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Internalization and Desensitization of the Oxytocin Receptor Is Inhibited by Dynamin and Clathrin Mutants in Human Embryonic Kidney 293 Cells

University of Bristol (M.P.S., V.J.A., A.L.B.) Clinical Sciences South Bristol, Division of Obstetrics and Gynaecology, St. Michael’s Hospital, Bristol BS2 8EG, United Kingdom; University of Bristol (S.J.M., E.K.), Department of Pharmacology, School of Medical Sciences, Bristol BS8 1TD, United Kingdom; and University of Bristol (C.A.M.), Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Bristol BS1 3NY, United Kingdom

Address all correspondence and requests for reprints to: M. P. Smith, University of Bristol, Clinical Sciences South Bristol, Division of Obstetrics and Gynaecology, St. Michael’s Hospital, Southwell Street, Bristol BS2 8EG, United Kingdom. E-mail: Mike.smith{at}bris.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca²⁺ calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, ß-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
OXYTOCIN (OT) and related peptides belong to a primitive hormone system involved in the process of birth in many vertebrate and invertebrate species (1). In mammals, OT is released in a pulsatile manner by the neurohypophysis and is thought to have a key role in the peripartum period by stimulating smooth muscle contractility in the uterus and mammary gland (2). OT is one of the most potent uterotonic agents known, and it has been used for many decades for the induction of labor in women (3). However, the practice of continuous iv infusion of OT to initiate and/or augment uterine contractions during labor may not be optimal, and the response of patients is often variable and unpredictable. Studies of myometrial cells, both in vivo and in vitro, have demonstrated that prolonged exposure to OT results in the down-regulation of the OT receptor (OTR) and its mRNA, coincident with a loss of cellular responsiveness (4, 5). Further, the OTR may rapidly desensitize upon exposure to OT, as is common with agonist stimulation among the rhodopsin-type or class I G protein-coupled receptors (GPCRs) to which the OTR and the closely related vasopressin receptor family belong (for recent and extensive reviews, see Refs.6 and 7).

Possible mechanisms of OTR internalization and desensitization have emerged from previous studies in mammalian cell lines. It is likely that, as with other class I GPCRs, the OTR becomes internalized and may desensitize via the classical clathrin-mediated pathway. Briefly, upon agonist stimulation, many GPCRs become phosphorylated by G protein-receptor kinase (GRK) or second messenger kinases such as protein kinase C (PKC) (8). The receptor then uncouples from the G protein through the actions of the arrestin protein family, which also facilitate internalization via clathrin-coated pits (9). Dynamin (Dyn), a large GTPase, is then able to pinch off the clathrin-coated vesicle (10), allowing the receptor within to be targeted either for recycling to the cell membrane or degradation by lysosomes, the precise fate being receptor specific (11). Both GRK2 and ß-arrestin have been shown to be involved in OTR internalization and desensitization (12, 13, 14); however, the roles of proteins of the clathrin-mediated pathway in these processes have yet to be investigated.

In this study, we aimed to further elucidate OTR internalization and desensitization through the use of recently available dominant-negative mutant cDNA constructs of key components of the clathrin pathway of receptor internalization and appropriate OTR agonists and inhibitors. Utilizing model cell line human embryonic kidney (HEK)-293, induced to express the receptor by transient transfection, we have first assessed the time- and concentration-dependent effects of agonist binding upon OTR internalization. Second, we have investigated the role of heterologous second messenger activation in receptor internalization through the use of selective kinase inhibitors. Finally, utilizing transfection of mutant cDNA constructs, we have evaluated the roles of G-protein receptor kinase 2 (GRK2), ß-arrestin2 (Arr), Dyn, and epidermal growth factor p-c2-Eps15 [Eps15 (15)], an essential component of clathrin-coated pit formation, in OTR internalization and desensitization. This approach has, for the first time, clarified the role of these proteins in the mechanisms of agonist-stimulated OTR internalization and desensitization and may now be used as a basis for further study in myometrial cells. It is anticipated that such research might deliver improved understanding of the physiological regulation of the OTR and may prove clinically beneficial in the use of OT during labor management.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Visualization of OT-Induced Hemagglutinin (HA)-OTR Internalization
Confocal microscopy, used to visualize fluorescently labeled HA-OTR, revealed internalization of the receptor upon agonist (OT) exposure. In the absence of agonist, HA-OTR immunostaining was predominantly confined to the plasma membrane (Fig. 1, A and B). After exposure to OT, however, the receptor appeared predominantly localized within the cytoplasm in a punctate manner consistent with endocytic vesicles (Fig. 1C). The distribution of OTR was unaffected by the presence of transfected dominant-negative mutants (Fig. 1, D and E). HA-OTR immunostaining was not apparent in nontransfected cells or in cells transfected with pcDNA3 alone (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 1. Confocal Photomicrographs Showing the Localization of HA-OTR in Transiently Transfected HEK-293 Cells A, After 30 min at 4 C; B, after 30 min at 37 C; C, after 30 min at 37 C in the presence of 100 nM OT; D, after cotransfection of HA-OTR and Arr-DNM, 30 min at 37 C; and E, after cotransfection of HA-OTR and Arr-DNM, 30 min at 37 C in the presence of 100 nM OT.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Time and OT Dependence of HA-OTR Internalization Cell surface immunoreactivity to HA-OTR in transiently transfected HEK-293 cells was assessed by ELISA. A, Raw absorbance units of cells transfected with HA-OTR or empty vector pcDNA3. B, Percentage surface receptor remaining after variable time exposure to 100 nM OT at 37 C () and 4 C (). C, Percentage surface receptor remaining after exposure to variable concentrations (0.1 nM to 0.1 µM) of OT over 30 min. Data presented are mean values ± SEM from at least three separate experiments in each group.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effects of Neurohypophyseal Agonist Hormone Analogs on OTR Loss from the Cell Surface Cell surface immunoreactivity to HA-OTR in transiently transfected HEK-293 cells was assessed by ELISA after exposure (30 min at 37 C) to 100 nM neurohypophyseal peptide receptor agonists. A, Highly potent OTR agonist, [HO1,Thr4]OT; B, selective OTR agonist, [HO1,Thr4,Gly7]OT; C, selective V2 agonist, [Val4,D-Arg8] vasopressin; D, [Arg8]-vasopressin. Data presented are mean values ± SEM of at least three separate experiments. , P < 0.05. Statistical analysis was by one-way ANOVA, utilizing Dunnet’s multiple comparison test.

Second Messenger-Dependent Protein Kinase-Mediated Endocytosis in OT-Induced OTR Internalization
Two selective kinase inhibitors were assessed for their ability to affect OT-induced HA-OTR internalization: PKC inhibitor, GF 109203X, and the Ca²⁺ calmodulin–dependent kinase II (CaMKII) inhibitor, KN-93. HEK-293 cells transiently transfected with HA-OTR were pretreated for 15 min at 37 C with 100 nM kinase inhibitor before incubation with either 100 nM or 1 nM OT for 30 min at 37 C. There was no significant effect upon HA-OTR internalization after cells were exposed to either inhibitory compound compared with control cells not exposed to inhibitor [% internalization after exposure to 100 nM OT being 70.5 ± 5.1 vs. 61.4 ± 9.1 (P > 0.05) and 70.5 ± 5.1 vs. 68.5 ± 4.8 (P > 0.05), respectively; n = 5 independent experiments per group].

Clathrin-Mediated Receptor Endocytocis in OT-Induced OTR Internalization
The clathrin-mediated pathway of internalization/desensitization has been implicated in OTR activation (12, 13) and recently, transiently transfected HA-OTR has been shown to colocalize rapidly with both GRK2 and ß-arrestin after activation by OT in HEK-293 cells (14). Our experiments used cDNA encoding dominant-negative mutant (DNM) forms of proteins that are key to clathrin-mediated receptor endocytosis to assess the potential role of this pathway in OTR internalization.

Experiments were performed to assess potential concentration-dependent effects of each DNM upon cells cotransfected with a constant level of HA-OTR cDNA. Cotransfection with each DNM, shown in Fig. 4A, inhibited agonist-induced HA-OTR internalization in a concentration-dependent manner and indicated that the optimal concentration for each was approximately 10 µg/25 cm³ flask. Because the Arr-DNM has previously been shown to inhibit HA-ß₂-adrenoceptor internalization (17), experiments were performed to assess the specificity of action of this DNM to inhibit HA-OTR internalization. As illustrated in Fig. 4B, cotransfection of Arr-DNM with HA-OTR and by comparison HA-ß₂-adrenoreceptor resulted in the inhibition of agonist-induced receptor internalization (P < 0.05) when compared with control cells cotransfected with the receptor and pcDNA3 alone.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Agonist-Induced Receptor Internalization in Transiently Transfected HEK-293 Cells A, Receptor internalization after cotransfection of HA-OTR with increasing concentrations of Arr-DNM, GRK2-DNM, Eps-15-DNM, and Dyn-DNM (n = 3 in each experimental group). B, Comparison of the effects of Arr-DNM in HEK-293 cells transfected with HA-OTR (left, challenged with 1 nM and 100 nM OT for 30 min) or with ß2-adrenoceptor (right, challenged with 100 nM or 10 µM isoproterenol for 30 min) (n = 3 in each experimental group). C, The effects of pcDNA3 (control), wild-type Arr (Arr WT), Arr-DNM, GRK2-DNM, Eps-15-DNM, and Dyn-DNM in OT-induced HA-OTR internalization. Cells were exposed to 100 nM OT for 30 min at 37 C (n 7 in each experimental group). Data shown are mean ± SEM. Statistical analysis was by one-way ANOVA, utilizing Dunnet’s multiple comparison test (, P < 0.05).

Agonist-Induced Ca²⁺ Mobilization
Visualization of Ca²⁺ mobilization in response to agonist exposure is a good way to measure receptor desensitization (18). We assessed the specificity of agonist-induced Ca²⁺ mobilization in HEK-293 cells transiently transfected with HA-OTR or pcDNA3. Control and HA-OTR-transfected cells were exposed to 100 nM OT and/or carbachol [an agonist of muscarinic acetylcholine receptors, endogenously expressed in HEK-293 cells (19)]. Control cells responded to carbachol with a rapid spike followed by a plateau phase in Ca²⁺ mobilization but did not respond to OT, whereas HA-OTR-transfected cells responded to both OT and carbachol, indicating that OT-induced Ca²⁺ mobilization was dependent upon HA-OTR expression in the cells (data not shown). We next assessed whether repeated exposure to OT would lead to a desensitization in Ca²⁺ mobilization. As illustrated in Fig. 5A, upon initial exposure to OT, Ca²⁺ concentrations reached a rapid peak of 146 ± 32 nM. On second exposure to OT, however, this peak reached just 72 ± 14 nM, a reduction of more than 50% in Ca²⁺ concentration (P < 0.01). This reduction was not apparent when cells were exposed to OT followed by carbachol (CBC) or vice versa (Fig. 5, B and C). Because the muscarinic acetylcholine receptor, activated by CBC, utilizes only intracellular Ca²⁺ stores, it is likely that the observed reduction in response after repeated exposure to OT is due to homologous desensitization of HA-OTR, rather than a loss of Ca²⁺ from intracellular stores.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Agonist-Induced Ca2+ Mobilization HEK-293 cells transiently transfected with HA-OTR were pretreated for 30 min with fura2/AM before extensive washing. Cells were mounted on the microscope stage and incubated with Ca2+ containing medium (PSS). After approximately 45 sec, a further 5 ml of PSS was added (as indicated) to ensure that sample addition did not induce Ca2+ mobilization. After approximately 90 sec, 5ml of agonist (100 nM OT or CBC) was added (as indicated). There then followed a series of extensive washes over approx. 5 min (indicated) to remove any remaining agonist before the above agonist addition protocol was repeated. A, Ca2+ mobilization in response to addition of OT followed by OT. B, Ca2+ mobilization in response to addition of OT followed by CBC. C, Ca2+ mobilization in response to addition of CBC followed by OT. Data presented are mean values (n = at least four cells per field over at least six separate experiments).

View larger version (31K): [in this window] [in a new window]   Fig. 6. The Effects of GRK2, Arrestin, Dyn, and Clathrin in Repeated OT-Induced Ca2+ Mobilization A and B, Ca2+ mobilization protocol as described in Fig. 5 legend; cells were transfected with HA-OTR ± 5–10 µg pcDNA3 (control), Arr-DNM, wild-type Arr (Arr-WT), Dyn-DNM, GRK2-DNM, or Eps-15-DNM as indicated. C, Secondary Ca2+ response of HEK-293 cells transfected with HA-OTR ± assorted cDNA constructs (Secondary response is expressed as the percentage difference in response compared with the initial response, where the initial response = 100%). Data are presented are mean values ± SEM (n 4 cells per field over at least six separate experiments). Statistical analysis was by one-way ANOVA, utilizing Dunnet’s multiple comparison test (, P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

In this study we have investigated HA-OTR internalization and desensitization in HEK-293 cells transiently transfected with the receptor and, for the first time, clearly demonstrated that these are Dyn- and clathrin-mediated events. First, HA-OTR internalization in response to OT was visualized by confocal microscopy. After agonist exposure, the receptor appeared predominantly localized within the cellular cytoplasm rather than at the plasma membrane as was the case with cells not exposed to OT. The punctate pattern of HA-OTR immunostaining observed after agonist exposure in this study is indicative of receptor accumulation within endocytic vesicles as previously reported in HEK-293 cells (13). Cell surface receptor loss was found to increase in a concentration-dependent manner and was also rapid, with greater than 50% of total receptor loss occurring within the first 5 min of agonist exposure. These findings are similar to previous data (6, 14) where rapid (15-min) HA-OTR internalization was reported in response to OT in HEK-293 cells. The specificity of OT-induced HA-OTR internalization was assessed using a selection of OT and vasopressin analogs. Each agonist compound was found to induce OTR internalization to a degree that reflected their known relative oxytocic potencies (6, 16), and internalization was blocked with selective OTR antagonists, indicating that HA-OTR internalization observed in this study was homologous, i.e. ligand-selective.

For the members of the - and ß-adrenoceptor and muscarinic families, desensitization involves phosphorylation of the receptors (20) by PKC and/or GRKs (21, 22). We therefore, assessed the potential roles of second messenger kinases, PKC and CaMKII, in OTR internalization. These enzymes have previously been shown to suppress GPCR internalization (23), and analysis of the amino acid sequence of the OTR suggests that there are at least eight potential PKC and four potential CaMKII phosphorylation sites within the intracellular loops and C-terminal tail (24). It was found that OT-induced HA-OTR internalization was not affected by the PKC inhibitor, GF 109203X, or by the CaMKII inhibitor, KN-93. Previously, PKC has been shown to physically associate with the OTR after exposure to micromolar concentrations of OT (12), but it appears that, at lower OT concentrations, this association does not affect agonist-induced HA-OTR internalization.

Recent experiments in COS-7 cells transfected with OTR have demonstrated that a rapid GRK2-mediated phosphorylation of the agonist-occupied OTR is a key first step leading to its desensitization, and that it precedes and is required for ß-arrestin-dependent internalization (14). We have explored mechanisms of OT-induced HA-OTR internalization and desensitization using dominant-negative mutant cDNA constructs corresponding to proteins involved in GPCR sequestration and internalization via GRK, arrestin- and clathrin-coated pit formation, and scission. Briefly, the GRK2-DNM construct reduces agonist-induced GPCR phosphorylation (25), Arr-DNM lacks a receptor-binding region and competes with wild-type arrestin for clathrin binding (26), Dyn-DNM inhibits Dyn-mediated scission of clathrin-coated vesicles from the plasma membrane (27), and Eps15-DNM inhibits the formation of clathrin-coated pits (15, 28). Transiently transfected HA-OTR has recently been shown to associate with both GRK2 and ß-arrestin upon agonist binding (14), and internalization of the structurally related vasopressin receptor has been shown to require Dyn (29). We found that cotransfection of each DNM construct led to a reduction in agonist-induced HA-OTR loss from the cell surface. Thus it would appear that each of these proteins plays a key role in the process of receptor internalization.

Upon agonist binding, many GPCRs are trafficked into clathrin-coated pits and internalized by endocytosis; however, some receptors are internalized preferentially through specialized lipid raft/caveolae microdomains of the plasma membrane (30). Interestingly G protein subunits are also internalized through lipid rafts and could regulate effectors at multiple cellular sites (31). Because Dyn is involved in both clathrin and nonclathrin pathways, our data using Dyn-DNM demonstrate the role of endocytosis in OTR desensitization but do not conclusively determine the pathway involved. Eps15 was originally identified as a substrate for receptor-activated epidermal growth factor tyrosine kinase and has since been found to be ubiquitously and constitutively associated with the activator protein 2 adaptor protein, and plays an important role in clathrin-mediated endocytosis (32, 33). Hence, the strong inhibitory effect on OTR internalization that we observed in our system suggests that the clathrin pathway is indeed involved as previously suggested (34).

To assess HA-OTR desensitization, we visualized Ca²⁺ mobilization in response to repeated agonist exposure. Cotransfection of HA-OTR with each DNM construct resulted in significantly greater Ca²⁺ mobilization upon repeated exposure to OT, compared with controls, indicating that desensitization was suppressed by inactivated forms of these proteins. These data strongly suggest that receptor internalization might play a major role in OTR desensitization, because inhibition of this process via the expression of key DNM proteins reinstates Ca²⁺ mobilization in response to repeat agonist exposure. An alternative explanation of these data, however, is that both agonist-bound and unbound (activated and resting) OTRs become internalized as a localized cohort of receptors. Thus, rather than affecting desensitization per se, blocking OTR internalization may increase the availability of, as yet, resting receptors and thereby raise the potential of response to further agonist stimulation. Radioligand binding studies measuring agonist occupancy of the OTR over time and in the presence/absence of the DNM constructs used in this study may clarify the relative importance of internalization to the receptor desensitization process.

In summary, these data provide strong evidence that agonist exposure induces OTR internalization and desensitization via the classical clathrin-mediated pathway. Our data fit well with the GPCR internalization model established for the ß-adrenoceptor (9, 39), suggesting that upon agonist binding the receptor becomes internalized and desensitized via GRK2 phosphorylation and subsequent targeting to, and scission of, clathrin-coated pits via ß-Arrestin, Eps-15, and Dyn. Moreover, the data presented here indicate that receptor internalization may be an essential factor in OTR desensitization and also provide support for the recent real-time observations of agonist-induced OTR interaction with GRK2 and ß-Arrestin (14). Interestingly, we have recently obtained preliminary data to suggest that OTR endogenously expressed in human myometrial cells undergoes a similarly extensive and rapid process of internalization and desensitization after agonist exposure. It is now important to further study the dynamics of endogenous OTR internalization/desensitization and resensitization in myometrial cells, to determine their physiological relevance to the process of uterine activation in normal and premature birth, and to help develop better protocols for the pharmacological use of OT in the peripartum period.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
DMEM, fetal calf serum, pcDNA3, and LipofectAMINE 2000 transfection reagent were purchased from Invitrogen (Paisley, Scotland, UK). HA-OTR cDNA was a kind gift from Professor Marc Caron (Duke University, Durham, NC). Anti-HA monoclonal antibody (HA-11) was from Covance Laboratories, Inc. (Madison, WI), anti-Fc specific goat antimouse fluorescein-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). OT was from Bachem Ltd. (Liverpool, UK). Kinase inhibitors were purchased from Tocris Cookson (Bristol, UK). All other reagents were purchased from Sigma-Aldrich Corp. (Poole, UK) or VWR International (Leicester, UK).

Cell Culture
The HEK-293 cell line was maintained in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate at 37 C in a humidified atmosphere of 95% air and 5% CO₂.

Transient Transfection
Transient transfections using HA-OTR cDNA with or without DNM cDNA constructs of GRK2 (25), Arr(319–418) (26), Dyn (K44A) (27), Eps15 (15), or Arr-WT or empty vector pcDNA3 (negative control), were performed with cells at approximately 90% confluence within a 50 ml/25 cm³ tissue culture flask, utilizing LipofectAMINE 2000 according to the manufacturer’s instructions. After 24 h exposure to the cDNA/LipofectAMINE complex, cells were transferred to each well of either a 24-well tissue culture plate for subsequent ELISA, or a six-well tissue culture plate, containing glass coverslips, pretreated with poly-l-lysine (100 mg/ml) to promote cell adhesion, for subsequent analyses. Cells were incubated for a further 24-h period at 37 C, 5% CO₂ in complete DMEM before further experimentation. The efficiency of HA-OTR transfection, based upon the percentage of cells that responded to OT exposure, was assessed during dynamic Ca²⁺ imaging and routinely found to be greater than 60%. In comparative experiments the effect of Arr-DNM was assessed under identical conditions in cells cotransfected with either HA-OTR or HA-ß₂-adrenoceptor (17).

Visualization of Receptor Internalization
The localization of receptors to the cytosolic or membranous compartments of transfected cells before and after agonist treatment was performed as described previously (17, 23). Briefly, receptors were labeled by incubation with a mouse anti-HA antibody and a subsequent incubation with an Fc-specific goat antimouse antibody. The coverslips to which the cells were attached were mounted on glass slides and stored at 4 C before microscopic analysis using an inverted Leica (Wetzler, Germany) TCS-NT confocal laser scanning microscope attached to a Leica DM IRBE epifluorescence microscope with phase contrast and a Plan-Apo x40 numerical aperture objective under oil immersion. All images were stored on Leica TCS-NT software.

Receptor Internalization Assay
The internalization of HA-tagged receptors was assessed by quantitation of cell surface HA immunoreactivity, using ELISA as described elsewhere (17, 23). Briefly, after transfection and a further 24-h period of incubation, cells were serum starved for 1 h before exposure to various concentrations of OT or OT analog over various time periods at 37 C. Reactions were terminated by fixation in 3.7% (vol/vol) formaldehyde in Tris-buffered solution. After fixation, cell surface immunoreactivity of HA-tagged receptor was assessed by ELISA; briefly, fixed cell samples were incubated with a mouse anti-HA monoclonal antibody for 1 h at room temperature. Unbound antibody was removed through a series of 5-min washes in Tris-buffered solution, and samples were then incubated for an additional hour with a secondary, Fc-specific, alkaline phosphatase-conjugated goat antimouse antibody. After a further series of washes, developer (Bio-Rad Laboratories Ltd. Hertfordshire, UK) was applied to each sample, the medium was mixed with NaOH, and the resulting color reaction was measured using a plate reader at 405 nm. Background absorbance from samples transfected with pcDNA3 alone was subtracted from all results. Results are expressed as the percentage of cell surface receptor loss, where 100% represents total absorbance minus background in control samples. Data were derived from at least four separate experiments in each case.

Dynamic Video Imaging of Cytosolic Ca²⁺
Dynamic video imaging to measure cytosolic Ca²⁺ in fura 2-loaded HEK-293 cells, with or without transfected vectors, was performed as previously described (40, 41). Briefly, cells were washed in a physiological salt solution (PSS, 127 mM NaCl, 1.8 mM CaCl₂, 5 mM KCl, 2 mM MgCl₂, 0.5 nM NaH₂PO₄, 5 mM NaHCO₃, 10 mM glucose, 0.1% BSA, and 10 mM HEPES, pH 7.4) before fura 2/AM (2.5 µM in PSS) loading by incubation for 30 min at 37 C. Excess fura 2/AM was then removed by washing the cells in PSS, and the coverslips were fitted into a stainless steel holder and placed into a heating chamber at 37 C. Imaging of the cells was performed within 5–20 min of placement into the heated chamber in approximately 500 µl PSS using MagiCal hardware, Tardis software, and a Nikon Diaphot microscope (Kingston-upon-Thames, Surrey, UK). Details of cell stimulations are given in the figures and figure legends. Cells were excited alternately at 340 and 380 nm, and emitted light was collected at 510 nm, averaging the data from eight video frames and subtracting background values before ratioing. The ratio of fluorescence at 340 and 380 nm was calculated on a pixel-by-pixel basis and used to determine ionized Ca²⁺ concentration assuming a dissociation constant of 225 nM for fura-2 and Ca²⁺ at 37 C.

Statistics
Data are expressed as the mean ± SE of at least four separate experiments with P < 0.05 considered significant. Data were analyzed by GraphPad Prism3 (GraphPad Software, Inc., San Diego, CA), and significance was determined by one-way ANOVA utilizing Dunnet’s multiple-comparison test.

	ACKNOWLEDGMENTS

 
HA-OTR cDNA was a gift from the laboratory of Professor M. G. Caron (Duke University, Durham, NC). Synthetic OT peptide analogs were kindly supplied by Professor M. Manning (Medical College of Ohio, Toledo, OH).

	FOOTNOTES

 
This work was supported by Wellbeing of Women (Grant PG/142).

First Published Online September 22, 2005

Abbreviations: Arr, ß-Arrestin2; CaMK, calmodulin-dependent kinase; DNM, dominant-negative mutant; Dyn, dynamin; Eps15, epidermal growth factor p-c2-Eps15; GPCR, G protein-coupled receptor; GRK2, G-protein receptor kinase 2; HA, hemagglutinin; HEK, human embryonic kidney; OT, oxytocin; OTR, oxytocin receptor; PKC, protein kinase C; PSS, physiological salt solution.

Received for publication January 12, 2005. Accepted for publication September 13, 2005.

	REFERENCES

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