In this study, we aimed to identify binding partners to the differentially-phosphorylated forms of the AQP2 COOH-terminus, the portion of the AQP2 molecule that has been implicated in the regulation of trafficking to the apical plasma membrane 2,5,6
. We used protein mass spectrometry as a tool to establish binding partners’ identities and relative abundances in the eluates of a cytosolic pull-down experiment using synthetic AQP2 COOH-terminal peptides.
Mass spectrometry was used as an initial screening tool, followed by confirmation by semi-quantitative immunoblotting and evaluation of identified interactions with native AQP2 by coimmunoprecipitation. Evaluation of mass spectra and subsequent verification by immunoblotting demonstrated the associations between hsp70/hsc70 and AQP2 shown by Lu et al 19
Our studies further verified and characterized the association between annexin II and AQP2 previously demonstrated from mass spectrometric analysis of AQP2 immunoprecipitates 44
Other proteins that have been shown previously to interact with AQP2, namely SPA-1 17
and MAL 20
, were not identified in the present study. SPA-1 is a RapGAP 17
. According to the IMCD transcriptome database (http://dir.nhlbi.nih.gov/papers/lkem/imcdtr
), the abundance of SPA-1 mRNA is very low in the IMCD 45
, suggesting that the protein abundance may be low as well, limiting our ability to detect it. MAL is an integral membrane protein. Our pull-downs utilized only the cytosolic fraction of IMCD cells for incubation with AQP2 COOH-terminal peptides. Thus, we would not expect to have identified MAL in these experiments.
Our studies, then, yielded seven candidate binding partners to the AQP2 COOH-terminus as demonstrated quantitatively by protein mass spectrometry and by immunoblotting of pull-downs: hsc70, hsp70 (isoforms 1 and 2), BiP (Grp78, or hsp70 isoform 5), annexin II, PP1c, GDI-2, and RAN. With the notable exception of BiP, which was most abundant in the eluates corresponding to the pS256 AQP2 COOH-terminal peptide pull-downs, the other candidate binding partners showed a preference for the nonphosphorylated peptide. Interestingly, hsc70 showed a small but statistically significant preference for the nonphosphorylated and pS261 COOH-terminal AQP2 tail peptides over the pS256 peptide.
Studies using circular dichroism, a spectroscopic technique capable of elucidating peptide and protein secondary structure, demonstrated that the nonphosphorylated form of the AQP2 COOH-terminus appeared most alpha−helical; phosphorylation at Ser-256 significantly altered this conformation, and phosphorylation at Ser-261 caused a less pronounced change. These findings are consistent with the hypothesis that a charge effect from phosphorylation is not the sole mediator of binding partner selectivity for the differentially phosphorylated AQP2 COOH-terminus. Thus, we may partially explain slight changes in binding partner selectivities to the AQP2 COOH-terminus by alteration of secondary structure due to phosphorylation. In the remainder of this discussion, we describe potential physiologic consequences for these demonstrated protein-protein interactions.
The direct interaction of hsp70 and hsc70 with AQP2 was initially characterized by Lu et al 19
. Our findings are consistent with their GST and native protein pull-down studies. Moreover, we were able to demonstrate different selectivities of hsp70 and hsc70 for the various in vitro
phosphophorylated AQP2 COOH-terminal peptides. Our in vitro
data suggested that hsc70 and hsp70 interact selectively with AQP2 that is not phosphorylated at Ser-256, which would be expected to be more abundant in the absence of vasopressin. Lu et al
. showed that functional knockdown of hsc70 induced membrane accumulation of AQP2, demonstrating potential involvement of hsc70 in clathrin-mediated endocytosis of AQP2 19
BiP is a unique member of the hsp70 family, whose primary function is believed to be in the quality control mechanism in the ER. Its unique KDEL sequence is known to be an ER localization signal 46
. However, a number of studies have shown BiP localization at the plasma membrane and cytosol in a variety of cell types 37-43
. We have confirmed these conclusions in collecting duct cells using immunogold EM and confocal immunofluorescence microscopy. BiP appeared in our cytosolic fractions of IMCD cell homogenates, despite stringent ultracentrifugation prior to the AQP2 COOH-terminal peptide pull-down experiments. We further confirmed an association between native AQP2 and BiP by immunoprecipitation of AQP2 from native IMCD cells and subsequent immunoblotting for BiP. BiP abundance has been shown to decrease in animals subjected to long-term vasopressin escape, a condition in which pS256-AQP2 theoretically decreases compared to control animals 47
. In contrast, BiP abundance has been shown to increase in the IMCD after long-term lithium treatment 48
. Our finding that BiP preferentially binds pS256 AQP2 COOH-terminal peptides suggests a trafficking role for this protein or possibly a counter-regulatory role in hsc70/hsp70-mediated endocytosis of AQP2. Alternatively, BiP binding to pS256 may alter the conformation of the C-terminal tail of AQP2, thereby promoting the association of kinases that phosphorylate AQP2 at neighboring sites. This could potentially explain the dependence of phosphorylation at S264 and S269 on prior phosphorylation at S256 as demonstrated by Hoffert et al. 12
Several studies have suggested an association between annexin II and AQP2 21,22
. Annexins have been a subject of interest in AQP2 trafficking since Hill et al.
identified an association—though not a functional one—between annexin IV and AQP2 49
. Barile et al.
found that immunoisolated AQP2-bearing vesicles contained annexins I, II, IV, and V 25
. Furthermore, Noda et al.
identified annexin II as part of a “multiprotein ‘motor’ complex” associated with immunoprecipitated AQP2 21
. We found preferential binding of annexin II to the nonphosphorylated AQP2 COOH-terminal peptide. A direct association was confirmed by co-immunoprecipitation of annexin II with an antibody against AQP2. Since annexin II has been shown to be involved in both endocytosis and exocytosis, it is possible that this protein functions in both processes in the collecting duct 50
. Finally, a recent study by Tamma et al.
demonstrates that inhibition of annexin II impairs water permeability in cultured cells 22
PKA has been demonstrated to bear responsibility for phosphorylation of Ser-256. To our knowledge, until the present study, no phosphatases have been proven to associate directly with the COOH-terminus of AQP2. A study by Valenti et al.
suggested a functional interaction between AQP2 and either PP2A or PP1 51
. That particular study found that the phosphatase inhibitor okadaic acid, at concentrations high enough to inhibit both phosphatases, increased osmotic water permeability and increased AQP2 trafficking to the plasma membrane. These findings, taken together with our findings that PP1c, but not PP2Ac, preferentially binds the nonphosphorylated AQP2 COOH-terminus in vitro
(by peptide pulldown and immunoblotting), suggests the hypothesis that PP1 may play a role in regulation of the phosphorylation state of the COOH-terminus of AQP2 in the unstimulated state.
The significance of the in vitro
association between the AQP2 COOH-terminus and both GDI-2 and RAN remains unclear. GDP dissociation inhibitors such as GDI-2 maintain small GTPases in their inactive state, preventing their insertion into membranes 52,53
. This finding suggests a potential role of Ras-like GTPase trafficking in regulation of AQP2. Unlike GDI-2, RAN is primarily known to function in nuclear-cytoplasmic trafficking (possibly bi-directionally) and in nuclear functions in mitosis, but it is also present in the cytosol 54,55
. In a recent proteomics study by Nielsen et al.,
RAN expression was increased in the IMCD after two weeks of lithium treatment 48
. However, no clear connection has been established between changes in RAN expression and lithium-induced down-regulation of AQP2.
In summary, our studies have used mass spectrometry to identify seven potential binding partners to the AQP2 COOH-terminus in rat IMCD cells. These proteins have differential selectivities for phosphorylated versus nonphosphorylated AQP2 COOH-terminal, synthetic peptides in vitro. Furthermore, using an antibody to AQP2, we have been able to co-immunoprecipitate three of the protein binding partners (i.e. annexin II, PP1c, and BiP) from native IMCD cells as well as from a stably-transfected kidney cell line. We also demonstrated, through immunofluorescence microscopy, that these proteins co-localize with endogenous AQP2 in native rat collecting duct.