1 Division of Nephrology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115; 2 Department of Biomedical Sciences, National Institute of Health, Seoul, Korea; 3 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; 4 Vollum Institute, Howard Hughes Medical Institute, and 5 Neurological Sciences Institute, Oregon Health Sciences University, Portland, Oregon 97201; 6 Tulane University Medical Center and Veterans Affairs Medical Center, New Orleans, Louisiana 70112; and 7 Maine Medical Center Research Institute, Portland, Maine 04102
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ABSTRACT |
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We have demonstrated
that inner medullary collecting duct (IMCD) heavy endosomes purified
from rat kidney IMCD contain the type II protein kinase A (PKA)
regulatory subunit (RII), protein phosphatase (PP)2B, PKC, and an
RII-binding protein (relative molecular mass ~90 kDa) representing a
putative A kinase anchoring protein (AKAP). Affinity chromatography of
detergent-solubilized endosomes on cAMP-agarose permits recovery of a
protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKC
.
With the use of small-particle flow cytometry, RII and PKC
were
localized to an identical population of endosomes, suggesting that
these proteins are components of an endosomal multiprotein complex. 32P-labeled aquaporin-2 (AQP2) present in these
PKA-phosphorylated endosomes was dephosphorylated in vitro by either
addition of exogenous PP2B or by an endogenous endosomal phosphatase
that was inhibited by the PP2B inhibitors EDTA and the
cyclophilin-cyclosporin A complex. We conclude that IMCD heavy
endosomes possess an AKAP multiprotein-signaling complex similar to
that described previously in hippocampal neurons. This signaling
complex potentially mediates the phosphorylation of AQP2 to regulate
its trafficking into the IMCD apical membrane. In addition, the PP2B
component of the AKAP-signaling complex could also dephosphorylate AQP2
in vivo.
kidney; water channel; adenosine 3',5'-cyclic monophosphate; protein phosphatase 2B; aquaporin-2; A kinase anchoring protein
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INTRODUCTION |
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TRANSEPITHELIAL WATER REABSORPTION across renal collecting duct cells is stimulated by arginine vasopressin (AVP). AVP acts by increasing the number of aquaporin-2 (AQP2) water channels in the apical membrane of inner medullary collecting ducts (IMCDs), thus increasing its permeability to water (reviewed in Refs. 19 and 27). Before AVP stimulation, AQP2 resides primarily in subapical membrane vesicles, and the subsequent stimulation increases the insertion of these AQP2-containing vesicles into the apical membrane itself. It is known that AVP binding to the V2 receptor stimulates cAMP formation and that the insertion of AQP2 vesicles into the apical membrane involves phosphorylation of AQP2 by cAMP-dependent protein kinase A (PKA). A present area of active investigation is how the activated PKA interacts with its AQP2 substrate and whether it is specifically targeted to it. In addition, although a number of studies have investigated PKA-mediated AQP2 phosphorylation, little is known regarding the dephosphorylation of AQP2.
The COOH-terminal region of AQP2 contains a single putative phosphorylation site for PKA (Ser256) (12, 26). In LLC-PK1 cells transfected with wild-type AQP2, addition of AVP and forskolin induces insertion of AQP2-containing vesicles into the cell membrane. However, when such expression studies are performed with an AQP2 mutant possessing an alanine instead of Ser256, this AQP2 insertion response is abolished (16). Studies using both purified AQP2-containing vesicles (22) and Xenopus laevis oocytes (21) suggest that PKA-mediated phosphorylation of AQP2 does not alter its intrinsic water permeability but rather may be a key event permitting AQP2 access to the plasma membrane. Recently, Klussmann et al. (20) have provided evidence for the involvement of A kinase anchoring proteins (AKAPs) in the translocation of AQP2 from intracellular vesicles to the IMCD membrane. Although these data demonstrate that compartmentalization of PKA by AKAPs is necessary for AQP2 translocation, questions remain regarding the structure of this PKA-AKAP complex and the location of the intracellular compartment to which it is bound.
The subcellular trafficking of vesicles has been studied extensively in neurons, which are also polarized and may be related to epithelial cells (17). The postsynaptic densities of hippocampal neurons have been demonstrated to contain a multiprotein-signaling complex consisting of a 79-kDa A kinase anchoring protein (150 kDa in mice) (7) associated with a number of signaling molecules, including the PKA regulatory subunit, protein phosphatase 2B (PP2B or calcineurin), and PKC (reviewed in Ref. 8). This hippocampal neuron-signaling complex is postulated to provide the molecular specificity for cycles of PKA-mediated phosphorylation and dephosphorylation of membrane protein components, including ion channels. In mice, a novel AKAP family has also been cloned that localizes to the apical membranes of renal proximal tubule cells (AKAP-KL) (9).
Given the postulated evolutionary and functional relationships between polarized neurons and epithelial cells (17), we investigated whether rat IMCD heavy endosomes, which we have previously shown to be apically derived and AQP2 containing (14, 22), also contain such an AKAP complex, because such a mechanism could help explain how AQP2 trafficking is regulated in response to AVP. Furthermore, given the presence of PP2B in the neuronal AKAP complex, we investigated whether it is also expressed in IMCD cells and whether it can dephosphorylate AQP2 previously phosphorylated by PKA.
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METHODS |
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Materials.
Various items were obtained from the following sources:
[-32P]ATP (10 Ci/mmol) was from NEN Life Science
Products, Sepharose 4B was from Pharmacia (Uppsala, Sweden),
polyvinylidene fluoride membrane was from MSI (Westborough, MA), and
SDS, acrylamide,
-mercaptoethanol, and ammonium persulfate were from
Bio-Rad. All other reagents were from either sources described
previously (22) or Sigma (St. Louis, MO). Male
Sprague-Dawley rats (200-250 g) were purchased from Charles River
Laboratories (Cambridge, MA).
Purification of IMCD endosomes.
Endosomes were prepared from rat kidney inner medullary papillae as
described previously (14). Briefly, papillae were
homogenized in buffer A (300 mM mannitol and 12 mM HEPES,
titrated to pH 7.6 with Tris) and centrifuged at 2,500 g to
remove nuclei and unlysed cells. The postnuclear supernatant was
centrifuged at 20,000 g for 20 min, and the new supernatant
and the lightly packed upper layer of the pellet were collected. This
postmitochondrial supernatant was then recentrifuged at 48,000 g for 30 min, and the resulting pellet was resuspended in
buffer A and fractionated on a self-forming Percoll gradient
(18% Percoll by weight). After centrifugation for 30 min at 48,000 g, the bottom one-third of the gradient was collected and
dispersed in an eightfold larger volume of buffer B [(in
mM) 300 mannitol, 100 KCl, 5 MgSO4, and 5 HEPES, adjusted to pH 7.6 with Tris] and left on ice for 15 min. This vesicle suspension was then recentrifuged at 48,000 g for 30 min,
and the resulting membrane pellet was resuspended in buffer
B. After centrifugation at 5,000 g for 15 min, the
endosomes appeared as a pearly white layer overlaying a darker membrane
pellet. These vesicles, previously characterized as apical endosomes,
were either used immediately or stored at 80°C until use. Endosomal
protein content was determined by the method of Bradford
(3).
Immunoblotting.
Western blot analysis of IMCD proteins was performed as described
previously (15) using the following antisera: anti-PKC and anti-PKC
rabbit polyclonal (Life Technologies, Gaithersburg, MD), and, anti-RII goat polyclonal and anti-PP2B (catalytic subunit) mouse monoclonal antibodies (Upstate Biotechnology, Lake Placid, NY).
Immunohistochemistry.
Rats were perfusion fixed as described previously (25),
using freshly prepared 4% paraformaldehyde followed by sucrose
cryoprotection. Tissue samples were then embedded in OCT compound
(Miles, Elkart, IN), snap-frozen in 2-methylbutane in liquid
N2, and stored at 70°C until further use.
Immunohistochemistry was then performed as previously described
(24) using frozen sections (4 µm) cut from the tip of
individual rat kidney inner medullae and stained with various antisera.
Counterstaining was performed with methyl green (Fisher, Pittsburgh, PA).
32P-RII overlay.
Rat tissue samples were subjected to SDS-PAGE electrophoresis and
transferred to a nitrocellulose membrane that was incubated in buffer
containing 1% (wt/vol) bovine serum albumin and recombinant RII
protein, which was phosphorylated using [-32P]ATP as
described previously (7). The membrane was incubated in
the resulting 32P-RII probe for 14 h at 4°C in the
presence or absence of the blocking peptide Ht31 (4). The
membrane was then washed free of unbound probe, and the presence of
bound 32P-RII was determined by autoradiography.
Affinity chromatography using cAMP-agarose. IMCD heavy endosomes were solubilized on ice for 2 h in hypotonic buffer [(mM) 10 HEPES (pH 7.9), 1.5 MgCl2, 10 KCl, 1 polymethylsulfonyl fluoride, 0.5 dithiothreitol, 1 benzamidine, and 0.01 IBMX] containing 0.5% (vol/vol) Nonidet P-40 and centrifuged at 15,000 g for 15 min. The detergent-soluble supernatant was mixed with cAMP-agarose equilibrated in hypotonic buffer containing 0.1% Nonidet P-40. After being mixed by rotation at 4°C for 14 h, the cAMP-agarose pellet was washed four times in hypotonic buffer and then analyzed for its protein content by SDS-PAGE and immunoblotting (6).
Localization of dual antibody labels by small-particle flow
cytometry.
Freshly prepared endosomes were incubated in 1:1 normal donkey serum
(clarified by centrifugation at 180,000 g for 20 min) for
1 h at room temperature, washed, and then incubated in primary antibodies produced in goats or rabbits at 4°C for 14 h. The
endosomes were washed again and incubated in fluorescently conjugated
anti-donkey secondary antibody (1:100 dilutions) for 2 h at room
temperature. After a final wash, endosomes were analyzed by
small-particle flow cytometry (14). Secondary antibodies
(Jackson Immunochemicals, Bar Harbor, ME) were donkey anti-goat
cyanine-5 (for anti-RII) and donkey anti-rabbit cyanine-3 (for
anti-AQP2 and anti-PKC). Cyanine-3 was excited with 100 mW of 488-nm
blue argon ion laser light, and cyanine-5 was excited with 100 mW of
647-nm ruby-red krypton laser light. The fluorescence of each of the
2,000 individual particles was collected in photomultipliers beyond a
575 ± 26- or 675 ± 20-nm (cyanine-3 or cyanine-5,
respectively) band-pass filter. Flow cytometry files were collected and
analyzed using Becton Dickinson Cell Quest flow cytometry software.
[32P] labeling and
immunoprecipitation of AQP2.
Endosomes were phosphorylated using the catalytic subunit of PKA in the
presence of [-32P]ATP, as described previously
(22). The 32P-labeled endosomes were then
subjected to various treatments during incubation at 37°C in buffer
containing (in mM) 20 Tris (pH 7.0), 50 KCl, 3 Mg2+, 0.1 Ca2+, 0.5 Ni2+, and 0.1 dithiothreitol as well
as 3,900 U PKI. The resulting [32P]AQP2 was then
collected by immunoprecipitation and assayed for its 32P
content. Briefly, after 32P labeling, endosomes were
solubilized using 1% (vol/vol) Triton X-100 (1 h on ice) and
centrifuged at 14,000 g for 30 min. The supernatant was
combined with rabbit anti-AQP2 antiserum coupled to Sepharose 4B and
incubated with continuous mixing at 4°C for 14 h. The
immunoprecipitate was pelleted by centrifugation, washed three times in
phosphate-buffered saline, and denatured with Laemmli buffer.
[32P]AQP2 was then resolved by SDS-PAGE, detected by
autoradiography, and quantified by densitometry.
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RESULTS |
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Purified IMCD heavy endosomes possess a 90-kDa AKAP, RII, PP2B,
and PKC. With the use of specific antisera, Fig.
1 shows that rat inner medulla contains a
55-kDa RII species and the 18-kDa PP2B
-subunit (Fig. 1,
A and B). Sands et al. (25) have
reported previously that purified rat IMCD heavy endosomes also possess PKC
. Because these three proteins represent the basic components of
an AKAP-signaling complex present in hippocampal neurons (10, 18), we sought to determine whether IMCD heavy endosomes also possess a similar AKAP-signaling complex. As detected using a 32P-RII overlay assay shown in Fig. 1, AQP2 endosomes also
possess AKAP(s). To demonstrate that the RII probe is binding
specifically to endogenous AKAP, we used the anchoring inhibitor
peptide Ht31 to ablate its binding (Fig. 1C, lane
6). Ht31 represents the peptide sequence within AKAP that is
responsible for RII binding (8). We have assigned the
single most prominent band a molecular mass of ~90 kDa based on data
from multiple experiments (n = 12). However, it should
be noted that the band is broad and in some experiments appears as a
doublet that included a smaller band of an ~75-kDa relative molecular
mass (Fig. 1C). More complete characterization will be
required to determine whether these multiple AKAP bands observed on
32P-RII overlay blots result from either proteolysis of a
larger AKAP protein or to posttranslational modification of an AKAP(s). Alternatively, these multiple bands may derive from two independent species of RII-binding protein. The data shown in Fig. 1 are consistent with and extend those reported by Klussman et al. (20).
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To verify that RII, PP2B, and PKC colocalized within collecting duct
cells, we immunostained sections of rat inner medulla using antisera
specific for each of the proteins. As shown in Fig.
2, rat IMCDs exhibited diffuse
immunoreactivity to each of the antibodies tested with the exception of
anti-PP2B antibody, which strongly stained the apical region of IMCD
cells (Fig. 2C).
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IMCD heavy endosomes possess a multiprotein
phosphorylation complex similar to that found in neurons.
With the employment of a methodology similar to that used previously to
demonstrate the existence of an AKAP-signaling complex in neurons
(7), detergent-solubilized IMCD heavy endosomes were mixed
with cAMP-agarose, with the resulting cAMP-agarose-bound proteins
identified by an anti-RII immunoblot or by a 32P-RII
overlay assay. As shown in Fig.
3A, cAMP-agarose retained both
RII and the 90-kDa putative AKAP, substantially clearing these proteins
from a mixture of detergent-soluble endosomal proteins.
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Colocalization of RII with AQP2 and
PKC in the same IMCD endosomal
population.
To further test the hypothesis that RII and PKC
are associated
together within a putative AKAP complex present in IMCD heavy endosomes, small-particle flow cytometry was utilized to examine whether anti-RII and anti-PKC
antibodies colocalize to the same population of heavy endosomes. Rat IMCD heavy endosomes, shown previously to contain abundant AQP2 (14), were incubated
with antisera against RII and PKC
, and their presence was determined with species-specific secondary antibodies coupled to either of the
fluorophores cyanine-3 or cyanine-5. Perfusion of the rats with
fluorescein-dextran (F-dextran) before kidney harvest results in the
uptake of the concentrated F-dextran by endocytic vesicles retrieved
from the apical membrane of the IMCD as previously described (14). Therefore, through the measurement of fluorescence
at the three different emission wavelengths corresponding to excitation of fluorescein, cyanine-3, and cyanine-5, it is possible to demonstrate whether a single F-dextran-containing endosome possesses one or two
specific proteins bound to its cytoplasmic surface.
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AQP2 is dephosphorylated by endogenous
PP2B activity and by exogenous PP2B in
vitro.
Previously, we have shown that the AQP2 present in IMCD heavy endosomes
is phosphorylated by endogenous PKA activity (22). To test
whether this endogenous AQP2 is also a substrate for exogenous PP2B in
vitro, endosomes were 32P labeled using the catalytic
subunit of PKA and incubated at 37°C for various intervals in the
presence or absence of an exogenous PP2B catalytic subunit. AQP2 was
then immunoprecipitated from the endosomes, and the 32P
content of AQP2 was determined by SDS-PAGE and autoradiography. As
shown in Fig. 5, addition of the PP2B
catalytic subunit increased the rate of [32P]AQP2
dephosphorylation.
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DISCUSSION |
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The phosphorylation of AQP2 by PKA is a crucial signal in the AVP-elicited water reabsorption pathway. The trafficking of AQP2 vesicles into the IMCD apical membrane is regulated by PKA activity (reviewed in Ref. 27) and AKAPs (19), although the exact subcellular location of the PKA responsible for AQP2 phosphorylation has not been determined. Studies of the postsynaptic densities of hippocampal neurons have revealed the existence of a signaling complex containing RII, PP2B, and PKC that is bound to a 79-kDa AKAP. This multiprotein complex permits the regulation of membrane receptors and ion transporters, such as AMPA/kainate receptors, Ca2+ channels, or N-methyl-D-aspartate receptors, via protein phosphorylation by PKA or PKC or via dephosphorylation by PP2B (11).
The data reported here show that a purified population of IMCD heavy
endosomes also contains the various protein components of a similar
AKAP complex. In previous work, these IMCD heavy endosomes have been
shown to 1) be of apical origin, 2) possess abundant AQP2 protein by immunoblotting analysis, and 3)
exhibit a very large mercury-sensitive osmotic water permeability as
determined by using stop-flow fluorimetry (14, 22). A
combination of Western blotting and immunocytochemistry suggests that
AKAP complex constituents, including RII, PP2B and PKC, are present
in IMCD tubules.
Studies using a 32P-RII probe demonstrate its binding to a protein band/doublet of 75-to 90-kDa approximate molecular mass in purified IMCD heavy endosomes (Fig. 3). The 75- to 90-kDa putative AKAP protein(s) reported on here awaits further characterization to determine whether it is a novel member of the AKAP family or represents a member of the AKAP-KL (expressed in kidney and lung) family of proteins, as reported by Dong et al. (9). Although AKAP-KL has been localized to the apical membranes of murine proximal tubules, it has not been reported as being expressed in IMCD.
Affinity chromatography using cAMP-agarose demonstrated that RII, PP2B,
PKC, and a single 90-kDa RII-binding protein (putative AKAP) were
all retained on the cAMP matrix whereas PKC
and AQP2 were not
absorbed. Data reported here suggest the presence of a multiprotein
AKAP-signaling complex, similar to that described in neurons, in rat
AQP2 apical endosomes. Because renal AQP2 expression is upregulated in
dehydration and after chronic AVP treatment, it would be interesting to
know whether these conditions also alter the relative expression of
these AKAP complex proteins.
To demonstrate that these proteins are actually associated with apical
endosomes rather than being present in contaminating membranes in some
portion of the purified IMCD heavy endosomal preparation, we performed
three-color, small-particle flow cytometry to colocalize constituents
of a putative AKAP complex on the surface of apical endosomes
containing entrapped F-dextran. The studies demonstrate that binding of
both RII and PKC is colocalized on the cytoplasmic surface of
F-dextran-containing vesicles, providing further evidence for the
presence of an AKAP complex in apically derived IMCD endosomes. A
significant number of these endosomes also contain immunoreactive AQP2 protein.
The data displayed in Figs. 1-4 provide the first evidence for the presence of an AKAP complex on the endocytic arm of AQP2-containing vesicles in IMCD cells. In contrast, studies by Klussmann et al. (20) provide strong support for the importance of AKAPs in the corresponding exocytic arm of AQP2 vesicle insertion into the IMCD apical membrane. Thus the presence of AKAPs in AQP2-containing vesicles may be similar to the situation in neurons, where the AKAP-signaling complex is a dynamic structure with successive phases of protein association and disassociation. It is possible that the AKAP-containing signaling complex described here would possess all the kinases and phosphatases necessary to potentially initiate and terminate the AVP-mediated insertion and removal of AQP2 water channels in the IMCD apical membrane. A similar mechanism might also mediate the function of other membrane transporter proteins. In this regard, ROMK1 channels expressed in Xenopus laevis oocytes are insensitive to forskolin treatment, but when ROMK1 is coexpressed with the 79-kDa AKAP, it exhibits cAMP-dependent activation (1).
We reported previously (22) that AQP2 can be both phosphorylated by an endogenous cAMP-dependent protein kinase and dephosphorylated by an endogenous phosphatase present in IMCD heavy endosomes. Data presented here extend these studies and show that endosomal [32P]AQP2 is dephosphorylated by an endogenous phosphatase that is inhibitable by EDTA or the cyclophilin-cyclosporin A complex. Moreover, AQP2 dephosphorylation also occurred after addition of the exogenous PP2B catalytic subunit (Fig. 5). Together, these data indicate that PKA-phosphorylated AQP2 is an in vitro substrate for PP2B and suggest that PP2B may also be present on the cytoplasmic surfaces of IMCD heavy endosomes in vivo. However, additional work is necessary to demonstrate whether AQP2 dephosphorylation will promote its retrieval from the apical IMCD membrane. Moreover, the incomplete inhibition of endogenous AQP2 dephosphorylation by the cyclophilin-cyclosporin A complex might also suggest the presence of another divalent cation-dependent phosphatase in IMCD heavy endosomes.
The functional role of PKC in the endosomal signaling complex is not
understood. In this regard, rat AQP2 contains a PKC phosphorylation
consensus sequence at Ser226 (Ser231 in human
AQP2). Although there is evidence to suggest that PKC activation may
have an inhibitory effect on AVP-induced water reabsorption in
collecting ducts (2), it is not known whether this effect
is caused by the PKC-mediated phosphorylation of AQP2 as occurs with
AQP4 (13). Alternatively, PKC may act indirectly, perhaps
promoting retrieval of AQP2 endosomes in a process similar to that
described for glucose transporters expressed in oocytes (28). The evidence linking PKC to water reabsorption is
presently based on the use of phorbol esters (1,2 diacylglycerol
analogs) that are unable to activate the atypical PKC
, whereas
PKC
is instead known to be activated via phosphatidylinositol
3-kinase. Thus, if the PKC
present within this AKAP-signaling
complex does play a role in water reabsorption, then the role could be
to modulate the cAMP-mediated ADH response by a
phosphatidylinositol 3-kinase-mediated hormonal or ionic signal.
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ACKNOWLEDGEMENTS |
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We thank Wendy Campbell and Edmund Benes for assistance in the flow cytometry experiments and Dr. Daniela Riccardi for a critical reading of the manuscript.
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FOOTNOTES |
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* I. Jo and D. T. Ward contributed equally to this work.
This work was supported in part by National Institutes of Health Grants DK-38874 (H. W. Harris) and GM-48231 (J. D. Scott).
Address for reprint requests and other correspondence: D. T. Ward, G38 Stopford Bldg., Univ. of Manchester, Oxford Rd., Manchester M13 9PT. UK (E-mail: d.ward{at}man.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 September 2000; accepted in final form 3 July 2001.
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