell migration, which is an important mechanism of tissue repair following injury, in both MDCK and LLC-PK1 cells, and that promigration effects requires both AQP2 and b1 integrin. Finally, they proceeded to show that AQP2 promotes epithelial cell migration by facilitating the turnover of b1 integrin in the focal adhesions.…”

“…In addition, Tamma et al 10 recently showed that integrin signaling modulates AQP2 trafficking. This suggested to Chen et al that an interaction between AQP2 and an integrin at this site may contribute to the tubular abnormalities seen in AQP2-null mice.…”

Glutathionylation of the Aquaporin-2 Water Channel

“…tert-Butyl hydroperoxide (tBHP) was kind gift from A. Signorile (University of Bari). To immunoprecipitate and detect the total amount of AQP2, we used antibodies (Pre-C-tail Ab) against the 20-amino acid residue segment just N-terminal from the polyphosphorylated region of AQP2 (CLKGLEPDTDWEEREVRRRQ) (11). Alternatively, AQP2 was detected using a specific antibody (C-tail Ab) raised against a synthetic peptide corresponding to the last 15 C-terminal amino acids of human AQP2 (12).…”