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Laboratory of Molecular Endocrinology, McGill University Health Centre, Montreal, Quebec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Hans H. Zingg, M.D., Ph.D., Laboratory of Molecular Endocrinology, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GPCRs are known to undergo desensitization after a sustained or prolonged stimulation by their respective agonists (5), a phenomenon characterized by a decrease in the response to further agonist stimulation (5, 6). It is now well established that this desensitization process involves different steps. The first one consists in receptor modification by phosphorylation, leading to the uncoupling between the receptor and its associated G protein (5, 6). This step involves either second messenger-dependent kinases, such as protein kinase A or protein kinase C, or GPCR-specific protein kinases, namely GRKs (5, 6, 7). In the latter case, the phosphorylated receptor becomes a target for other intracellular proteins, such as the arrestins. Arrestins will bind the GRK-mediated phosphorylated and agonist-occupied GPCR, uncoupling the G protein from the receptor. More than participating in the desensitization of GPCR, the arrestins act also as adapter proteins for the internalization of receptors by targeting the desensitized receptor to the clathrin pathway (8, 9).
The cell-specific changes in OT binding and OTR mRNA abundance during pregnancy and in response to steroids has been extensively studied. Thus, myometrial OTR expression is highly up-regulated at the end of pregnancy and this regulation may be involved in mediating parturition under physiological conditions (1, 10, 11). Moreover, uncontrolled OTR up-regulation may be a contributing cause to preterm labor (1). In addition, the efficiency of OT signaling may be dependent on OTR desensitization, intracellular trafficking of the receptor and its resensitization (1, 12, 13). However, the precise mechanisms mediating OTR regulation are not well understood. Studies using the expression of the human OTR in human embryonic kidney (HEK)-293 or Madin-Darby canine kidney cells have shown that agonist-stimulation of OTR lead to receptor desensitization (14) and internalization (12, 13). Attempts to coimmunoprecipitate any of the existing GRKs with the OTR have so far been unsuccessful (15), although the recruitment of ß-arrestins and their intracellular trafficking with the OTR are compatible with a role for GRK-mediated phosphorylation (12).
In an attempt to gain more insight into the mechanisms underlying OTR regulation and to address the question specifically whether GRKs are involved in mediating OTR function, we investigated the role of phosphorylation in OTR desensitization and internalization. Using two different cell systems, HEK-293 cells and COS-7 cells, we show that GRK2 regulates phosphorylation, arrestin-mediated internalization, and desensitization of the OTR. Most importantly, we demonstrate, in live cells and in real time, the formation of an agonist-mediated complex between the OTR and GRK2 using an adapted bioluminescence resonance energy transfer (BRET) assay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), indicating that, in both cell systems, the OTR was efficiently coupled to the classical phospholipase C/inositol triphosphate/ calcium pathway as in intact myometrial cells (17).
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Internalization of OTR Is Dependent on GRK2 and ß-Arrestin
We next assessed to what extent the observed agonist-induced desensitization of OTR signaling was accompanied by decreased OT surface binding. As shown in Fig. 2A, OT treatment of OTR expressing HEK-293 cells for 15 or 30 min led to a decrease of [3H] OT binding by 35% and 55%, respectively, suggesting rapid agonist-induced receptor internalization. To test for the involvement of GRK-mediated receptor phosphorylation, we suppressed endogenous GRK-2 activity by cotransfecting HEK-293 cells with GRKK220M, a dominant-negative GRK2 mutant that specifically inhibits GRK2 action (18). As illustrated in Fig. 2A, expression of GRKK220M strongly diminished agonist-induced loss of cell surface binding.
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). Internalization was further reduced by cotransfecting COS-7 cells with GRK2K220M. On the other hand, transfection with GRK2 and ß-arrestin restored receptor internalization in COS-7 cells to levels observed in HEK-293 cells (Fig. 2B). Transfection with either vector alone had only a moderate effect. This suggested that the weak agonist-induced receptor internalization in COS-7 cells was due to low GRK and ß-arrestin abundance and that OTR internalization was both GRK and ß-arrestin dependent.
Agonist-Induced OTR Phosphorylation
We next examined OTR phosphorylation directly by metabolic in vivo labeling in HEK-293 and COS-7 cells transfected with a hemagglutinin-tagged OTR (HA-OTR). Autoradiographic analysis of HA-immunoprecipitates showed an agonist-dependent phosphorylation of the glycosylated form of the HA-OTR (Fig. 3A). Two additional smaller bands were also detected in the autoradiogram, likely corresponding to the nonglycosylated and a partially glycosylated form of the OTR. The phosphorylation of these bands was not affected by agonist exposure. No specific bands were detected in immunoprecipitates of cells transfected with vector alone (lanes 1 and 2).
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, in HEK-293 cells, agonist exposure led within 10 min to an 8-fold increase in the amount of receptor phosphorylation. In contrast, in COS-7 cells, only a 3-fold increase was noted within the same time frame. In keeping with the idea that differing amounts of GRK2 and ß-arrestin expression accounted for the observed difference in OTR phosphorylation in the two cell lines, we tested the effect of transfecting COS-7 cells with expression vector for GRK2. As shown in Fig. 3B, in COS-7 cells transfected with a GRK2 expression vector, agonist-induced OTR phosphorylation was increased to levels similar to the ones observed in HEK-293 cells.
Effect of GRK-Dominant Negatives on OTR Phosphorylation
To evaluate the involvement of endogenous GRK2 in OTR phosphorylation, we assessed the effect of transfecting dominant-negative GRK2 mutants on agonist-induced OTR phosphorylation. The dominant-negative GRK2 mutants used included: the GRK2K220M mutant in which the kinase domain was inactivated, the prenylated GRK mutant GRK2C20-K220M, and ß-adrenergic receptor kinase (ßARKct), the C-terminal fragment of GRK2. Transfection with any of the mutants totally abolished agonist-induced OTR phosphorylation, indicating the involvement of GRK2 in agonist-triggered phosphorylation of the OTR (Fig. 3B).
Endogenous Expression Levels of GRK2
To further corroborate the hypothesis that the observed disparity in the efficiency of OTR desensitization was due to differences in the endogenous expression levels of GRK2 in COS-7 vs. HEK-293 cells, GRK2 immunoreactivity present in the two cell lines was assessed by Western blotting using a specific GRK2 antibody. As shown in Fig. 4, there was indeed a striking difference in the GRK2 expression levels between the two cell lines. However, even in HEK-293 cells, the endogenous expression levels of GRK2 did not reach the high levels present in rat uterus, one of the main in vivo sites of OTR expression.
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). This enrichment was, however, very transient and lasted less than 5 min.
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). A band corresponding to GRK2 immunoreactivity was readily detectable after 1 min of OT addition, indicating a rapid agonist-induced physical association between HA-OTR and GRK2. A decrease in band intensity was observed after 5 min of agonist exposure, compatible with the transient nature of GRK translocation described above (Fig. 5, B and C).
Real-Time Detection of GRK2/OTR and ß-Arrestin/OTR Interactions by BRET
The BRET technique offers the advantage to detect protein/protein interaction in intact living cells. We used this technique to follow such interactions in real time. This technique is based on a nonradiative transfer of energy between the luminescent donor Renilla luciferase (RL) and the fluorescent acceptor yellow fluorescent protein (YFP) (19). Whereas light emission by RL is induced by substrate addition, the characteristic fluorescence emission of YFP is strictly dependent on its close proximity to RL upon luminescence emission. We first applied this approach to assess the role of GRK2 on OTR/ß-arrestin interactions, using an OTR tagged with RL (OTR-RL) at its C terminus in conjunction with an YFP-tagged ß-arrestin construct. Preliminary experiments showed that in COS-7 cells transfected with OTR-RL, the RL tag did not interfere with ligand binding and receptor signaling, as assessed by agonist-induced calcium mobilization (data not shown). In cells transfected with both OTR-RL and ß-arrestin-YFP, addition of OT led to a rapid and sustained rise in the BRET ratio (Fig. 6, A and B). Levels reached a plateau phase at 2 min and declined only slowly, with an 11% decrease measured at 8 min. These data indicated a rapid and sustained agonist-induced association between OTR and ß-arrestin. The following controls attested to the specificity of this observation: no significant BRET was observed when the vector ß-arrestin-YFP was replaced by the parent vector pEYFP-N1, no ligand-induced change in BRET was observed with the specific OT antagonist OTA (20), and the agonist-induced BRET was fully suppressed in presence of an excess of the antagonist OTA (Fig. 6C). Inhibition of OTR phosphorylation by cotransfection of the GRK-2 dominant-negative mutant GRK2K220M led to a significant 45% suppression of the agonist-induced increase of the BRET ratio (Fig. 6C). This observation lent further support to the hypothesis that specific GRK2-mediated OTR phosphorylation is of functional importance for efficient OTR/arrestin association and thus OTR desensitization.
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, A and B). However, in contrast to the OTR/ß-arrestin association, the BRET ratio declined rapidly and decreased by 65% after 8 min of agonist addition. The specificity of this observation was assessed by repeating the experiments in cells transfected with an excess of a vector encoding wild-type GRK2 (without the YFP moiety). As shown in Fig. 7C, competition with wild-type GRK2 inhibited agonist-induced changes in the BRET ratio.
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): GRK/OTR interaction occurred immediately after agonist addition within the first 4 sec, and maximum levels were reached within 10 sec. By contrast, ß-arrestin/OTR interactions only took off after a lag time of 10 sec. This was followed by a constant linear rise over the next 20 sec to reach near plateau levels 30 sec after ligand addition.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using the BRET technique, we were able to dissect precisely the kinetics of GRK2 interaction with the OTR. GRK2/OTR interactions occurred immediately upon agonist stimulation but were transient. Comparison of the kinetics demonstrated that GRK2/OTR interactions peaked at exactly the time point at which ß-arrestin/OTR interactions started to increase. Moreover, and in contrast to the ß-arrestin/OTR interactions, the GRK2/OTR association was very short lasting, compatible with the idea that a rapid but transient association of GRK2 with the receptor is necessary and sufficient to induce receptor phosphorylation. Receptor phosphorylation then induces a more stable association with ß-arrestin, which associates with the OTR for a longer time. These data not only further corroborate the concept that the OTR is phosphorylated via an interaction with GRK2 but also represent the first direct demonstration of a GRK/GPCR interaction in real time in a living cell. Taken together, our observations strongly suggest that the OTR is phosphorylated by GRK2 upon OT stimulation, and that this covalent modification is causally linked to the observed OTR desensitization. However, we cannot exclude the possibility that other GRK subtypes might interact with the OTR and may be involved in the physiological regulation of the receptor. A conclusive answer to this question will require the use of the recently developed GRK transgenic and knockout mouse models that represent important tools to determine the in vivo specificity of different GRKs in the desensitization of different GPCRs (22).
For many GPCRs, it has been shown that the receptor phosphorylation alone was not sufficient to fully desensitize the receptor. The presence of ß-arrestin is required to quench the receptor signal and to enhance the desensitization process (23, 24, 25, 26). For the OTR, our results showed that ß-arrestin, when coexpressed with GRK2, enhanced both OTR desensitization and internalization in COS-7 cells. Furthermore, our data obtained with the real-time BRET assay showed that ß-arrestin interacted with the OTR, and that this interaction was stable. This is in contrast to the transient interaction between GRK2 and OTR that we observed. The sustained signal observed by BRET with OTR and ß-arrestin might reflect the persisting association of the two proteins. Recent data have shown that GPCRs could be separated into two distinct classes according to the stability of receptor/ß-arrestin interactions (27, 28). The members of one class (class A) have more affinity for ß-arrestin-2 than ß-arrestin-1, and interact only in a transient fashion with ß-arrestins at the plasma membrane, whereas the members of the second class (class B) bind both ß-arrestin-1 and –2 with equal affinity and their interaction with arrestins is more stable. Members of class A do not cointernalize with ß-arrestin and are rapidly dephosphorylated and recycled back to the plasma membrane, whereas the members of class B internalize into endosomes with ß-arrestin and are poorly dephosphorylated and retargeted to the cell surface. The stability of the OTR/arrestin complex observed by real-time BRET is in agreement with a class B phenotype where the receptor, as recently reported, internalizes with arrestin in endosomes and slowly recycles back to the plasma membrane (12, 27, 29, 30).
In summary, our results strongly support that a rapid GRK2-mediated phosphorylation of the agonist-occupied OTR is an important first step leading to its desensitization as well as to its ß-arrestin-dependent internalization. Moreover, our real-time BRET analysis does not only represent the first demonstration of a GRK2/GPCR physical interaction in real time in living cells, but it also offers insights into the dynamics of this process. Our studies show clearly in real time that GRK2/OTR interactions precede the ß-arrestin/OTR association, and that the latter interaction is dependent on the former one. Although additional kinases may be involved in mediating OTR phosphorylation under physiological conditions, the present data clearly establish that in the present model systems used, GRK-2 is able to mediate agonist-induced OTR phosphorylation and the GRK-2/OTR interaction is a prerequisite for ß-arrestin-mediated OTR desensitization. Finally, our system of real-time detection of GRK/receptor interactions may also represent a useful tool for the screening of agents designed to modulate GPCR signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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cDNA Constructs
Expression vectors for HA-OTR and for a ß-arrestin-YFP were provided by Marc G. Caron (Duke University, Durham, NC) (12). A vector encoding RL fused to the C-terminal domain of OTR (OTR-RL) was previously described by our laboratory (31). GRK2 with YFP added to its C-terminal domain (GRK-YFP) was generated using PCR. Briefly, a PCR fragment encoding the full-length bovine GRK2 was inserted in frame in the expression vector pEYFP-N1 (CLONTECH, Palo Alto, CA). Several dominant-negative mutants of GRK2 (GRK2K220M, GRK2C20-K220M, and ßARKct) were generously provided by Marc G. Caron and Robert J. Lefkowitz (Duke University). GRK2K220M cDNA encodes a disabled GRK with respect to its kinase activity (32, 33). The GRK2C20-K220M mutant corresponds to GRK2K220M with a geranygeranyl moiety added. This modification allows the protein to be constitutively anchored at the cell membrane without the need of interacting with Gß
subunits (32, 34). ßARKct corresponds to the C-terminal domain of GRK2. This molecule prevents GRK2/Gß interactions and thus GRK membrane translocation by competing with GRK2 for the interaction with the Gß subunit (35).
Cell Transfection
HEK-293 and COS-7 cells were transfected with the HA-OTR cDNA in the presence or the absence of cDNA constructs for GRK2, and/or rat ß-arrestin2 and/or GRK2 dominant-negative mutants. A total amount of 4 µg/100 mm dish of DNA was used for each condition. The empty vector pcDNA3.1 (Invitrogen, Carlsbad, CA) was used as control or to supplement DNA to reach 4 µg/dish. Transfections were performed using the Effectene reagent kit (QIAGEN, Valencia, CA). Twenty-four hours after transfection, cells were transferred to 6-, 24-, or 96-well plates and experiments were performed 48 h after transfection.
Intracellular Calcium Measurements
Twenty-four hours after transfection, cells were seeded into 96-well plates (Corning, Corning, NY) and were grown for another 24 h. Cells were then washed with serum-free DMEM and loaded with 10 µM of esterified Fura-2 (Calbiochem, La Jolla, CA) in serum-free DMEM for 30 min at 37 C. Fura-2 loaded cells were washed twice with Hanks’ medium [137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 0.34 mM NaH2PO4, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 20 mM HEPES, 2.02 mM glucose (pH 7.4)] and incubated in the same medium supplemented or not with 1.5 mM CaCl2 for 30 min at 37 C in the dark. Calcium measurements were performed at room temperature using a plate reader spectrofluorometer equipped with an injector (Fluostar Optima, BMG LabTechnologies, Durham, NC). Excitation occurred at wavelengths of 340 and 380 nm, and emission was measured at the wavelength of 520 nm. The ratio (F340/F380) between the emission in response to excitation at 340 nm (F340), and the emission in response to excitation at 380 nm (F380) was recorded and used as an indicator for the intracellular calcium concentration, as described (36). Data were plotted as means of 10 consecutive measurements taken at 0.4-sec intervals. For desensitization studies, cells were first exposed to 200 nM OT (Sigma, St. Louis, MO) for 100 sec, washed three times with Hanks’ medium, and were then allowed to recover for 5 min before a second agonist application.
Binding Studies
Ligand binding studies were performed on attached cells as described (37, 38), with minor modifications. Briefly, cells were first treated with 100 nM of OT for different times (0, 15, and 30 min), then washed once with cold buffer containing 5 mM Tris-HCl, 1 mM EDTA (pH 7.5) to remove the ligand (39), and were finally washed three times with PBS containing 0.2% BSA before performing the radioligand binding assay. Ligand binding was allowed to occur in 200 µl final volume of binding buffer [25 mM HEPES, 20 mM NaHCO3, 1% BSA (pH 7.4)] at 30 C for 1 h in the presence of 10 nM [3H]OT (NEN-Dupont, Boston, MA), and in the presence (nonspecific binding) or in the absence (total binding) of 10 µM unlabeled OT. After three washes with PBS/0.2% BSA, cells were scraped in 250 µl of 0.2 N NaOH and radioactivity was determined by scintillation counting. Results were analyzed using the Kell program (version 6, Biosoft, Cambridge, UK). Routinely, we found a maximal binding capacity ranging between 500–700 fmol/mg of total protein. No specific binding was observed in nontransfected cells or cells transfected with vector alone.
Cell Treatment and Solubilization
HEK-293 and COS-7 cells were treated, when indicated, with 100 nM OT (Sigma) for different times. Cells were then washed twice with PBS (pH 7.4). For the preparation of crude membrane and cytosol fractions, cells were scraped in 1 mM EDTA, 5 mM Tris HCl (pH 7.4), and pelleted by centrifugation at 500 x g for 5 min. Cells were resuspended in the same buffer supplemented with a mixture of protein inhibitors (1 µg/ml pepstatin A, 10 µg/ml leupeptine, 5 µg/ml aprotinine, and 0.1 mM phenylmethylsulfonyl fluoride) and sonicated three times for 15 sec. Homogenates were centrifuged at 140,000 x g for 1 h. Protein content of pellets (crude membranes) and supernatants (crude cytosol fraction) was measured using a bicinchoninic acid kit reagent (Pierce) and BSA as standard.
Immunoprecipitation and Immunoblotting
An anti-HA mouse monoclonal antibody (12CA5, Roche Diagnostics, Indianapolis, IN) was used to immunoprecipitate the HA-OTR from cell lysates. The lysates were incubated with the antibody at 4 C overnight, and Protein-G Sepharose (Amersham Biosciences, Baie d’Urfé, Quebec, Canada) was added 1 h before immunoprecipitation. Beads were washed three times with cold lysis buffer [25 mM HEPES (pH 7.4), 5 mM EDTA, 50 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, and a cocktail of protein inhibitors as indicated above], and Laemmli’s buffer (40) was added to each sample. Proteins were resolved by electrophoresis on polyacrylamide gels under denaturing conditions (SDS-PAGE) and transferred onto activated polyvinylidene fluoride membranes (Millipore, Billerica, MA) using a semi-dry transfer system (Bio-Rad, Hercules, CA). Membranes were washed twice with PBS-0.1% Tween 20 (PBS-T) and incubated in PBS-T/10% nonfat milk for 1 h. After two washes in PBS, membranes were incubated with a GRK2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h. Membranes were washed (2 x 5 min in PBS-T, 10 min in PBS-T/10% milk and 2 x 5 min in PBS) and incubated with a horseradish peroxidase-conjugated polyclonal secondary antibody (Santa Cruz) for 2 h. After washes as indicated above, the horseradish peroxidase-conjugated secondary antibody was detected using a chemiluminescence-based kit (Pierce).
For coimmunoprecipitation experiments, a permeable cross-linker agent was used as described by Zhang et al. (26). Briefly, after treatment with OT, 800 µl of 25 mM DSP in dimethylsulfoxide were added to the medium (final concentration 2.5 mM). Cells were incubated at room temperature in DSP for 30 min with constant rocking. They were then scraped and centrifuged at 500 x g for 5 min at 4 C. Cells were washed twice with cold PBS and solubilized for 30 min at 4 C with lysis buffer. The solubilized cells were cleared by centrifugation at 21,000 x g for 35 min at 4 C, and supernatants were recovered for immunoprecipitation or immunoblotting experiments.
Metabolic Labeling
Cells were incubated in phosphate-free DMEM containing 20 mM HEPES buffer (pH 7.4) with 75 µCi/ml of [32P] orthophosphoric acid (NEN-Dupont) for 4 h at 37 C. Cells were washed twice with phosphate-free DMEM. Treatments with 100 nM OT were performed at 37 C for different times in the same medium, and cells were washed twice with cold PBS before solubilization (as described above).
BRET Assay
To detect and analyze interactions between OTR and ß-arrestin or GRK2, BRET studies were performed. For this purpose, cells were transfected with an OTR construct tagged at its C terminus with RL (OTR-RL), in the absence or in the presence of cotransfection with plasmids encoding GRK2 or ß-arrestin, both tagged at their C terminus with YFP (GRK2-YFP, ß-arrestin-YFP). Cells were seeded into 96-well plates at a density of 105 cells/well. After the induction of RL-mediated light emission by the addition of the substrate coelenterazine h (Molecular Probes, Eugene, OR), emission was measured using an injector-equipped plate-reader spectrofluorometer (Fluostar Optima, BMG LabTechnologies) at the wavelengths of 475 and 535 nm, corresponding to the maxima of the emission spectra for RL and YFP, respectively. The BRET ratio was calculated using the equation described by Angers et al. (21): BRET ratio = [(emission at 510–590 nm) – (emission at 440–500 nm) x Cf]/(emission at 440–500), where Cf corresponded to (emission at 510–590 nm/(emission at 440–500 nm) for cells expressing OTR-RL alone. BRET ratios were plotted as means of 10 or 40 consecutive measurements taken at 0.4- to 0.5-sec intervals. Cells were treated, when indicated, with OT (100 nM) and/or the specific OT antagonist OTA (10 µM; Peninsula Laboratories Inc., San Carlos, CA) (21).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ß-ARK, ß-Adrenergic receptor kinase; BRET, bioluminescence resonance energy transfer; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; HEK, human embryonic kidney; OT, oxytocin; OTA, oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT; OTR, oxytocin receptor; RL, Renilla luciferase; YFP, yellow fluorescent protein.
Received for publication November 13, 2003. Accepted for publication February 10, 2004.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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q/11. Endocrinology 136:1509–1515[Abstract]
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), or coexpressing OTR and the GRK dominant-negative mutant GRK2K220M (
). B, COS-7 cells expressing OTR alone (
), OTR and ß-arrestin-2 (
), or OTR and both GRK2 and ß-arrestin-2 (
). Results are means ± SEM of three independent experiments, each performed in triplicate. Values significantly different from the corresponding values for cells expressing OTR alone are marked by an asterisks (P < 0.05).
) and ß-Arrestin (