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Originally published In Press as doi:10.1074/jbc.M710340200 on March 26, 2008

J. Biol. Chem., Vol. 283, Issue 21, 14516-14523, May 23, 2008
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Protein Kinase A Phosphorylation of Spinophilin Modulates Its Interaction with the {alpha}2A-Adrenergic Receptor (AR) and Alters Temporal Properties of {alpha}2AAR Internalization*

Jianmin Xu{ddagger}, Yunjia Chen{ddagger}, Roujian Lu{ddagger}, Christopher Cottingham{ddagger}, Kai Jiao§, and Qin Wang{ddagger}1

From the Departments of {ddagger}Physiology and Biophysics and §Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, December 19, 2007 , and in revised form, March 20, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Spinophilin plays critical roles in regulating trafficking and signaling of the {alpha}2-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940–1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-{alpha}2AAR interaction to regulate {alpha}2AAR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the {alpha}2AAR, and this event can be blocked by Ser -> Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser -> Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the {alpha}2AAR binding region of spinophilin, is sufficient to block the spinophilin-{alpha}2AAR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the {alpha}2AAR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the {alpha}2AAR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating {alpha}2AAR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}2 adrenergic receptor (AR)2 belongs to the G protein-coupled receptor superfamily and couples to the Gi/o subfamily of G proteins. In native cells, activation of the {alpha}2AR leads to inhibition of adenylyl cyclase and voltage-gated Ca2+ channels and activation of inward rectifying K+ channels and mitogen-activated protein kinases (1, 2). Ligand stimulation also causes internalization of the {alpha}2AR, a process that is important in regulating the sensitivity and duration of receptor-mediated signaling (3–5). {alpha}2AR internalization is mediated by β-arrestins (6), which bind to the {alpha}2AR (7–9) after the receptor is phosphorylated by G protein-coupled receptor kinases (10, 11). In previous studies, we identified spinophilin as a competitor of G protein-coupled receptor kinase and β-arrestin 2 for binding to the third intracellular loop (3iloop) of the {alpha}2AR (9, 12) and showed that interaction of spinophilin with the {alpha}2AR is enhanced by agonist stimulation of the receptor (13). As a result, spinophilin stabilizes the {alpha}2AR at the cell surface, as evident in cells without spinophilin expression, where internalization of the {alpha}2AR is significantly accelerated and enhanced (12, 14). In addition to all three{alpha}2AR subtypes ({alpha}2A, {alpha}2B, and {alpha}2CAR) (13, 14), spinophilin interacts with several other G protein-coupled receptors, including the D2 dopamine receptor (15) and the {alpha}1AR (16).

Spinophilin is a multidomain protein containing an actin binding domain (amino acids (aa) 1–151), a protein phosphatase 1 binding sequence (aa 427–470), a PDZ domain (aa 496-586), and three coiled-coil domains at the C terminus (aa 607–817) (17, 18). The {alpha}2AR interaction region of spinophilin is mapped to aa 151–444, which is adjacent to the actin binding domain (13). Spinophilin is a substrate of several protein kinases, including PKA (19), CaMKII (20), and ERK (21), suggesting that activities of spinophilin can be regulated by multiple intracellular pathways. PKA phosphorylation of spinophilin can be detected both in cultured cells and in the brain, and the target sites were mapped at Ser-94 and Ser-177 (19). In the present study, we addressed whether phosphorylation of spinophilin by PKA affects its interaction with the {alpha}2AAR and investigated the functional relevance of phosphorylation of spinophilin at Ser-177 on the temporal properties of{alpha}2AAR internalization. Our studies suggest that PKA regulates {alpha}2AAR trafficking through phosphorylation of spinophilin and subsequent blockade of the spinophilin-{alpha}2AAR interaction, which represents a potential novel mechanism by which other intracellular pathways regulate {alpha}2AAR function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Dulbecco's modified Eagle's medium (Invitrogen), fetal bovine serum, Lipofectamine 2000, and calcium phosphate transfection kit were purchased from Invitrogen. Restriction enzymes and T4 ligase were from New England Biolab. O-Phenylenediamine dihydrochloride was from Pierce. Dodecyl-β-D-maltoside was from Calbiochem. QuikChange II site-directed mutagenesis kit was from Stratagene. Rat anti-HA monoclonal antibody, mouse anti-GFP monoclonal antibody, and immobilized protein G-agarose were from Roche Applied Science. Mouse HA.11 monoclonal antibody was from Covance. Mouse anti-Myc monoclonal antibody was from Clontech. Rabbit anti-spinophilin polyclonal antibody was from Upstate. TnT in vitro translation kit was from Promega. [35S]Methionine and [32P]orthophosphate were from GE Healthcare. All other chemicals were from Sigma-Aldrich or Fisher.

Plasmid Constructs
Construction of pCMV4-Myc-Sp94A,177A—Ser to Ala mutations were introduced at Ser-94 and Ser-177 by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene) following the manufacturer's instruction. The template used was pCMV4-Myc-spinophilin (22), and mutagenesis primers used for generating 94A and 177A were 5'-CGGCGTGCGCCTTGCCCTGCCGCG-3' and 5'-GCAGGAGCGCGCAGCGCTGCAGGACCGG-3', respectively. The entire coding region of spinophilin in the final construct was sequenced at the Genomic Core Facility of University of Alabama (UAB), Heflin Center for Human Genetics. Introduced mutations were confirmed, and no additional mutations were identified.

Construction of pLEGFP-Sp177D—Ser to Asp mutation was introduced at Ser-177 by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene). The template used was pLEGFP-spinophilin (pLEFGP-SpWT) (23), and the mutagenesis primer used was 5'-GCAGGAGCGCGCAGATCTGCAGGACCGG-3'. Sequencing of the entire coding sequence of spinophilin in the final construct verified the mutation at Ser-177 and confirmed that no additional mutations were introduced. Sequencing was performed at the Genomic Core Facility of UAB, Heflin Center for Human Genetics.

Construction of GST-Sp151–444(177D)—Spinophilin region aa 151–444 carrying the S177D mutation was amplified by PCR using pLEGFP-Sp177D as a template and cloned into pGEX2T vector.

METHODS
Cell Culture—Immortalized mouse embryo fibroblasts (MEFs) were derived from WT (Sp+/+) or spinophilin knockout (Sp-/-) mice with identical genetic background (23). CosM6 cells, BOSC 293 cells, and MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 10 µg/ml streptomycin at 37 °C/5% CO2.

Coimmunoprecipitation of Spinophilin with the {alpha}2AAR—CosM6 cells were transfected with pCMV4-HA-{alpha}2AAR (0.3 µg/100-mm plate) together with pCMV4-Myc-SpWT or pCMV4-Myc-Sp94A,177A or pLEGFP-Sp177D (6 µg/100-mm plate) by Lipofectamine 2000 (Invitrogen). 48 h post-transfection, cells were pretreated with 10 µM forskolin or vehicle for 15 min and then stimulated with 100 µM epinephrine or vehicle for 3 min. Co-immunoprecipitation assays were performed as described previously (9).

Intact Cell Protein Phosphorylation—Phosphorylation of HA-{alpha}2AAR and Myc-spinophilin in intact cells was examined as described before (9). In brief, CosM6 cells transiently transfected with cDNA encoding HA-{alpha}2AAR or Myc-SpWT were labeled with 0.1 mCi/ml [32P]orthophosphate for 1 h. Cells were then treated with 10 µM forskolin or vehicle for 15 min. HA-{alpha}2AAR or Myc-SpWT was immunoisolated from cell lysates using a rat anti-HA antibody or a mouse anti-Myc antibody. Samples were separated on SDS-PAGE and analyzed by autoradiography and Western analysis.

In Vitro GST Pulldown Assay—Interaction of the {alpha}2AAR 3iloop with GST-Sp151–444 or GST-Sp151–444(177D) was examined by GST pulldown assays. Preparation of GST fusion proteins, synthesis of 35S-labeled {alpha}2AAR 3iloop, and pulldown assays were performed as described before (9). 25 µg of GST or 2.5 µg of GST-Sp151–444 or GST-Sp151–444(177D) and an equivalent amount of 35S-{alpha}2AAR 3iloop were used in each reaction.

Packaging of Retroviral Constructs and Transduction of MEFs— BOSC 293 cells were transfected with pLEGFP vector, pLEGFP-SpWT, or pLEGFP-Sp177D using calcium phosphate transfection kit (Invitrogen) following the manufacturer's protocol. 48 h post-transfection, supernatants were harvested and used to transduce MEFs as described previously (23). 72 h post-transduction, MEFs stably expressing GFP or GFP-fused proteins were selected by fluorescence-activated cell sorting in the UAB Flow Cytometry Core Facility.

Antibody Labeling and Quantitative Fluorescence Analyses— To specifically detect trafficking of cell surface HA-{alpha}2AAR, MEFs cultured on coverslips were incubated with HA.11 antibody for 12 min at room temperature, followed by extensive wash to remove unbound antibody. Cells were then stimulated at 37 °C with 100 µM epinephrine or vehicle (together with 1 µM prazosin to block {alpha}1ARs and 1 µM propranolol to block βARs) for various time periods. Stimulation was stopped by putting coverslips into ice-cold PBS followed by fixation with 4% paraformaldehyde for 15 min. After three washes with PBS-0.1% Triton X-100 (PBST), coverslips were blocked with 2% bovine serum albumin in PBST for 1 h and then incubated with Alexa Fluor 594 conjugated anti-mouse secondary antibody for 1 h at room temperature. Coverslips were then washed three times with PBST and mounted for microscopy. Fluorescent images were taken using a Leica confocal microscope in the UAB High Resolution Imaging Facility and quantified using MetaMorph software. Relative internalization of HA-{alpha}2AAR was measured as internal/total fluorescent intensity normalized to untreated controls. Three independent experiments were performed, and at least 15 cells were analyzed for each time point in each experiment.

Intact Cell Surface ELISA—Intact cell surface ELISA was performed as described previously (14) to examine internalization of HA-{alpha}2AAR in response to agonist stimulation for various time periods.

Statistical Analysis—Student's t tests (2-tail, unpaired) were performed to determine the difference between two experimental groups. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Forskolin Treatment Eliminates Agonist-promoted Association of Spinophilin and the {alpha}2AAR—Spinophilin has been shown to be a PKA substrate in both cultured cells and murine brains (19). We first addressed whether activation of the PKA pathway would affect the spinophilin-{alpha}2AAR interaction. Forskolin has been widely used to activate PKA through elevating intracellular cAMP levels. In a previous study, forskolin was used to induce spinophilin phosphorylation at its PKA target sites (19). Cells were pretreated with 10 µM forskolin (or vehicle) and then stimulated with 100 µM epinephrine (an {alpha}2-agonist). As we demonstrated previously, agonist stimulation enhances interaction of spinophilin and the {alpha}2AAR (13) (Fig. 1A). Strikingly, forskolin pretreatment abolishes this agonist-enhanced interaction between Myc-tagged spinophilin and HA-tagged {alpha}2AAR in immunoprecipitation assays (Fig. 1A). This conclusion is confirmed by quantitative analysis (Fig. 1B). Similarly, preincubation of cells with another PKA activator, dibutyryl cAMP, also blocks the agonist-enhanced spinophilin-{alpha}2AAR interaction (data not shown). These data suggest that activation of PKA eliminates agonist-enhanced spinophilin-{alpha}2AAR association in intact cells.


Figure 1
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  		FIGURE 1.
Forskolin treatment blocks agonist-enhanced spinophilin-{alpha}2AAR interaction. A, representative blots of co-immunoprecipitation assays. CosM6 cells transiently transfected with HA-{alpha}2AAR and Myc-spinophilin were pretreated with 10 µM forskolin or vehicle for 15 min and then stimulated with 100 µM epinephrine for 3 min. Top, proteins in anti-HA immunoisolates were separated by SDS-PAGE and blotted with anti-Myc antibody. Middle, proteins in anti-HA immunoisolates were separated by SDS-PAGE and blotted with anti-HA antibody. Bottom, total protein in cell lysates was blotted with anti-Myc antibody. B, quantitative data of agonist-induced -fold changes of Myc-spinophilin associated with HA-{alpha}2AAR with or without pretreatment of forskolin. Values represent mean ± S.E., n = 3. **, p < 0.01. n.s., not significant.

 


Figure 2
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  		FIGURE 2.
Forskolin treatment enhances phosphorylation of spinophilin, but not the {alpha}2AAR, in intact cells. CosM6 cells transiently transfected with HA-{alpha}2AAR (A) or Myc-spinophilin (B) were treated with 10 µM forskolin or vehicle for 15 min. Upper, autoradiograph of immunoisolated HA-{alpha}2AAR (A) or Myc-spinophilin (B) separated by SDS-PAGE shows phosphorylated {alpha}2AAR or spinophilin. Lower, Western blots show total immunoisolated HA-{alpha}2AAR (A) or Myc-spinophilin (B). No phosphorylation of HA-{alpha}2AAR could be detected in response to forskolin treatment in four independent experiments. C, quantitative data of -fold change of spinophilin phosphorylation in response to forskolin treatment. Values represent mean ± S.E. n = 7. *, p < 0.05.

 
Forskolin Treatment Enhances Phosphorylation of Spinophilin, but Not the {alpha}2AAR, in Intact Cells—Co-incubation of purified {alpha}2AAR with the PKA catalytic subunit leads to receptor phosphorylation in vitro (24). Therefore, forskolin treatment may lead to {alpha}2AAR phosphorylation by PKA and thereby affect its interaction with spinophilin. To test this possibility, we examined whether the {alpha}2AAR can be phosphorylated in response to forskolin treatment in intact cells. As shown in Fig. 2A, no apparent phosphorylation of the {alpha}2AAR could be detected either with or without 10 µM forskolin treatment, even after 24 h of exposure in autoradiography. By contrast, as reported previously (19), the same forskolin treatment markedly enhanced the phosphorylation level of spinophilin, which was readily detectable by autoradiography after only 2 h of exposure (Fig. 2B). Quantitative data revealed a significant enhancement of spinophilin phosphorylation in response to forskolin treatment (Fig. 2C). From these data, we conclude that in intact cells forskolin treatment enhances phosphorylation of spinophilin, but not the {alpha}2AAR.

Phosphorylation of Spinophilin at the PKA Target Sites Is Required for Forskolin Blockade of the Spinophilin-{alpha}2AAR Interaction—PKA phosphorylation of spinophilin primarily occurs at Ser-94 and Ser-177 (19). To test whether forskolin-induced blockade of the spinophilin-{alpha}2AAR interaction is due to PKA phosphorylation of spinophilin at these sites, we generated a Myc-tagged mutant spinophilin carrying Ala at aa 94 and aa 177 (designated Myc-Sp94A,177A, Fig. 3A). We first confirmed that Myc-Sp94A,177A cannot be effectively phosphorylated in response to PKA activation by forskolin (Fig. 3B) and then examined the interaction of the {alpha}2AAR with this spinophilin mutant. We found that Myc-Sp94A,177A was able to interact with HA-{alpha}2AAR in an agonist-promoted manner (Fig. 3C) and that the level of enhancement of the Myc-Sp94A,177A-HA-{alpha}2AAR interaction by agonist stimulation was comparable with that of the Myc-SpWT-HA-{alpha}2AAR interaction (Fig. 3D). As shown previously (Fig. 1), forskolin pretreatment abolishes co-immunoisolation of the HA-{alpha}2AAR with Myc-tagged WT spinophilin (Myc-SpWT) in response to agonist (p < 0.05); however, such treatment does not significantly alter agonist-enhanced association of HA-{alpha}2AAR with the Myc-Sp94A,177A mutant (p = 0.39) (Fig. 3, C and D). These data suggest that phosphorylation of spinophilin at the PKA target sites Ser-94 and Ser-177 is required for blockade of the spinophilin-{alpha}2AAR interaction by forskolin.


Figure 3
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  		FIGURE 3.
Phosphorylation of spinophilin Ser-94 and Ser-177 is required for blockade of the agonist-enhanced spinophilin-{alpha}2AAR interaction by forskolin. A, scheme of spinophilin structure. Ser-94 and Ser-177 are the two major PKA phosphorylation sites. In Sp94A,177A, these two sites were mutated to Ala. B, forskolin treatment fails to promote phosphorylation of the Myc-Sp94A,177A mutant. Left, representative images from autoradiograph and Western blot of Myc-Sp94A,177A. Right, quantitative data of -fold change of spinophilin phosphorylation in response to forskolin treatment. Values represent mean ± S.E. n = 3. n.s., not significant. C, representative blots of co-immunoprecipitation assays. CosM6 cells transiently transfected by HA-{alpha}2AAR together with Myc-SpWT or Myc-Sp94A,177A were pretreated with forskolin or vehicle for 15 min and then stimulated with epinephrine for 3 min. D, quantitative data of agonist-induced -fold changes (no agonist added defined as 1.0-fold) of Myc-SpWT or Myc-Sp94A,177A associated with HA-{alpha}2AAR with or without forskolin. Values represent mean ± S.E. n = 4. *, p < 0.05. n.s., not significant.

 
A Ser to Asp Mutation at Spinophilin aa 177 Is Sufficient to Cause Loss of Interaction of Spinophilin with the {alpha}2AAR in Intact Cells—Ser-177 is located within the region of spinophilin that directly binds to the {alpha}2AAR 3iloop (13) (Fig. 4A). We therefore addressed whether phosphorylation of spinophilin at Ser-177 is sufficient to alter interaction of spinophilin with the {alpha}2AAR. We generated a GFP-tagged mutant spinophilin carrying Asp at aa 177 (designated GFP-Sp177D, Fig. 4A), which mimics the phosphorylated state at this site by introducing a negative charge, and examined its interaction with the HA-{alpha}2AAR in response to agonist stimulation. Although agonist treatment enhances association of GFP-tagged WT spinophilin (GFP-SpWT) with HA-{alpha}2AAR, it fails to promote interaction of the receptor with GFP-Sp177D (Fig. 4B), suggesting that mimicking the phosphorylated state at Ser-177 of spinophilin is sufficient to cause the loss of agonist-enhanced spinophilin-{alpha}2AAR interaction in intact cells.


Figure 4
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  		FIGURE 4.
A Ser to Asp mutation at spinophilin aa 177 disrupts the spinophilin-{alpha}2AAR interaction. A, scheme of spinophilin structure used in co-immunoprecipitation and GST pulldown assays. B, loss of agonist-enhanced association between the HA-{alpha}2AAR and GFP-Sp177D mutant in intact cells. Representative blots of co-immunoprecipitation assays performed in CosM6 cells transiently expressing HA-{alpha}2AAR with GFP-SpWT or GFP-Sp177D. Similar results were obtained in four independent experiments. C, the receptor binding region of spinophilin (Sp151–444) carrying a Ser to Asp mutation at aa 177 exhibits reduced binding to the {alpha}2AAR 3iloop. In vitro GST pulldown assay was performed using GST-Sp151–444WT or Sp151–444(177D) and 35S-labeled {alpha}2AAR 3iloop. Similar results were obtained in three independent experiments. Upper, autoradiography of 35S-{alpha}2AAR 3iloop pulled down by glutathione-agarose in the presence of GST, GST-Sp151–444WT, or GST-Sp151–444(177D). Input lane represents the total amount of 35S-{alpha}2AAR 3iloop added in each reaction. Lower, Coomassie blue staining of GST, GST-Sp151–444WT, or GST-Sp151–444(177D) in the reaction.

 
We further tested whether the S177D mutation affects direct interaction between spinophilin aa 151–444 (Sp151–444) and the {alpha}2AAR 3iloop. We generated GST-fused mutant Sp151–444 carrying Asp at aa 177 (GST-Sp151–444(177D), Fig. 4A) and examined its interaction with the radiolabeled {alpha}2AAR 3iloop by pulldown assays. Compared with GST-Sp151–444WT, the GST-Sp151–444(177D) mutant has a remarkably reduced interaction with the {alpha}2AAR 3iloop (Fig. 4C), suggesting that mimicking the phosphorylated state at Ser-177 reduces the direct interaction between spinophilin and the {alpha}2AAR.


Figure 5
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  		FIGURE 5.
Spinophilin carrying the S177D mutation has reduced membrane localization. Sp-/- MEFs stably expressing HA-{alpha}2AAR were transduced with retrovirus encoding GFP alone, GFP-SpWT, or GFP-Sp177D. A, representative images of GFP, GFP-SpWT, and GFP-Sp177D expressed in Sp-/- MEFs. B, quantification of percentage of green fluorescence on plasma membrane in MEFs expressing GFP-SpWT or GFP-Sp177D. Values represent mean ± S.E. n = 23. **, p < 0.01.

 
Mutant Spinophilin Carrying 177D Exhibits Reduced Localization at the Plasma Membrane—It has been reported that phosphorylation at Ser-177 attenuates spinophilin interaction with actin (19), which plays an important role in targeting spinophilin to cellular compartments, including the plasma membrane (23). Therefore, we compared the cellular distribution of GFP, GFP-SpWT, and GFP-Sp177D. Both GFP-SpWT and GFP-Sp177D, but not GFP alone, can be detected at the plasma membrane and in cytosolic compartments (Fig. 5A). Quantitative analysis revealed that ~55% of GFP-SpWT and 38% of GFP-Sp177D are localized at the plasma membrane (Fig. 5B), suggesting a reduction in surface localization of spinophilin caused by mimicking the phosphorylated state at Ser-177. The reduction is statistically significant (p < 0.01). Because the {alpha}2AAR is localized at the cell surface, reduced localization of Sp177D at this compartment would also contribute to loss of interaction between the {alpha}2AAR and this spinophilin mutant in intact cells.

Agonist-induced Internalization of the {alpha}2AAR Is Accelerated in Cells Expressing Sp177D—To further understand the functional relevance of phosphorylation of spinophilin at Ser-177 on {alpha}2AAR trafficking and signaling, we evaluated the impact of the S177D mutant spinophilin on the rate of {alpha}2AAR internalization. We used retroviral transduction to introduce GFP, GFP-SpWT, or GFP-Sp177D into Sp-/- MEFs stably expressing HA-{alpha}2AAR. Cells stably co-expressing HA-{alpha}2AAR with GFP or GFP-tagged proteins were selected by fluorescence-activated cell sorting for use in the following experiments. Western analysis confirmed that exogenous GFP-SpWT or GFP-Sp177D was expressed in Sp-/- cells at levels comparable with that of endogenous spinophilin in Sp+/+ MEFs (Fig. 6A). We compared {alpha}2AAR internalization in Sp-/- cells expressing different GFP fusion proteins with that in Sp+/+ cells by intact cell surface ELISA. Consistent with our previous report (14), internalization of {alpha}2AAR is accelerated and enhanced in Sp-/- cells as compared with that in Sp+/+ cells (Fig. 6B). Reintroduction of GFP-SpWT into Sp-/- cells completely rescues this phenotype, whereas expression of GFP-Sp177D in Sp-/- cells fails to do so (Fig. 6B). These data suggest that mimicking the phosphorylated state at Ser-177 of spinophilin leads to acceleration and enhancement of agonist-induced internalization of the {alpha}2AAR.


Figure 6
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  		FIGURE 6.
Intact cell ELISAs reveal accelerated turnover of HA-{alpha}2AAR from the surface of GFP-Sp177D-expressing Sp-/- cells compared with WT or GFP-SpWT-expressing Sp-/- cells. A, total cell lysates of Sp+/+ cells or Sp-/- cells stably expressing HA-{alpha}2AAR with GFP alone, GFP-SpWT, or GFP-Sp177D were separated by SDS-PAGE and blotted with anti-spinophilin antibody to show endogenous spinophilin or exogenous GFP-SpWT or GFP-Sp177D expression (Upper). Actin in cell lysates is shown as loading controls (Lower). B, intact cell surface ELISAs performed with different types of cells. Values represent mean ± S.E. n = 12 for each time point in each type of cell. **, p < 0.01.

 
We also exploited quantitative immunofluorescent studies to examine redistribution of cell surface {alpha}2AAR in response to agonist stimulation in Sp-/- cells expressing GFP, GFP-SpWT, or GFP-Sp177D. Cell surface HA-{alpha}2AARs were first labeled with an anti-HA antibody and then stimulated by agonist for various time periods. The receptor-antibody complexes were internalized in response to agonist treatment and visualized using a fluorescent conjugated secondary antibody and confocal microscopy. In cells expressing GFP alone or GFP-Sp177D, 10 min of treatment with epinephrine caused internalization of surface HA-{alpha}2AAR to the perinuclear region, whereas in cells expressing GFP-SpWT, the majority of HA-{alpha}2AAR was still localized at the cell surface at this time point (Fig. 7A). After 30 min of agonist treatment, internalization of {alpha}2AAR to the perinuclear area could be readily detected in all cell types (Fig. 7A). Quantification of receptor fluorescence revealed that internalization of the HA-{alpha}2AAR in cells expressing GFP-Sp177D occurs at a level similar to that observed in cells expressing GFP alone, which is significantly higher than that in cells expressing GFP-SpWT at both 10- and 30-min time points (Fig. 7B). These data provide additional evidence that {alpha}2AAR internalization is accelerated and enhanced by expression of spinophilin carrying the S177D mutation.


Figure 7
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  		FIGURE 7.
Agonist-induced internalization of the HA-{alpha}2AAR occurs to a greater extent in cells expressing GFP-Sp177D than in cells expressing GFP-SpWT. Sp-/- MEFs stably expressing HA-{alpha}2AAR with GFP alone, GFP-WT spinophilin, or GFP-Sp177D were stimulated with 100 µM epinephrine (together with 1 µM prazosin to block {alpha}1AR and 1 µM propranolol to block βAR) for various times. A, representative images showing distribution of HA-{alpha}2AAR at different time points. B, quantification of fluorescent images of HA-{alpha}2AAR revealing relative internalization of HA-{alpha}2AAR in response to agonist stimulation. Values represent mean ± S.E. n = 3 independent experiments, and 15–18 cells were analyzed for each time point in each experiment. *, p < 0.05. **, p < 0.01. n.s., not significant.

 
Forskolin Treatment Accelerates Agonist-induced Internalization of the {alpha}2AAR in Cells Expressing WT Spinophilin, but Not in Cells Lacking Spinophilin or Expressing Sp177D—The studies described above demonstrate that a spinophilin mutation mimicking the phosphorylated state at one of the PKA target sites, Ser-177, leads to alteration of {alpha}2AAR trafficking. We sought to directly address whether activation of the PKA pathway changes the temporal properties of agonist-induced {alpha}2AAR internalization and whether the phosphorylation state of spinophilin is important in this process. Therefore, we examined {alpha}2AAR internalization in Sp-/- cells expressing GFP alone or GFP-tagged SpWT or Sp177D with or without forskolin pretreatment using a quantitative immunofluorescent approach. In the absence of forskolin, we again observed accelerated {alpha}2AAR internalization in cells expressing GFP alone or GFP-Sp177D as compared with that in cells expressing GFP-SpWT (Fig. 8, A, C, and E, left panels). In cells expressing GFP-SpWT, forskolin pretreatment dramatically enhanced agonist-induced internalization of the {alpha}2AAR at both 10- and 30-min time points (Fig. 8, C and D), indicating that activation of the PKA pathway accelerates and enhances agonist-induced internalization of the {alpha}2AAR in these cells. By contrast, in cells expressing GFP alone (Fig. 8, A and B) or GFP-Sp177D (Fig. 8, E and F), forskolin pretreatment did not lead to significant changes in {alpha}2AAR internalization as compared with the no-treatment control, demonstrating the importance of spinophilin and its phosphorylation state in PKA-mediated regulation of {alpha}2AAR trafficking. Taken together, these data suggest that activation of PKA indeed accelerates and enhances agonist-induced {alpha}2AAR trafficking and that phosphorylation of spinophilin at the PKA target site, Ser-177, is critical for such regulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PKA is an important intracellular Ser/Thr kinase involved in numerous cellular processes. PKA can directly phosphorylate some G protein-coupled receptors, including β1 (25, 26) and β2AR (27, 28), M1 muscarinic acetylcholine receptor (29), D1 dopamine receptor (30), and chemokine receptor (31). PKA phosphorylation of these receptors induces heterologous desensitization (25–31) and receptor internalization (29, 32), leading to reduced responsiveness to agonist stimulation. Although the {alpha}2AAR can be phosphorylated by the PKA catalytic subunit in vitro (24), we failed to detect phosphorylation of {alpha}2AAR in response to forskolin treatment in intact cells despite repetitive trials (Fig. 2A), suggesting that the {alpha}2AAR may not be a direct target for PKA-mediated regulation in cells. Instead, the present study has revealed a potential novel mechanism by which PKA regulates {alpha}2AAR. We showed that activation of PKA by forskolin treatment attenuates the interaction between {alpha}2AAR and spinophilin and thereby alters {alpha}2AAR trafficking. Our further studies suggest that PKA phosphorylation of spinophilin is required and sufficient for this regulation.


Figure 8
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  		FIGURE 8.
Forskolin pretreatment enhances agonist-induced HA-{alpha}2AAR internalization in cells expressing GFP-SpWT, but not in cells expressing GFP alone or GFP-Sp177D. Sp-/- MEFs stably expressing HA-{alpha}2AAR with GFP alone (A and B), GFP-WT spinophilin (C and D), or GFP-Sp177D (E and F) were pretreated with 10 µM forskolin or vehicle for 15 min and then stimulated with epinephrine (together with 1 µM prazosin and 1 µM propranolol) for various times. A, C, E, representative images showing distribution of HA-{alpha}2AAR at different time points. B, D, F, quantification of fluorescent images of HA-{alpha}2AAR in A, C, E, respectively, revealing relative internalization of HA-{alpha}2AAR in response to agonist stimulation. Values represent mean ± S.E. n = 3 independent experiments, and 12–15 cells were analyzed for each time point in each experiment. *, p < 0.05. n.s., not significant.

 
Spinophilin has two major PKA phosphorylation sites, Ser-94 and Ser-177. Mutation of these two sites to Ala leads to loss of forskolin blockade of the agonist-enhanced spinophilin-{alpha}2AAR interaction (Fig. 3, C and D), suggesting that phosphorylation of spinophilin at the PKA target sites is required for this process. Ser-177 is located within the region of spinophilin (Sp151–444) that directly interacts with the {alpha}2AAR 3iloop (13). A Ser to Asp mutation at this site, which mimics the phosphorylated state by introducing negative charge, remarkably attenuated direct binding of Sp151–444 to the {alpha}2AAR 3iloop (Fig. 4C). In addition, full-length spinophilin carrying the 177D mutation exhibits a significantly reduced plasma membrane localization (Fig. 5B), presumably due to attenuated association with actin (19). Therefore, we conclude that the inability of {alpha}2-agonist to promote interaction of the {alpha}2AAR with Sp177D in cells (Fig. 4B) is due to a decreased ability of this spinophilin mutant to bind to the {alpha}2AAR as well as its altered cellular distribution.

In cells expressing the Sp177D mutant, agonist-induced internalization of the {alpha}2AAR is enhanced and accelerated (Figs. 6 and 7), conceivably due to loss of the spinophilin-{alpha}2AAR interaction and spinophilin-dependent regulation. Indeed, the kinetics of {alpha}2AAR internalization in cells expressing Sp177D resembles that observed in Sp-/- cells (Fig. 6). These data suggest that PKA phosphorylation of spinophilin is functionally relevant in regulating {alpha}2AAR trafficking. In further support of this notion, we found that activation of PKA accelerates and enhances agonist-induced {alpha}2AAR internalization in cells expressing WT spinophilin, but not in cells lacking spinophilin or expressing the Sp177D mutant (Fig. 8).

In addition to PKA, spinophilin is a substrate of several other kinases, including CaMKII (20) and ERK (21). Whether phosphorylation of spinophilin by these kinases also affects spinophilin interaction with {alpha}2AAR remains to be determined. In summary, this study suggests that modulation of the spinophilin-{alpha}2AAR interaction represents a potential novel pathway where cross-talk between G protein-coupled receptors and other intracellular signaling pathways may occur.


   FOOTNOTES
 
* This work was supported by an Alabama state fund and an American Heart Association Scientist Development grant (to Q. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 958 MCLM, 1918 University Blvd., Birmingham, AL 35294. Tel.: 205-996-5099; Fax: 205-975-9028; E-mail: qinwang{at}uab.edu.

2 The abbreviations used are: AR, adrenergic receptor; PKA, protein kinase A; ERK, extracellular signal-regulated kinase; MEF, mouse embryonic fibroblast; HA, hemagglutinin; Sp, spinophilin; WT, wild type; 3iloop, third intracellular loop; GFP, green fluorescent protein; GST, glutathione S-transferase; aa, amino acid; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Paul Greengard for generously allowing use of the MEFs derived from Sp+/+ and Sp-/- mice and Dr. Lee Limbird for providing valuable cells as well as the cDNA encoding GFP-SpWT. We thank Dr. Roger Colbran for providing the cDNAs encoding Myc-spinophilin and GST-Sp151–444. We also thank Drs. Lee Limbird and Ashley Brady for critically reading this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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R. Lu, Y. Li, Y. Zhang, Y. Chen, A. D. Shields, D. G. Winder, T. Angelotti, K. Jiao, L. E. Limbird, Y. Zhou, et al.
Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of {alpha}2-Adrenergic Responses Is because of Ligand-selective Internalization
J. Biol. Chem., May 8, 2009; 284(19): 13233 - 13243.
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