Regulation of AQP2 localization by S256 and S261 phosphorylation and ubiquitination

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.     

Aquaporin-2 regulates cell volume recovery via tropomyosin

Cell volume regulation is particularly important for kidney collecting duct cells. These cells are the site of water reabsorption regulated by vasopressin and aquaporin-2 (AQP2) trafficking to the apical membrane, and subject to changes in osmolality. Here, we examined the role of AQP2 in regulatory volume decrease (RVD), which is a cellular defensive process against hypotonic stress. Stable expression of AQP2 increases RVD in MDCK cells and its phosphorylation levels decrease during the RVD process. We then examined the involvement of AQP2 phosphorylation at serine 256 and serine 261 in RVD using cells stably expressing the phosphorylation mutants. Both S256A- and S256D-AQP2 decrease RVD compared to wild type (WT)-AQP2 although only S256A mutation decreases the initial osmotic swelling, indicating that AQP2-enhanced RVD is independent of osmotic swelling induced by the water permeability of AQP2. S261A and S261D mutations do not induce changes compared with WT-AQP2. These findings indicate that switching between phosphorylation and dephosphorylation at S256 is important for RVD. We previously reported that AQP2 interacts with tropomyosin 5b (TM5b), which regulates actin stability. AQP2 interactions with TM5b are rapidly increased by hypotonicity and then decreased, which are consistent with AQP2 phosphorylation levels. Knockdown and overexpression of TM5b show its essential role in WT-AQP2-enhanced RVD. RVD in S256A- and S256D-AQP2-expressing cells is not changed by TM5b knockdown or overexpression. The present study shows that AQP2 regulates RVD via TM5b and switching between phosphorylation and dephosphorylation at S256 in AQP2 is critical for this process.     

The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.     

Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser(256), Ser(261), Ser(264), and Ser(269)) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser(269) and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)(269)-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser(256) phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser(256), but not Ser(269). Phosphorylation of AQP2 at Ser(269) did not occur when Ser(256) was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser(269) phosphorylation depends upon prior phosphorylation at Ser(256). Immunogold electron microscopy localized Ser(P)(269)-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D "phosphomimic" AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser(269):(a) depends on prior PKA-mediated phosphorylation of Ser(256) and (b) enhances apical plasma membrane retention of AQP2.     

Phosphorylation of aquaporin-2 regulates its water permeability

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.     

A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2

Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK(1) cells by a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by addition of an 18-amino acid signal peptide from hen egg white lysozyme. Immunofluorescence labeling, together with analysis of Alexa 555-dextran internalization, revealed that ssYFP is exclusively located in the secretory pathway. Immunofluorescence and immunogold electron microscopy showed significant colocalization of ssYFP and AQP2. Fluorimetry and Western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK(1) and AQP2-expressing cells. In AQP2-expressing cells, a twofold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89 and was not observed in native cells. The endocytotic inhibitor methyl-beta-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. Although cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP induced a significant increase in exocytosis. Our data indicate that 1) this assay can monitor exocytosis in cultured epithelial cells, 2) VP has an acute stimulatory effect on ssYFP secretion in AQP2-expressing, but not native, cells, and 3) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis and play only a minor role in the VP-induced burst. These results support the idea that, in addition to its role in reducing AQP2 endocytosis, VP increases AQP2 exocytosis.     

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.     

Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in 'endocytosis-resistant' membrane domains after vasopressin treatment

BACKGROUND INFORMATION: Aquaporin 2 (AQP2) plays an important, VP (vasopressin)-regulated role in water reabsorption by the kidney. The amount of AQP2 expressed at the surface of principal cells results from an equilibrium between the AQP2 in intracellular vesicles and the AQP2 on the plasma membrane. VP shifts the equilibrium in favour of the plasma membrane and this allows osmotic equilibration to occur between the collecting duct lumen and the interstitial space. Membrane accumulation of AQP2 could result from a VP-induced increase in exocytosis, a decrease in endocytosis, or both. In the present study, we further investigated AQP2 accumulation at the cell surface, and compared it with V2R (VP type 2 receptor) trafficking using cells that express epitope-tagged AQP2 and V2R. RESULTS: Endocytosis of V2R and of AQP2 are independent events that can be separated temporally and spatially. The burst of endocytosis seen after VP addition to target cells, when AQP2 accumulates at the cell surface, is primarily due to internalization of the V2R. Increased endocytosis is not induced by forskolin, which also induces membrane accumulation of AQP2 by direct stimulation of adenylate cyclase. This indicates that cAMP elevation is not the primary cause of the initial, VP-induced endocytic process. After VP exposure, AQP2 is not located in endosomes with internalized V2R. Instead, it remains at the cell surface in 'endocytosis-resistant' membrane domains, visualized by confocal imaging. After VP washout, AQP2 is progressively internalized with the fluid-phase marker FITC-dextran, indicating that VP washout releases an endocytotic block that maintains AQP2 at the cell surface. Finally, polarized application of VP to filter-grown cells shows that apical VP can induce basolateral endocytosis and V2R down-regulation, and vice versa. CONCLUSIONS: After VP stimulation of renal epithelial cells, AQP2 accumulates at the cell surface, while the V2R is actively internalized. This endocytotic block may involve a reduced capacity of phosphorylated AQP2 to interact with components of the endocytotic machinery. In addition, a complex cross-talk exists between the apical and basolateral plasma-membrane domains with respect to endocytosis and V2R down-regulation. This may be of physiological significance in down-regulating the VP response in the kidney in vivo.     

cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.     

NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip

Vasopressin increases urine concentration through activation of aquaporin-2 (AQP2) in the collecting duct. Nonsteroidal anti-inflammatory drugs (NSAIDs) block prostaglandin E2 synthesis, and may suppress AQP2 producing a urine concentrating defect. There are four serines in AQP2 that are phosphorylated by vasopressin. To determine if chronic use of NSAIDs changes AQP2’s phosphorylation at any of these residues, the effects of a non-selective NSAID, ibuprofen, and a COX-2-selective NSAID, meloxicam, were investigated. Daily ibuprofen or meloxicam increased the urine output and decreased the urine osmolality significantly by days 7 through 14. Concomitantly, meloxicam significantly reduced total AQP2 protein abundance in inner medulla (IM) tip to 64% of control and base to 63%, respectively. Ibuprofen significantly decreased total AQP2 in IM tip to 70% of control, with no change in base. Meloxicam significantly increased the ratios of p²⁵⁶-AQP2 and p²⁶¹-AQP2 to total AQP2 in IM tip (to 44% and 40%, respectively). Ibuprofen increased the ratio of p²⁵⁶-AQP2 to total AQP2 in IM tip but did not affect p²⁶¹-AQP2/total AQP2 in tip or base. Both ibuprofen and meloxicam increased p²⁶⁴-AQP2 and p²⁶⁹-AQP2 ratios in both tip and base. Ibuprofen increased UT-A1 levels in IM tip, but not in base. We conclude that NSAIDs reduce AQP2 abundance, contributing to decreased urine concentrating ability. They also increase some phosphorylated forms of AQP2. These changes may partially compensate for the decrease in AQP2 abundance, thereby lessening the decrease in urine osmolality.     

Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.     

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.     

Mu-opioid receptor desensitization: role of receptor phosphorylation,internalization, and representation

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.     

Trafficking and phosphorylation dynamics of AQP4 in histamine-treated human gastric cells

BACKGROUND INFORMATION: AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells. RESULTS: In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide. CONCLUSIONS: Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.     

Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.     

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.     

Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (hek) cells: identification of Key Ser/Thr (S/T) amino acids

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.