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. 2009 Jun 25;23(9):1455–1465. doi: 10.1210/me.2009-0084

Inhibition of WNT Signaling by G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2)

Liming Wang 1, Diane Gesty-Palmer 1, Timothy A Fields 1, Robert F Spurney 1
PMCID: PMC2737558  PMID: 19556343

Abstract

Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator β-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of β-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/β-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

GRK2 inhibits Wnt/β-catenin canonical signaling by mechanisms requiring GRK2 enzymatic activity and a protein-protein interaction between GRK2 and the adenomatous polyposis coli (APC) protein.

Osteoporosis is a significant medical problem affecting more than 75 million people in Europe, the United States, and Japan (1) The disease is characterized by a reduction in bone mass and an increase in the susceptibility to fractures as a result of loss of skeletal architecture (1,2). For every 10% decrease in bone mass, the risk of fracture doubles (2). In the United States alone, it is estimated that 16.8% of postmenopausal women have lost more than 10% of their maximum bone mass and 4.8 million have suffered fractures (2). The majority of therapies for osteoporosis are largely aimed at preventing bone loss by inhibiting osteoclast-mediated bone resorption (1,2,3). Because regeneration of trabecular architecture by bone-forming osteoblasts (OBs) is required to fully restore the mechanical integrity of bone, there is much interest in developing anabolic agents to stimulate de novo bone formation without increasing osteoclast-mediated bone resorption. Understanding the molecular mechanisms that regulate OB-mediated bone formation might lead to novel therapies for treating disorders of bone loss.

The function of bone-forming OBs is modulated by the large superfamily of heptahelical receptors that are largely coupled to G protein activation (3). Belonging to this superfamily are frizzled (Frz) receptors that are activated by secreted glycoproteins termed Wnts (4,5,6). After Frz receptor activation, multiple intracellular signaling pathways are activated including a pathway leading to stabilization of the transcription regulator β-catenin (canonical signaling pathway) (1,6,7,8). Stabilizing β-catenin increases the cytoplasmic free pool of β-catenin (6,9), and a portion of stabilized β-catenin then translocates to the nucleus where it binds to responsive elements in a member of the lymphoid enhancer factor-1/T-cell factor (Lef-1/Tcf) family and promotes target gene transcription (1,6). The importance of this signaling pathway (canonical signaling cascade) in OB physiology became apparent when genetic diseases that alter bone mass were found to result from mutations that either increase or decrease the activity of the canonical signaling pathway in OBs (10,11). As result, there is increasing interest in determining the factors that regulate Wnt signaling.

Discovering ways to selectively manipulate Frz receptor signaling to promote OB-mediated bone formation represents an important area of research. In classical G protein-coupled receptor (GPCR) systems, receptor activity is regulated by a family of seven kinases termed GPCR kinases (GRKs) (12,13,14,15), which directly phosphorylate GPCRs and attenuate receptor signaling after agonist stimulation (12,13,14,15). Receptor phosphorylation is followed by binding of a second group of protein cofactors termed “arrestins”, which sterically inhibit receptor-G protein coupling (12,13,15,16). As mentioned above, Frz receptors belong to the GPCR superfamily (4,5,6), and accumulating evidence suggests that Frz receptor activation may be modulated by some of the same enzymes and protein cofactors that regulate classical GPCR systems (4,17,18,19,20,21,22).

In the absence of Wnt signaling, β-catenin is phosphorylated by casein kinase I and glycogen synthase kinase 3β (GSK3β) (6,9). Phosphorylated β-catenin is recognized by βTrCP (β-transducin repeat-containing protein 2) (6,7), which then targets β-catenin for degradation. Phosphorylation of β-catenin occurs in a large multiprotein complex that includes casein kinase I, GSK3β, the scaffolding protein axin, and the tumor suppressor gene product adenomatous polyposis coli (APC) (6,9). Formation of the complex is facilitated by a protein-protein interaction between the regulator of G protein signaling (RGS) domain in axin and the midportion of APC known as the serine-alanine-methionine-proline (SAMP) repeat region (23,24). Because previous studies have identified an RGS domain in GRK2 that is closely related to the RGS domain in axin (25), we hypothesized that the GRK2 RGS domain might promote an association of GRK2 with the axin-APC complex and, in turn, modulate canonical signaling. To test this hypothesis, we assessed the effects of either overexpression of GRK2 or depletion of GRK2 on Wnt signaling. We found that GRK2 negatively regulates canonical signaling though mechanisms that require enzymatic activity and a protein-protein interaction between the SAMP repeat region in APC and the RGS domain in GRK2. We speculate that inhibiting GRK2 activity in bone-forming OBs and, in turn, enhancing canonical signaling might be a useful therapeutic strategy for increasing bone mass.

Results

GRK2 inhibits canonical signaling

To determine whether GRK2 modulated Wnt/β-catenin signaling, we first coexpressed GRK2 with Wnt1 in human embryonic kidney (HEK)293 cells and measured activity of the Wnt-responsive luciferase reporter TOPFlash. Cells were harvested 2 d after transfection, and reporter activity was monitored by measuring luciferase activity (see Materials and Methods). As shown in Fig. 1A, transfection of Wnt1 enhanced canonical signaling, and this stimulatory effect was inhibited by cotransfection of GRK2. The inset shows that GRK2 expression was enhanced in cells transfected with GRK2 compared with cells transfected with the pRK5 control vector.

Figure 1.

Figure 1

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GRK2 inhibits canonical signaling. A, Transfection of GRK2 inhibited Wnt1-dependent activation of the reporter construct in HEK293 cells. B, Transfection with GRK2 siRNA enhanced activation of the reporter construct in HEK293 cells. C, Calvarial OBs were treated overnight with TAT proteins or DMEM vehicle in the presence or absence of Wnt3a conditioned medium. Treatment with GRK2-TAT (+) inhibited Wnt3a-dependent activation of the reporter construct. D, Treatment with Wnt3a enhanced cytosolic levels of β-catenin in cells treated with GRK2-TAT (−), and this effect was inhibited by treatment with GRK2-TAT (+) in calvarial OBs. In panels A–C, results are from three to five separate experiments. In panel D, similar results were observed in three separate experiments. *, P < 0.01 vs. basal; †, P < 0.025 vs. either Wnt1 control or Wnt3a control. B-cat, β-Catenin; CTL, control.

We next determined whether knockdown of endogenous GRK2 using RNA interference modulated Wnt/β-catenin signaling. For the studies, cells were transfected with the TOPflash reporter construct and either Wnt1 or control vectors as well as GRK2 small interfering RNA (siRNA) or control siRNA as described in Materials and Methods. Cells were then harvested for measurement of luciferase activity (see Materials and Methods). As shown in the inset of Fig. 1B, GRK2 siRNA reduced expression of GRK2 by approximately 90% compared with cells transfected with control siRNA. The reduction in GRK2 protein levels caused a significant increase in Wnt1-induced activation of the reporter construct. Taken together with the GRK2 overexpression studies (Fig. 1A), these data suggest that GRK2 negatively modulates the Wnt/β-catenin signaling pathways.

To determine whether GRK2 inhibited canonical signaling in a cell model relevant to bone biology, we used protein transduction to introduce GRK2 into calvarial OBs by tagging the GRK2 protein with the TAT HIV protein sequence. For the studies, calvarial OBs were transfected with the TOPflash reporter construct and/or control vectors, after which the cells were treated overnight with Wnt3a-conditioned medium or control medium as well as either the GRK2-TAT protein [GRK2-TAT (+)] or a GRK2 protein lacking the TAT sequence [GRK-TAT (−)]. As shown in Fig. 1C, GRK2-TAT (+) potently inhibited Wnt-induced activation of the reporter construct. In contrast, GRK-TAT (−), which is unable to enter cells, did not significantly affect Wnt3a-dependent activation of canonical signaling. As shown in the inset of Fig. 1C, immunoblot analysis revealed that GRK2-TAT (+) was effectively transduced into calvarial OBs. In contrast, hemagglutinin (HA)-tagged GRK2 was not detected in cells treated with GRK2-TAT (−). To complement the reporter studies, we examined the effect the TAT proteins on Wnt3a-induced stabilization of β-catenin. As shown in Fig. 1D, Wnt3a enhanced cytosolic levels of β-catenin, and this effect was inhibited by GRK2-TAT (+). These data suggest that GRK2 inhibits Wnt/β-catenin signaling in a cell system relevant to bone biology.

GRK2 enzymatic activity is required to inhibit canonical signaling

The ability of GRK2 to regulate GPCR signaling depends, in part, on its kinase activity (12,13,14,15). To determine whether GRK2 kinase activity was required to inhibit Wnt/β-catenin signaling, we first determined whether a GRK2 construct lacking kinase activity (GRK2-K220R) altered activation of the TOPflash reporter construct. For the studies, HEK293 cells were transfected with TOPflash reporter construct and either Wnt1, GRK2, GRK2-K220R, or control vectors. Cells were harvested 2 d after transfection for measurement of luciferase activity. As shown in Fig. 2A, GRK2, but not GRK2-K220R, inhibited canonical signaling induced by Wnt 1. We next examined the effects of GRK2 and GRK2-K220R on Wnt3a-induced stabilization and nuclear translocation of β-catenin. For these studies, HEK293 cells were transfected with either GRK2, GRK2-K220R, or control vectors. The next day, cells were treated overnight with Wnt3a-conditioned medium or control medium, after which cells were harvested and stabilization of β-catenin was assessed as described in Materials and Methods. As shown in Fig. 2B, Wnt3a significantly enhanced cytosolic levels of β-catenin. Consistent with its inhibitory effects in the reporter studies, GRK2 also inhibited Wnt3a-dependent β-catenin stabilization (Fig. 2B, lane 4). Moreover, in agreement with its lack of effect on Wnt-dependent activation of the TOPflash reporter construct (Fig. 2A), the kinase-dead GRK2-K220R had no significant effect on Wnt3a-dependent β-catenin stabilization. Figure 2, C and D, shows nuclear translocation of β-catenin by immunoblotting of nuclear fractions (Fig. 2C) as well as fluorescence microscopy (Fig. 2D). Treatment with Wnt3a enhanced nuclear accumulation of β-catenin. As was seen with both Wnt-induced activation of the reporter construct and β-catenin stabilization, Wnt3a-dependent β-catenin nuclear accumulation was inhibited by expression of GRK2 but not the kinase-dead GRK2-K220R. Using several complementary strategies, these data suggest that GRK2 inhibits canonical signaling and that GRK2 enzymatic activity is required for the inhibitory effect.

Figure 2.

Figure 2

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GRK2 enzymatic activity is required to inhibit canonical signaling. A, GRK2, but not GRK2–K220R, inhibited Wnt1-dependent activation of the reporter construct in HEK293 cells. B, Wnt3a increased stabilization of β-catenin, and this Wnt3a-dependent effect was inhibited by GRK2 but not GRK2–K220R in HEK293 cells. Actin was used as loading control for immunoblotting of cytosolic fractions. C and D, Wnt3a enhanced nuclear translocation of β-catenin, and this Wnt3a-dependent effect was inhibited by GRK2 but not GRK2–K200R in HEK293 cells. Histone 3 was used as a loading control for immunoblotting of nuclear fractions. In panel D, 80–100% of the cells transfected with either vector or GRK2–K220R exhibited nuclear translocation of β-catenin. Consistent with transfection efficiency, nuclear translocation of β-catenin was inhibited in 50–70% of cells transfected with GRK2. In panel A, results are from five separate experiments. In panels B–D, similar results were seen in three separate experiments. *, P < 0.01vs. basal; †, P < 0.25 vs. either Wnt1 control or Wnt3a control. B-cat, β-Catenin.

GRK2 interacts with APC though its N-terminal RGS domain

Formation of the axin-APC destruction complex is facilitated by a protein-protein interaction between the RGS domain in axin and the SAMP repeat region of APC (23,24). Because the RGS domain in GRK2 is closely related to the RGS domain in APC (25), we hypothesized that an interaction between GRK2 and APC may be critical for the GRK2-inhibitory effect on canonical signaling. To test whether GRK2 can interact with APC in cells, we expressed GRK2 in HEK293 cells and immunoprecipitated endogenous APC. As shown in Fig. 3A, GRK2 coimmunoprecipitated with APC. To determine the region of APC that mediated its protein-protein interaction with GRK2, we transfected cells with a FLAG-tagged APC construct that encoded amino acids 1342-1887 of the SAMP repeat region as well as either GRK2 or control vectors. As shown in Fig. 3B, GRK2 coimmunoprecipitated with this axin-binding region of APC (Fig. 3B, lane 3). In addition, the interaction between GRK2 and the APC fragment was enhanced by pretreatment with Wnt3a-conditioned media (Fig. 3B, lane 4). These data suggest that GRK2 interacts with the APC and that this interaction is enhanced by activation of canonical signaling.

Figure 3.

Figure 3

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GRK2 interacts with APC through an N-terminal RGS domain. A, GRK2 immunoprecipitates with endogenous APC in HEK293 cells. B, GRK2 immunoprecipitated with the FLAG-tagged APC protein in HEK293 cells. Treatment with Wnt3a increased the amount of GRK2 in the immunoprecipitates. C, Approximate locations of the N- and C-terminal truncations in the GRK2 mutants GRK2ΔCT and GRK2ΔRGS. D, Both GRK2 and GRK2ΔCT immunoprecipitated with the FLAG-tagged APC protein. In contrast, GRK2ΔRGS was not detected in the immunoprecipitates. In panels A, B, and D, similar results were seen in three separate experiments. IP, Immunoprecipitation.

We next determined whether the RGS domain of GRK2 was required for the GRK2-APC interaction. For these studies, HEK293 cells were transfected with the FLAG-tagged APC construct and either wild-type GRK2 or the GRK2 mutants shown schematically in Fig. 3C. GRK2ΔRGS lacked amino acids 1-105 in its N terminus (EIFD-106) and, therefore, lacked portions of the GRK2 RGS domain that correspond to the APC-binding domain in axin (25). GRK2ΔCT was truncated at amino acid 562 (CIMH-562) to remove domains of GRK2 critical for interacting with the βγ-subunits of G proteins (26). The FLAG-tagged APC protein was immunoprecipitated 2 d after the transfection, and the immunoprecipitates were probed for wild-type GRK2, GRK2ΔRGS, or GRK2ΔCT by immunoblotting for GRK2. Neither GRK2 mutant coimmunoprecipitated with the FLAG-tagged APC construct in the absence of Wnt3a (data not shown). As shown in Fig. 3D, however, after treatment overnight with Wnt3a, both wild-type GRK2 and C-terminally truncated mutant (GRK2ΔCT) immunoprecipitated with the FLAG-tagged APC protein. In contrast, the GRK2 mutant lacking amino acids 1-125 in the N terminus was not detected in the immunprecipitates by immunoblotting. These data are consistent with the notion that the RGS domain of GRK2 is required to interact with the axin-APC destruction complex.

The GRK2-APC interaction is required to inhibit canonical signaling

Targeting of GRK2 to its substrate is critical for the enzymatic function of GRK2 (26). In this regard, GRK2 is targeted to membrane-bound GPCR substrate by a protein-protein interaction between βγ-subunits of G proteins and βγ-binding domains in the C terminus of GRK2 (26). This protein-protein interaction is critical for GRK2-dependent inhibition of GPCR signaling. We determined, therefore, whether or not the RGS domain in GRK2 was required for GRK2-dependent inhibition of canonical signaling. For these studies, HEK293 cells were transfected with TOPflash and either Wnt1, wild-type GRK2, GRK2ΔRGS, GRK2ΔCT, or control vectors. Cells were then harvested and reporter activity was monitored by measuring luciferase activity as described in Materials and Methods. Figure 4A shows Wnt1-dependent activation of the reporter construct in cells transfected with control vectors, wild-type GRK2, GRK2ΔRGS, or GRK2ΔCT. As shown in Fig. 4A, expression of either GRK2 or GRK2ΔCT inhibited Wnt-stimulated activation of the reporter. In contrast, transfection of the GRK2ΔRGS construct had little effect on canonical signaling. Figure 4B shows that GRK2 and the GRK2 mutants were effectively expressed in HEK293 cells. Basal activation of the reporter construct was not affected by transfection of either GRK2, GRK2ΔRGS, or GRK2ΔCT (data not shown).

Figure 4.

Figure 4

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An intact GRK2 RGS domain is required to inhibit canonical signaling. A, Both GRK2 and GRK2ΔCT inhibited Wnt1-dependent activation of the reporter construct. Basal reporter activation was similar in the different treatment groups (not shown). B, The lysates were probed for expression of GRK2, GRK2ΔCT, and GRK2ΔRGS. C, The enzymatic activity of GRK2ΔRGS was examined in HEK293 cells by determining the ability of GRK2ΔRGS to phosphorylate the PTH receptor. Treatment with PTH enhanced PTH receptor phosphorylation, and this PTH-dependent effect was similarly enhanced by both GRK2 and GRK2NT. In panel A, results are from four separate experiments. In panels B and C, similar results were obtained in three separate experiments. CT, C-terminal; IP, immunoprecipitation; NT-N-terminal.

Previous studies have suggested that GRK2ΔCT has enzymatic activity when targeted to its substrate (26). It is possible, however, that the GRK2ΔRGS was enzymatically inactive and, as a result, was unable to inhibit canonical signaling due to a loss of enzymatic activity. We determined, therefore, whether GRK2ΔRGS could phosphorylate GPCR substrate in HEK293 cells. For these studies, cells were transfected with either GRK2, GRK2ΔRGS, or control vector as well as a HA-tagged PTH receptor, which we have previously shown is effectively phosphorylated by GRK2 (27). Cells were loaded with 32P 2 d after transfection and stimulated with PTH. HA-tagged PTH receptors were then immunoprecipitated as described in Materials and Methods. As shown in Fig. 4C, both wild-type GRK2 and GRK2ΔRGS enhanced agonist-dependent phosphorylation of the PTH receptor. These data suggest that the GRK2ΔRGS is enzymatically active. Taken together with the immunoprecipitation data, these findings suggest that targeting of GRK2 to the axin-APC destruction complex through its RGS domain is required for GRK2-dependent inhibition of canonical signaling.

The GRK2-APC interaction requires GRK2 enzymatic activity

Targeting of GRK2 to the destruction complex may require GRK2 enzymatic activity to either promote the association or stabilize the interaction. To test this possibility, we determined whether GRK2-K220R immunoprecipitated with APC. For these studies, HEK293 cells were transfected with the FLAG-tagged APC construct and either wild-type GRK2 or the GRK-K220R. After treatment overnight with Wnt3a-conditioned medium or control medium, the FLAG-tagged APC protein was immunoprecipitated, and the immunoprecipitates were probed for wild-type GRK2 or GRK2-K220R. As shown in Fig. 5, GRK2, but not GRK2-K220R, was detected in the immunoprecipitates. These data suggest that GRK2 enzymatic activity promotes the GRK2-APC interaction.

Figure 5.

Figure 5

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Effect of GRK2 enzymatic activity on the GRK2-APC interaction. GRK2 immunoprecipitated with the FLAG-tagged APC construct. In contrast, GRK2–K220R was not detected in the immunoprecipitates. Similar results were obtained in three experiments. IP, Immunoprecipitation.

GRK2 does not enhance β-catenin phosphorylation

Given the role of phosphorylation in β-catenin degradation, it is possible that recruitment of GRK2 to the destruction complex enhances β-catenin phosphorylation. To test this possibility, we transfected HEK293 cells with either GRK2 or empty vector. Cells were then loaded with 32P overnight in the presence or absence of Wnt3a, and β-catenin was immunoprecipitated as described in Materials and Methods. As shown in Fig. 6, transfection of GRK2 did not affect the extent of β-catenin phosphorylation.

Figure 6.

Figure 6

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Effect of GRK2 on phosphorylation of β-catenin. The autoradiograph in the upper panel shows that transfection of GRK2 did not increase β-catenin phosphorylation in 32P-loaded HEK293 cells. In the lower panel, the lysates were probed for expression of GRK2 by immunoblotting. Similar results were obtained in three experiments. B-cat, β-Catenin; IP, immunoprecipitation.

Discussion

The Wnt/β-catenin signaling pathway regulates diverse biological processes including embryonic patterning, cell fate decisions, cellular proliferation, and apoptosis (28). In mammals, Wnt/β-catenin signaling plays a key role in organogenesis, cancer, and stem cell renewal (28,29,30). With regard to bone, Wnt proteins regulate multiple events during skeletal development including limb patterning and joint formation (5,28). A large body of evidence also implicates the Wnt signaling cascade in critical aspects of skeletal biology postnatally. For example, inactivating mutations in the Wnt/β-catenin signaling cascade cause osteoporosis-pseudoglioma syndrome (11,31). Conversely, mutations that activate canonical signaling cause high bone mass (10,31,32). As a result, there is much interest in understanding the factors that regulate activity of the canonical signaling pathway. The present studies suggest that GRK2 negatively modulates Wnt/β-catenin signaling. In this regard, knockdown of GRK2 using siRNA increased Wnt1-dependent activation of canonical signaling. Because enhanced canonical signaling has anabolic effects in bone (10,31,32), selectively inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

The ability of GRK2 to both inhibit canonical signaling and associate with the destruction complex requires enzymatic activity as well as its N-terminal RGS domain. Our working hypothesis is that the RGS domain in GRK2 interacts with the SAMP repeat region of APC in a fashion analogous to the interaction between APC and the RGS domain in axin. The resulting interaction targets GRK2 enzymatic activity to the β-catenin destruction complex, resulting in phosphorylation of a component of the destruction complex and stabilization of the APC-GRK2 interaction. The following observations support this hypothesis: 1) The RGS domain in GRK2 is closely related to the RGS domain of axin (25); 2) GRK2 coimmunoprecipitates with endogenous APC; 3) Coimmunoprecipitation of GRK2 and APC requires the N-terminal RGS domain of GRK2 as well as GRK2 enzymatic activity; and 4) GRK2 coimmunoprecipitates with a portion of APC encoding amino acids 1342-1887 of the SAMP repeat region (23,24). Although our working hypothesis implies a direct interaction of GRK2 with APC, it is possible that the protein-protein interaction is mediated indirectly by binding of GRK2 to other proteins in the multiprotein destruction complex. Although additional experiments will be necessary to investigate this possibility, these data suggest that an intact RGS domain is required for GRK2-dependent inhibition of Wnt/β-catenin signaling.

Consistent with the notion that GRK2 plays a key role in regulating bone mass, we previously found that overexpression of GRK2 in bone-forming OBs decreased bone mass (33). Moreover, expression of a GRK2 inhibitor specifically in OBs had anabolic effect on bone (34). These latter studies, however, used a construct that lacks GRK2 enzymatic activity and acts as a βγ-sequestrant (26). Based on the present studies, it is unlikely that this construct would enhance activity of the canonical signaling pathway. Indeed, expression of the βγ-sequestrant has been reported to decrease canonical signaling by inhibiting βγ-dependent activation of Akt/protein kinase B and, therefore, preventing Akt-dependent inhibition of GSK3 β activity (Ref. 35; and Wang, L., and R. F. Spurney, unpublished observation). As mentioned above, we found that expression of the βγ-sequestrant in OBs inhibited GRK activity and enhanced bone mass (34); this anabolic effect, however, was seen only in younger animals and was absent in adult mice (36). In contrast, overexpression of GRK2 in OBs decreased bone mass, and this effect persisted throughout adulthood (33). One possible explanation for the differing results is that GPCR-dependent signaling cascades play a predominant role in regulating bone mass in young mice, but both GPCR and Wnt signaling, which would be stimulated and inhibited by the βγ-sequestrant, respectively, may contribute to maintenance of bone mass in adult animals. In support of this hypothesis, Bodine et al. (37) reported that deletion of the Wnt antagonist sFRP-1, and presumably enhanced canonical signaling, increased bone mass only in adult mice.

As mentioned above, GRK2 enzymatic activity is required both for association with the β-catenin destruction complex and for inhibition of canonical signaling. These data suggest that GRK2 may phosphorylate a component of the multiprotein destruction complex. In this regard, GRKs are capable of phosphorylating nonreceptor substrates. For example, we found that GRK6 phosphorylates the sodium hydrogen ion exchanger-regulatory factor 1 via a PDZ (postsynaptic density protein 95, Drosophila disc large tumor suppressor, zona occludens-1 protein) domain-mediated interaction (38). Since then, a number of additional nonreceptor substrates have been described including: the cytoskeletal proteins tubulin (39) and α-actinin (40), the ribosomal protein P2 (41), and the central nervous system proteins synucleins (42). Given the role of phosphorylation in β-catenin degradation, one possibility is that recruitment of GRK2 to the destruction complex phosphorylates β-catenin. Transfection of GRK2 in HEK293 cells, however, did not increase the phosphorylation state of β-catenin (Fig. 6).

It is also possible that GRK2 may phosphorylate components of the canonical signaling pathway that do not participate in formation of the destruction complex. In this regard, GRK2 plays an important role in regulating signaling by seven-transmembrane-spanning receptors (12,13,14,15). Belonging to this superfamily are frizzled (Frz) receptors (4,5,6), which raises the possibility that GRK2 might phosphorylate Frz receptors and inhibit canonical signaling. Although additional studies will be necessary to investigate this possibility, we were unable to demonstrate an effect of GRK2 on phosphorylation of Frz 5 (supplemental Fig. 1S published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). In contrast, Yanfeng et al. (43) found that Frz 3 is phosphorylated in a Dsh-dependent fashion. Although Frz 3 activates noncanonical signaling pathways (44) and the kinase mediating Frz 3 phosphorylation has not been identified (43), this finding raises the possibility that phosphorylation may play a role in regulating the activity of other Frz receptors including receptors coupled to canonical signaling pathways, perhaps in a GRK-dependent fashion.

Treatment with Wnt3a appeared to promote the association of GRK2 with the β-catenin destruction complex. Moreover, we were unable to coimmunoprecipitate either GRK2ΔRGS or GRK2ΔCT with the FLAG-tagged APC construct in the absence of Wnt3a. These data suggest that GRK2 may be recruited to the destruction complex after activation of the canonical signaling pathway. A similar recruitment of GRK2 to membrane-bound GPCRs has been reported through a protein-protein interaction between the βγ-binding domains in the C terminus of GRK2 and a protein complex composed of the βγ-subunits of G proteins and agonist-activated GPCRs (26). Our inability to coimmunoprecipitate either GRK2ΔRGS or GRK2ΔCT with the FLAG-tagged APC construct in the absence of Wnt3a suggests that the GRK2 mutations may have affected the affinity of the protein-protein interaction, which was enhanced in the presence of Wnt3a. Taken together, these data suggest a model similar to GRK2-dependent inhibition of GPCR signaling (12,13,14,15). In the presence of Wnts, GSK3β is inhibited and β-catenin is released from the destruction complex, translocates to the nucleus, and promotes gene transcription. This Wnt-dependent activation of the canonical signaling pathways, however, is dampened in a fashion analogous to recruitment of GRK2 to membrane-bound GPCR substrate and inhibition of GPCR signaling (12,13,14,15). In this scenario, after activation of canonical signaling, GRK2 is recruited to the β-catenin destruction complex and, in turn, attenuates canonical signaling.

Lastly, the GRK2 mutant lacking amino acids 1-105 in the N terminus (GRK2ΔRGS) was capable of phosphorylating the PTH receptor in transfected cells despite the absence of acidic amino acids in the N terminus that are required to phosphorylate GPCR substrate (45). GRK2ΔRGS, however, was modified by the addition of an N-terminal HA epitope that contains two acidic amino acid residues (46). It is possible that these acidic N-terminal amino acids restored the ability of GRK2 to phosphorylate GPCR substrate despite truncation of the GRK2 N terminus in GRK2ΔRGS.

In summary, we found that GRK2 inhibits canonical signaling through mechanisms requiring GRK2 enzymatic activity and involving recruitment of GRK2 to the β-catenin destruction through a protein-protein interaction between the RGS domain in the N terminus of GRK2 and the axin-APC destruction complex. We speculate that GRK2 may phosphorylate a component of the axin-APC protein complex and modulate canonical Wnt signaling. Moreover, treatments that inhibit GRK2 in bone-forming OBs may be a useful therapeutic strategy for increasing bone mass.

Materials and Methods

DNA constructs

The GRK2 construct and the HA (hemagglutinin)-tagged PTH receptor construct have been described elsewhere (27). The dominant-negative GRK2 (K220R) and the C-terminally truncated GRK2 (GRK2ΔCT) were gifts of Dr. Neil Freedman (47) and Dr. Robert Lefkowitz (26), respectively, at our institution. The Wnt1 construct (10) was a gift from Dr. Dianqing Wu (University of Connecticut Health Center). The TOPflash Tcf reporter construct and its control vector FOPflash were obtained from Upstate Biotechnology (Lake Placid, NY). A construct encoding a fusion between green fluorescent protein and β-catenin (48) was kindly provided by Dr. Randall Moon (University of Washington, Seattle, WA). The pRL-TK Renilla luciferase control vector was obtained from Promega Corp. (Madison, WI). The pTAT-HA vector (46) was provided Dr. Becter-Hapak (Washington University School of Medicine, St. Louis, MO). To create the N-terminally truncated GRK2 construct (GRK2ΔRGS), GRK2 (26) was cut with BglII, and the recessed ends were filled in with Pfu DNA polymerase. The resulting fragment was then cut with XbaI, and the GRK2 coding sequence was isolated by gel purification. The GRK2 fragment was then ligated into pBK-CMV (Stratagene) which had been prepared using the following protocol. A portion of the vector pTAT-HA (46) containing an HA epitope and the polylinker sequence was cut with the restriction enzymes BamHI and EcoRI and ligated into the polylinker of pBK-CMV after cutting with the same restriction enzymes. The resulting vector was cut with XhoI, and the recessed ends were filled in by treating with Pfu DNA polymerase (Agilent Technologies, Santa Clara, CA). The resulting fragment was then cut with XbaI and gel purified. The GRK2 and pBK-CMV fragments were then ligated together. The PCR was used to generate the FLAG APC construct. A fragment of human APC that contains two of the axin-binding SAMP repeat domains (amino acids 1342-1887) was amplified by PCR, and the BglII-SalI fragment was cloned into the pFLAG-6a (Sigma, St. Louis, MO) for expression in mammalian cells. All constructs were sequenced at the DNA Analysis Facility at Duke University Medical Center (Durham, NC).

Wnt3a conditioned medium

Wnt3a conditioned medium was prepared from mouse L-cells stably transfected with mouse Wnt3a (American Type Culture Collection, Manassas, VA) as described (49). Briefly, cells were split 1:10 to 1:20 and grown in DMEM for 4 d supplemented with 10% fetal calf serum. After harvesting, the medium was filtered through a 0.2-μm filter and either stored at 4 C for 2–3 months or frozen at −70 C for long-term storage.

Lymphoid enhancer factor-1/T-cell factor (Lef-1/Tcf) reporter assays

HEK293 cells were plated in 12-well tissue culture clusters (Evergreen Scientific, Los Angeles, CA) and transfected with the indicated constructs as well as either the reporter construct TOPflash (0.5 μg per well; Upstate Biotechnology) or its control FOPflash (0.5 μg per well; Upstate) using Lipofectamine (Invitrogen, Carlsbad, CA) according to the directions of the manufacturer. To control for transfection efficiency, cells were also transfected with a vector encoding Renilla luciferase, pRL-TK (0.25 μg per well; Promega). Appropriate empty vector constructs were added to equalize the amount of DNA in each transfection (2 μg per well). The cells were harvested 2 d after transfection using the Promega Dual Luciferase Reporter Assay System, and both firefly and Renilla luciferase intensity were measured with a luminometer (MGM Instruments, Hamden, CT) according to the directions of the manufacturer (Promega). To correct for transfection efficiency, the firefly luciferase values were divided by the Renilla luciferase values after subtracting the background light intensity.

For the siRNA experiments (see below), HEK293 cells were initially plated in 100-mm dishes and transfected with the indicated constructs as well as either TOPflash or the control vectors as described above (24 μg per dish). The following day, the cells were plated in 24-well tissue culture clusters (Evergreen Scientific) at a confluency of approximately 50% and then, after 24 h, transfected with siRNA as described below.

Nuclear translocation of β-catenin

For these studies, HEK293 cells were treated overnight with Wnt3a conditioned medium or control medium after which nuclei were isolated using the method of Andrews and Faller (50). Briefly cells were washed with ice-cold PBS and then scraped into PBS, pelleted, and resuspended in 400 μl 10 mm HEPES-potassium hydroxide (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonylfluoride. After incubation on ice for 10 min, the nuclei are pelleted by centrifugation for 10 sec. The nuclear pellet is resuspended in 50 μl of 20 mm HEPES-potassium hydroxide (pH 7.9), 25% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonylfluoride. After incubation on ice for 20 min, the sample is centrifuged to clear cellular debris and the supernatant saved at −70 C for immunoblot analysis.

To complement the immunoblotting studies, additional microscopic studies were performed after cells had been transfected with a β-catenin-green fluorescent protein construct (48) and either GRK2 or empty vector. After transfection, cells were treated overnight with Wnt3a or control medium, and then cellular localization β-catenin was performed using a Nikon Eclipse TB-2000 microscope (Nikon, Inc., Melville, NY). Pictures were acquired at room temperature using a magnification of ×400 with a Roper Scientific Photometric digital camera (Roper Scientific, Bogart, GA). The images were then converted to electronic files using Adobe Photoshop-CS software (Adobe Systems, Inc., San Jose, CA).

Isolation of cytoplasmic and particulate proteins

For these studies, HEK293 cells were treated overnight with Wnt3a conditioned medium or control medium after which the cells were scraped into ice-cold PBS and then sonicated. Samples were centrifuged at 16,000 × g at 4 C for 15 min. The supernatant (cytosolic fractions) was removed and mixed 1:1 (vol/vol) with 2× Laemmli sample buffer (51). The pellet (particulate fraction) was solubulized in 1× Laemmli sample buffer by sonication. Both particulate and cytosolic fractions were then frozen at −70 C until immunoblotting was performed using an antibody to β-catenin (Transduction Laboratories, Lexington, KY) as described below.

Source of antibodies

The source of the antibodies was as follows: 1) The anti-β-catenin antibody was from BD Biosciences (Franklin Lakes, NJ); 2) The anti-FLAG M2 monoclonal antibody was from Sigma; 3) The anti-GRK2 mouse monoclonal antibody and the anti-HA rabbit monoclonal antibody were from Upstate Biotechnology; 4) The anti-HA mouse monoclonal antibody was from Roche Diagnostics (Indianapolis, IN); 5) The antiactin mouse monoclonal antibody was from Chemicon International (Temecula, CA); and 6) The anti-APC mouse monoclonal antibody (c-APC 28.9) and the mouse monoclonal antihistone 3 antibody (mAbcam 10799) were from Abcam (Cambridge, MA). The anti-GRK2 antibody recognizes a minimal epitope encompassing amino acids 483–485 of GRK2 and is, therefore, suitable for recognizing the GRK2 mutants used in this manuscript.

GRK2 knockdown using siRNA

Knockdown of GRK2 in HEK 293 cells was performed as described (52). Briefly, GRK2 siRNA and a nonsilencing control (52) were synthesized by Xeragon (Germantown, MD). The GRK2 siRNA sequence has been previously shown to specifically reduce GRK2 expression without affecting expression of other GRK proteins (52). After transfecting HEK293 cells with Lef/Tcf reporter constructs described above, cells were split into 24-well tissue culture clusters (Evergreen Scientific) at a confluency of approximately 50% and, the following day, were transfected with siRNA (500 ng per well) using the GeneSilencer transfection reagent (Gelantis, San Diego, CA) according to the directions of the manufacturer.

TAT proteins

Protein transduction was used to introduce GRK2 into calvarial OBs by tagging GRK2 with the HIV TAT sequence (46). To create the TAT-tagged GRK2, GRK2 was ligated into pTAT-HA (46) after the following purification procedure. A portion of the vector pCR2.1 (Invitrogen) containing the polylinker sequence was cut with the restriction enzymes KpnI and XhoI and ligated into the polylinker of the vector pTAT-HA (46) after cutting with the same restriction enzymes. The resulting vector was cut with BstBI, and the recessed ends were partially filled in by treating with Pfu DNA polymerase (Stratagene) in the presence of 2.5 mm deoxy-CTP. The resulting fragment was then cut with EcoRI and gel purified. To insert GRK2 in sequence with the TAT coding sequence in pTAT-HA, the GRK2 construct (27) was cut with BamHI and the recessed ends were partially filled in with Pfu DNA polymerase (Agilent Technologies) in the presence of 2.5 mm each deoxy-GTP, deoxy-ATP, and deoxy-TTP. The resulting fragment was then cut with EcoRI, and the fragment containing the GRK2 sequence was isolated by gel purification and then ligated into the pTAT-HA fragment prepared as described above. A GRK2 construct lacking the TAT sequence was prepared by treating the GRK2-TAT construct with BamHI and the religating the resulting fragment. All constructs were sequenced at the DNA Analysis Facility at Duke University Medical Center.

The GRK2 DNA constructs were transfected into BL21(DE3)pLysS bacteria (Novagen, Madison, WI) after which the TAT proteins were purified sequentially on Ni-NTA columns (QIAGEN, Valencia, CA) followed by PD-10 desalting columns (Amersham Pharmacia Biotech, Piscataway, NJ) as described elsewhere (46). TAT proteins were then dialyzed overnight in DMEM in preparation for use in cultured cells.

Immunoblotting

Proteins were generally separated on 4–12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride (PVDF) membranes according to the manufacturer’s recommendations (Invitrogen). For APC, however, proteins were separated on 3–8% Tris-acetate gels and transferred overnight to PVDF membranes according to the manufacturer’s recommendations (Invitrogen). PVDF membranes were blocked overnight in Tris-saline buffer (20 mm Tris-HCl; 137 mm NaCl, pH 7.6) [Tris-buffered saline (TBS)] with 0.2% Tween 20 (T-TBS) and 5% nonfat dry milk. After blocking, the primary antibody was added at a dilution of 1:2000. The blot was incubated at room temperature for 1 h with gentle rocking followed by three washes with T-TBS. The horseradish peroxidase-labeled secondary antibody was added at a dilution of 1:2000. After rocking for 1 h at room temperature, the blot was washed once with T-TBS and twice with TBS. Proteins were detected by enhanced chemiluminescence according to the manufacturer’s specifications (Amersham, Buckinghamshire, UK). The immunoblots were converted into a digital format using an Epson Perfection scanner 1670 (Seiko Epsom Corp., Nagano, Japan) and then analyzed using ScanAnalysis 2.5 software (Biosoft, Ferguson, MO).

Immunoprecipitation experiments

Immunoprecipitation of PTH receptors was performed as described elsewhere (27). Briefly, HEK293 cells were transfected with a HA-tagged PTH receptor (27) as well as either GRK2, GRK2ΔRGS, or control vectors. Cells were washed with phosphate-free DMEM (Life Technologies, Inc., Gaithersburg, MD) 48 h after transfection and then placed in phosphate-free DMEM containing 0.1–0.2 mCi 32P (New England Nuclear, Boston, MA). After 90 min, cells were stimulated with agonist for 10 min at the indicated concentrations of agonist or vehicle. The reaction was stopped by washing the cells with ice-cold Dulbecco’s PBS after which immunoprecipitation of HA-tagged PTH receptors was performed as previously described (27). Proteins were separated 4–12% Bis-Tris polyacrylamide gels (Invitrogen) as described above. After the gels were dried, phosphorylated proteins were detected by autoradiography.

For immunoprecipitation of β-catenin and FLAG-tagged Frz receptor 5 (Frz 5), cells were transfected with either GRK2, FLAG-Frz 5, or empty vectors. Cells were loaded overnight 24 h later in the presence or absence of Wnt3a conditioned medium containing 0.1–0.2 mCi 32P (New England Nuclear, Boston, MA), and either β-catenin or FLAG Frz 5 was immunoprecipitated as described above. Proteins were separated 4–12% Bis-Tris polyacrylamide gels (Invitrogen) as described above. After the gels were dried, phosphorylated proteins were detected by autoradiography.

The remainder of the immunoprecipitation experiments were performed using previously described techniques (53) with the modification that the buffer was Tris-saline buffer (50 mm Tris-HCl; 150 mm NaCl; 2 mm MgCl2, pH 7.5) supplemented with 1% Nonidet P-40, 10% glycerol, and protease inhibitors (53). Immunoprecipitated proteins were separated on Bis-Tris polyacryalamide gels (Invitrogen), and immunopreciptated proteins were detected by immunoblotting as described above.

Calvarial OB cultures

Isolation of calvarial cells was performed as previously described (54). Briefly, calvaria were removed aseptically from 6- to 8-d-old mice, minced, and then subjected to three sequential digestions for 20, 40, and 90 min in αMEM with 25 mm HEPES, 0.05% trypsin (Life Technologies, Inc.), and 0.1% collagenase (Sigma) at 37 C. The first two digests were discarded, and the last digest was collected, washed in αMEM, and cultured in αMEM containing 100 U/ml penicillin G, 100 μg/ml streptomycin, and 10% fetal bovine serum. For differentiation, cells were cultured in medium supplemented with ascorbic acid (25 μg/ml) b-glycerol phosphate (10 mm) as previously described (54).

To determine whether GRK2 modulated canonical signaling in OBs, calvarial OBs were plated in six-well tissue culture clusters (Evergreen Scientific) after which OBs were transfected with pRL-TK (0.5 μg per well) and either TOPflash (3.5 μg per well) or FOPflash (3.5 μg per well) using Lipofectamine (Invitrogen) as described above. Thirty-six hours after transfection, cells were treated overnight with 100–200 nm GRK2-TAT proteins [GRK2-TAT (+)] or an identical amount of GRK2 protein lacking the TAT sequence [GRK2-TAT (−)] in the presence or absence of Wnt3a conditioned medium. After the overnight incubation, calvarial OBs were harvested after which both firefly and Renilla luciferase intensities were measured using the Promega Dual Luciferase Reporter Assay System as described above. To assess uptake of GRK2-TAT proteins by calvarial OBs, we immunoblotted for the HA epitope in the HA-tagged GRK2-TAT (+) and GRK2-TAT (−) proteins. Pilot studies suggested that this concentration of TAT protein optimized uptake of GRK2-TAT(+) by calvarial OBs with maximal uptake after 10–15 min.

In separate experiments, calvarial OBs were treated overnight with either GRK2-TAT (−) or GRK2-TAT (+), and then cytosolic fractions were isolated as described above. The cytosolic lysate was frozen at −70 C until immunoblotting was performed using an antibody to β-catenin (Transduction Laboratories) as described above.

Statistical analysis

Data are presented as the mean ± sem. For graphical presentation, data are shown as the fold change relative to the unstimulated vector control. For comparisons between two groups, statistical significance was assessed by a Student’s t test using the InStat computer program (GraphPad Software, Inc.).

Supplementary Material

[Supplemental Data]
me.2009-0084_index.html (688B, html)

Acknowledgments

We thank Patrick Flannery for his assistance in maintaining the cultured cells for the experiments.

Footnotes

These studies were supported by National Institutes of Health Grant R01-AR04672 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Dr. Spurney and Dr. Fields also received salary support from Grant RO1 DK075688 (to R.F.S) and Grant RO1 DK062883 (to T.A.F.) from the National Institute of Diabetes, Digestive and Kidney Diseases. Dr. Gesty-Palmer received salary support from the Arthritis Foundation.

Disclosure Summary: The authors have nothing to disclose.

First Published Online June 25, 2009

Abbreviations: APC, Adenomatous polyposis coli; Frz, frizzled; GPCR, G protein-coupled receptor; GRK, GPCR kinase; GSK3β, glycogen synthase kinase 3β; HA, hemagglutinin; HEK, human embryonic kidney; OB, osteoblast; PVDF, polyvinylidene difluoride; RGS, regulator of G protein signaling; SAMP, serine-alanine-methionine-proline; siRNA, small interfering RNA; TBS, Tris-buffered saline; T-TBS, TBS with Tween 20.

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