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Originally published In Press as doi:10.1074/jbc.M608871200 on December 14, 2006

J. Biol. Chem., Vol. 282, Issue 7, 5085-5099, February 16, 2007
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Assembly of an SAP97-AKAP79-cAMP-dependent Protein Kinase Scaffold at the Type 1 PSD-95/DLG/ZO1 Motif of the Human beta1-Adrenergic Receptor Generates a Receptosome Involved in Receptor Recycling and Networking*

Lidia A. Gardner{ddagger}, Anjaparavanda P. Naren§1, and Suleiman W. Bahouth{ddagger}2

From the {ddagger}Departments of Pharmacology and §Physiology, the University of Tennessee Health Sciences Center, Memphis, Tennessee 38163

Received for publication, September 15, 2006 , and in revised form, November 27, 2006.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Appropriate trafficking of the beta1-adrenergic receptor (beta1-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the beta1-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the beta1-AR (Gardner, L. A., Tavalin, S. A., Goehring, A., Scott, J. D., and Bahouth, S. W. (2006) J. Biol. Chem. 281, 33537-33553). In this study we show that AKAP79 forms a complex with the type 1 PDZ-binding sequence (ESKV) at the extreme carboxyl terminus of the beta1-AR, which is mediated by the membrane-associated guanylate kinase (MAGUK) protein SAP97. Thus, the PDZ and its associated SAP97-AKAP79 complex are involved in targeting the cyclic AMP-dependent protein kinase (PKA) to the beta1-AR. The PDZ and its scaffold were required for efficient recycling of the beta1-AR and for PKA-mediated phosphorylation of the beta1-AR at Ser312. Overexpression of the catalytic subunit of PKA or mutagenesis of Ser312 to the phosphoserine mimic aspartic acid both rescued the recycling of the trafficking-defective beta1-AR{Delta} PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the beta1-AR to Ser312 in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the beta1-AR. The data presented here show that a novel beta1-adrenergic receptosome is organized at the beta1-AR PDZ to generate a scaffold essential for trafficking and networking of the beta1-AR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sympathetic nervous system mediates its regulatory effects through G protein-coupled receptors (GPCR)3 related to the family of {alpha}- and beta-adrenergic receptors. Among these receptors is the beta1-AR, which is coupled to the Gs-cyclic AMP axis and plays a major role in transmitting sympathetic regulation to cardiac, renal, vascular, and other organs (2, 3).

Persistent activation of the beta1-AR or other GPCR causes their desensitization and internalization via clathrin-coated pits or caveolae into early endosomes (4-6). Internalized GPCR are either recycled back to the cell surface for another round of signaling or retained for degradation by lysosomal or proteasomal pathways (7-9). Characterization of the players involved in these distinct outcomes is the purpose of this study.

Recycling and resensitization of the beta1-AR are dependent upon two motifs; one is the ESKV sequence in the carboxyl-terminal tail, and the other is the region surrounding Ser312 in the 3rd IC of the beta1-AR (10, 11). The ESKV tetrapeptide conforms to a type I (PSD-95/DLG/ZO1) PDZ ligand (i.e. X(S/T)X{Phi}, where X at positions -1 and -3 is any amino acid, and {Phi} at position 0 is a hydrophobic amino acid) (12, 13). Mutagenesis of the type 1 PDZ or Ser312 to alanine prevented the recycling and resensitization of the beta1-AR (10, 11). Concerning Ser312, we determined that this residue is specifically phosphorylated by PKA and that the activity of PKA was required for recycling and resensitization of the human beta1-AR (11).

These results indicate that two distinct motifs are involved in recycling of the beta1-AR, but they do not explain how they cross-talk to one another to coordinate the sequence of events involved in recycling of this GPCR. A major breakthrough in identifying the mechanism of cross-talk between these two motifs was the identification of AKAP79 as the AKAP involved in recycling of the beta1-AR in HEK-293 and other cell lines (1). AKAP79 promoted the targeting of PKA to the beta1-AR by binding to the carboxyl-terminal 53 amino acids (between residues 425 and 477) of the beta1-AR (1). Here we report that the binding domain of AKAP79 to the beta1-AR overlaps with its type 1 PDZ motif. However, the binding between the PDZ and AKAP79 is indirect and involves the MAGUK protein SAP97 that simultaneously binds to AKAP79 and type 1 PDZ to target PKA to the beta1-AR. By scaffolding PKA to the beta1-AR, SAP97 facilitates PKA-mediated phosphorylation of Ser312, which is critical for trafficking of the internalized beta1-AR to membranes. These results indicate that a novel beta1-adrenergic receptosome is involved in recycling and resensitization of the beta1-AR as well as in its other physiological effects.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of FLAG- or Myc-tagged Full-length and Truncated beta1-AR—To allow rapid assessment of cell surface expression of the beta1-AR, the amino-terminal initiator methionine was replaced either by the FLAG tag sequence (DYKDDDDK) or by the Myc tag (EQKLISEEDL) sequence, resulting in N-FLAG/Myc-tagged WT beta1-AR or the S312D mutant in the mammalian expression vector pcDNA3.1 (Invitrogen) (14). To generate the beta1-AR({Delta}425-441) and beta1-AR({Delta}425-463) constructs, the full-length beta1-AR cDNA was cut with SmaI, and the resulting 1.3-kb cDNA encoding beta1-AR-(1-424) was cloned into pcDNA3.1. The 162-bp SmaI-EcoRI cDNA between bases 1272 and 1434 was used as a template for PCRs that generated cDNAs encoding the sequences between amino acids 441-477 and 463-477, which were then ligated in-frame into the beta1-AR-(1-424). The WT beta1-AR or S312D was used as PCR templates with the sense primer described in Gardner et al. (1) and the antisense primer 5'-TGGATCCTACGCTGCTGCTGCCGAGGCGAAGCCGGGGCGGCAC-3' to generate the beta1-AR{Delta}PDZ and the S312D-beta1-AR{Delta}PDZ, respectively. Sequences of all the epitope-tagged beta1-AR constructs were verified by automated dideoxy sequencing.

Construction of Fluorescently Tagged beta1-AR, SAP97, AKAP79, and RII{alpha} Subunit—Amino-terminal fusions of the WTbeta1-AR and AKAP79 to CFP and YFP were described (1). CFP- or YFP-beta1-AR{Delta}PDZ was generated using the forward primer described earlier and the reverse primer (5'-TGGATCCGCTGCTGCTGCCGAGGCGAAGCCGGGGCGGCAC). For SAP97, the coding sequence of rat myc-SAP97 in GW1-CMV was amplified by PCR using a forward primer (5'-AAGCTTATGCCGGTCCGGAAGCAAGATACC) and a reverse primer (5'-CGGTACCGTTAATTTTTCTTTTGCTGGGACCCAG) to generate the 2.8-kb HindIII-KpnI cDNA, which was fused in-frame into pECFP-N1 and pEYFP-N1. Expression of these fusion proteins was confirmed by sequencing, fluorescence microscopy, and Western blot analysis.

Cell Cultures and Radioligand Binding Parameters—HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum until they were ~90% confluent. The WT beta1-AR or its point mutants in pcDNA 3.1 were transiently transfected into HEK-293 cells using the Cytofectene reagent (Bio-Rad) as follows. Plasmid DNA (1 µg) was diluted into 200 µl of DMEM and then mixed with an equal volume of DMEM containing 12 µl of Cytofectene at room temperature for 20 min. Then 4 ml of DMEM was added, and the DNA-lipid complex was layered over the cells for 5 h at 37 °C, followed by the addition of an equal volume of DMEM + 10% fetal bovine serum. G-418-stable cell lines for the constructs described in Table 1 were generated and used where indicated. Binding of [125I]iodocyanopindolol (ICYP) to 0.5 µg of membranes prepared from the cells described in Table 1 was measured in 50 mM Tris-HCl, pH 7.4, plus 10 mM MgCl2 binding buffer containing 0.1 mM ascorbic acid for 2 h at 25 °C. For saturation binding experiments, ICYP concentrations ranging between 5 and 300 pM were used to calculate the KD and the Bmax values for ICYP binding by parametric fitting of the data by using the Prism 4 software (GraphPad Corp.).


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TABLE 1
Ligand binding properties of [125I] iodocyanopindolol to wild-type and mutated beta1-AR stably expressed in HEK-293 cells

Binding of ICYP to 0.5 µg of membranes derived from HEK-293 cells expressing the various beta1-AR constructs was measured as described under "Experimental Procedures." For saturation binding experiments, ICYP concentrations ranging between 5 and 300 pM were used. Saturation isotherms were analyzed by one- or two-site models to determine the KD and Bmax values of ICYP. Dose-response curves of isoproterenol-mediated stimulation of adenylyl cyclase were determined in membranes prepared from these cells, as described under "Experimental Procedures." These curves were analyzed by nonlinear regression (Prism 4.0) to calculate the EC50 ± S.E. for each construct (n = 3).

 
Antibodies, siRNA, Peptides, and Additional Reagents—The monoclonal antibodies against FLAG (M2) and Myc (9E-10) epitopes were purchased from Sigma and Upstate (Charlottesville, VA), respectively. The antibodies to human AKAP79 and to the various subunits of PKA were from Clontech; anti-human SAP97 monoclonal antibody was from StressGen (VAM-PS005, Nventa Corp., San Jose, CA); anti-SAP97 polyclonal antibody was from Novus Biologicals (NB 600-1229); anti-human beta1-AR antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-PSD-95 antibodies (anti-pan-PDZ monoclonal antibody 05-427 directed against the PDZ domain (residues 77-299) of human PSD-95 and anti-PSD-95 antibody 05-494) were from Upstate%20Biotechnology">Upstate Biotechnology, Inc. The siRNA to AKAP79 5'-AAgagaucagcagaagguagu-3' and its scrambled control AAggcaacaaaggcuaaguca were described (1). The siRNA sequence to human SAP97, 5'-GATATCCAGGAACATAAAT-3', or its control, 5'-CCATAATACAAGGTATAA-3', were cloned into the pSuperTM plasmid (OligoEngine Corp., Seattle, WA).

Acid Strip Confocal Recycling Microscopy Protocol—HEK-293 cells expressing the FLAG- or Myc-tagged WT beta1-AR or beta1-AR{Delta}PDZ were grown on poly-L-lysine-coated glass coverslips and serum-starved at 37 °C for 1 h in DMEM supplemented with 25 mM HEPES, pH 7.4. The receptors were labeled with fluorescein isothiocyanate-conjugated anti-FLAG M2 IgG (10 µg/ml) for 1 h at 37 °C. Cells were treated with 10 µM isoproterenol for 30 min at 37 °C to promote agonist-mediated beta1-AR internalization. Then the cells were chilled to stop endocytosis and exposed to 0.5 M NaCl, 0.2 M acetic acid, pH 3.5, for 4 min on ice to remove antibody bound to extracellular beta1-AR (1, 15, 16). Cultures were then incubated with culture medium supplemented with 100 µM of the beta-antagonist alprenolol at 37 °C for 10, 20, 30, or 45 min to establish the recycle time. After each time period, the coverslips were rinsed and fixed in 4% paraformaldehyde with 4% sucrose in PBS, pH 7.4, for 10 min at room temperature. Confocal fluorescence microscopy was performed on coded slides using a Zeiss Axiovert LSM 510 (100 x 1.4 DIC oil immersion objective), and the immunocytochemical data were analyzed to determine the recycle time (1).

Dual Confocal Microscopy—HEK-293 cells stably expressing the YFP-tagged beta1-AR were transfected with Myc-SAP97. The cells were exposed to 10 µM isoproterenol for 30 min, acid-washed, and then exposed to 100 µM alprenolol for 10, 20, 30, or 45 min. The coverslips were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 in PBS, and stained with Cy-3 conjugated to anti-Myc 9E-10 monoclonal antibody and visualized by dual confocal microscopy (YFP, {lambda}ex = 514 nm, {lambda}em = 530LP; Cy3, {lambda}ex = 543 nm, {lambda}em = 560 BP) using the LSM-510 multitracking configuration.

FRET Microscopy—Double stable cell lines expressing AKAP79-CFP and beta1-AR-YFP or SAP97-CFP and beta1-AR-YFP were established. In some cases, HEK-293 cells were transfected with the desired plasmids using the Lipofectamine reagent (Invitrogen) for 24-36 h. After transfection, cells were plated on poly-L-lysine-covered coverslips for 24 h, fixed with 4% paraformaldehyde, pH 7.4, and mounted onto glass slides in Fluoromount G mounting media (Electron Microscopy Sciences, Hatfield, PA). Coverslips were sealed with clear nail polish and imaged using the sensitized emission or the acceptor photobleaching methods described in Gardner et al. (1). After image acquisition, the LSM 510 FRET macro tool was used to calculate FRETN values. FRETN is a measure of FRET that is normalized for the concentrations of donor and acceptor fluorophores and therefore represents a fully corrected measure of FRET (17-19). In this method the corrected FRET value for each pixel is calculated and then divided by concentration values for donor and acceptor (18-20). FRETN was calculated on a pixel-by-pixel basis for the entire image.

In addition to FRETN microscopy, we performed acceptor photobleaching FRET microscopy. This method measures changes in the intensity of the donor channel that are observed upon complete photobleaching of the acceptor (YFP) by a 514 nm argon laser (1, 16-18). From each photobleaching session, an image set consisting of time-lapse recordings of donor and acceptor channel intensities was obtained. FRET was recorded by examining the loss of quenching of CFP during YFP photobleaching, followed by an analysis of these images by the LSM FRET tool version 1.5 (AIM software release 3.2) to calculate the FRET efficiencies using selected area averages for donor CFP before and after bleaching. FRET efficiencies (%) are presented as the means ± S.E. from 3 to 10 separate acquisition experiments on 5-10 images per experiment.

Co-immunoprecipitations and Pulldown Assays—Co-immunoprecipitations between FLAG- or Myc-tagged beta1-AR, SAP97, AKAP79, or the RII {alpha}-subunit of PKA were performed as follows. Cells stably expressing the indicated FLAG-tagged or Myc-tagged beta1-AR were lysed in radioimmune precipitation assay buffer (1), and the insoluble cellular debris was removed by centrifugation at 14,000 x gav for 15 min at 4 °C. After equalizing protein concentrations across all samples, lysates were added to M2 anti-FLAG- or anti-Myc-agarose beads at 4 °C with gentle rotation for 4 h. Control experiments were performed by incubating lysates with preimmune IgG at the same concentration for 4 h at 4 °C. The immune complexes were washed three times in radioimmune precipitation assay buffer and eluted from the beads with 40 µl of 2x Laemmli sample buffer containing 20 mM dithiothreitol. Resolved proteins and lysate inputs were separated by SDS-PAGE under denaturing conditions and electroblotted to nitrocellulose. Identical gels were run and transferred for separate detection of receptor, AKAP79, SAP97, or the RII{alpha} subunit of PKA by Western blotting.

Pulldown Assays—The human beta1-AR cDNA was digested with SmaI and XhoI to isolate the carboxyl-terminal fragment encoding the amino acids between 425 and 477. This fragment and the corresponding carboxyl-terminal tail fragment of the beta2-AR were cloned into the pGEX-4T-2 GST vector (GE Healthcare) and amplified in BL-21 Escherichia coli cells. HEK-293 cells that were transfected either with empty or with myc-SAP97 expressing vector were lysed with in 0.2% Triton X-100 in PBS supplemented with protease inhibitors. After 16,000 x gav centrifugation of cell lysates, GST orbeta1-ARc-tail orbeta2-ARc-tail-GST fusion proteins were added to aliquots of the supernatants. Twenty µl of glutathione-agarose beads (50% slurry in H2O) were added after mixing for 30 min at 4 °C. The mixture was mixed for another 2 h at 4 °C. After washing three times with the same lysis buffer, the proteins were eluted from beads with sample buffer (containing 2.5% beta-mercaptoethanol). Eluates were separated on a 4-15% gel and analyzed for SAP97 by immunoblotting. Far Western blots were performed to detect the interaction between FLAG-beta1-AR (WT and {Delta}PDZ) and SAP97 according to Hall (21) with a few modifications. In brief, the purified receptor was slot-blotted onto a dry nitrocellulose membrane in a volume of 100 µl (20 µl at a time was added) under vacuum (manifold II). The membrane was wetted in 5 ml of Tris-buffered saline containing 0.2% Tween 20 (TBST) and then blocked with TBST containing 10 mg/ml bovine serum albumin (TBST-BSA) for 1 h at 22 °C by gentle shaking. Affinity purified Myc-SAP97 was hybridized with the membranes in TBST-BSA for 16 h at 4 °C with gentle rocking. The membrane was washed five times (5 min each with TBST) and probed with 1:1000 dilution of anti-Myc IgG (9E-10 monoclonal antibody) for 1 h at 22 °C in TBST-BSA. After washing the blot five times with TBST, it was probed with horseradish peroxidase-conjugated anti-mouse IgG (Pierce) in TBST-BSA for 20 min at room temperature. The blot was washed five times in TBST (5 min each) and then developed using enhanced chemiluminescence.

Cyclic AMP Accumulation and Adenylyl Cyclase Assays—HEK-293 cells stably expressing the various beta1-AR constructs in 6-well plates were switched to DMEM + 25 mM HEPES for 2 h. Appropriate drugs in DMEM/HEPES, supplemented with 300 µM of the phosphodiesterase inhibitor isobutylmethylxanthine, were added to the cells for 10 min at 37 °C. The reaction was stopped, and 1 ml of 0.1 N cold HCl was added followed by freezing of the entire plate in liquid nitrogen. Frozen plates were quickly thawed at 65 °C to break the cells, and the cell extract was lyophilized. The dry pellet was resuspended in assay buffer, and cyclic AMP was quantified by radioimmunoassay (RIANEN Assay System; PerkinElmer Life Sciences). For the determination of adenylyl cyclase activity, membranes were prepared from cells without phenylmethylsulfonyl fluoride, and the activity of adenylyl cyclase in response to increasing concentrations of isoproterenol was determined (14, 16). The concentration-response curves to isoproterenol were fitted by nonlinear regression using Prism 4.1 software (GraphPad Corp.) in order to determine the concentration of isoproterenol that generated 50% of the maximal response (EC50) for each beta1-AR construct.

Adenylyl Cyclase Assays for beta-AR Desensitization and Resensitization—HEK-293 cells stably expressing the various beta1-AR constructs were divided into four sets. The first and second sets were used as control for desensitization and the third and fourth sets for resensitization assays. Cells for desensitization were exposed to 1 mM ascorbic acid (control) or 10 µM isoproterenol for 10 min at 37 °C and then processed for the preparation of membranes. The third set was used as the control for resensitization and the fourth set for resensitization assays. Cells for resensitization were exposed either to 1 mM ascorbic acid (control) or to 10 µM isoproterenol for 3 h at 37 °C and then incubated with 100 µM alprenolol for 1.5 h at 37 °C, followed by the preparation of membranes. Adenylyl cyclase activities in these membranes were determined (14, 16), and the Kact ± S.E. for each beta1-AR was calculated using the Prism 4 program, and statistical comparisons were analyzed using Prism 4 and Instat programs (GraphPad Corp.).


Figure 1
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FIGURE 1.
Characterization of the site of AKAP79 binding to the beta1-AR and its role in beta1-AR recycling. A, co-immunoprecipitation of various Myc-tagged beta1-AR constructs and FLAG-AKAP79 in transiently transfected HEK-293 cells were conducted as described under "Experimental Procedures." Lysates represent 5% of the total extract, whereas immunoprecipitations (IP) represent 30% of the total volume. B, HEK-293 cells stably expressing FLAG-WT beta1-AR (images a-e) or FLAG-beta1-AR{Delta}PDZ, in which the last four amino acids in the carboxyl terminus of the beta1-AR were mutated to alanine (images f-j), were cultured on glass bottom slides. Recycling of the WT beta1-AR and the beta1-AR{Delta}PDZ in response to 10 µM isoproterenol (n = 3) was conducted as described under "Experimental Procedures." Each scale bar represents 5 µm.

 
Phosphorylation and Phosphopeptide Mapping of the beta1-AR—To determine the effect of disrupting the beta1-AR PDZ or down-regulation of SAP97 on isoproterenol-mediated phosphorylation of the beta1-AR, HEK-293 cells expressing the WT beta1-AR were transfected with the SAP97 siRNA vector or its scrambled control. On the day of the experiment, the ATP pools were labeled with 200 µCi of 32PO4/ml for 1.5 h, and the cells were stimulated with either 1 mM ascorbic acid or 10 µM isoproterenol in 1 mM ascorbic acid for 10 min at 37 °C. After cell lysis, equivalent amounts of proteins in each supernatant were incubated with M2 anti-FLAG-agarose beads at 4 °C for 5 h. The resins were washed in radioimmune precipitation assay buffer, and the eluted proteins were resolved by SDS-PAGE. The gels were transferred to nitrocellulose, and amounts of 32P incorporated into the beta1-AR were determined by electronic counting with Packard InstantimagerTM. The bands corresponding to phosphorylated beta1-AR protein on the filter were cut out and submerged in 70% (v/v) formic acid containing 100 mg per ml of cyanogen bromide (Science Lab Chemicals, Kingswood, TX) for 1.5 h at room temperature (1). At the end of the digestion, the samples were lyophilized and dissolved in Tricine sample buffer. Then 5 µl from each sample was spotted onto a GF/C filter pre-moistened with 10% trichloroacetic acid. The filters were mounted on a filtration manifold and washed three times with 5 ml of 10% trichloroacetic acid to remove the free 32P. After drying, the counts/min of 32P/filter were determined by liquid scintillation spectrometry. Equal counts/min (1,200 ± 50) of 32P were loaded per lane and subjected to electrophoresis on 16% acrylamide gels in Tricine cathode buffer. At the end of the run the gel was electroblotted to nitrocellulose, and the filters were counted by the InstantimagerTM and then exposed to an x-ray film overnight.

Biotinylation Assay of beta1-AR Recycling with Cleavable Biotin—Cells expressing the WT beta1-AR with siRNA to SAP97 or its control were surface-biotinylated with 1.5 mg/ml sulfo-NHSSS-biotin (Pierce) in Hanks' balanced salt solution with Ca2+ and Mg2+ at 4 °C (1). Biotinylated cells were exposed to isoproterenol for 30 min and then cooled to 4 °C to stop membrane trafficking, and the remaining surface biotin was quantitatively cleaved with glutathione. After cleavage, warm DMEM was added, and cells were incubated at 37 °C for 15, 30, and 60 min to allow internalized receptor to recycle before the cells were cooled to 4 °C and incubated with glutathione cleavage buffer for a second time to ensure complete cleavage of any newly appearing surface biotin. At the end of each time point, the cells were scraped into detergent-free lysis buffer, sonicated, and then centrifuged at 100,000 x gav for 20 min at 2 °C. The membrane pellet was dissolved in lysis buffer supplemented with detergents and recentrifuged at 100,000 x gav for 20 min at 2 °C. The supernatant was collected, and equal amounts of protein from all samples were mixed with 50 µl of BSA-blocked ultralink-neutravidin beads (Pierce) to isolate the biotinylated proteins. The resin was extracted, and the extracts were subjected to immunoblotting with anti-FLAG antibody to determine the density of beta1-AR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of AKAP79 Binding to the beta1-AR—Myc-tagged beta1-AR constructs with progressive deletions within their carboxyl termini were co-expressed with FLAG-AKAP79 in order to localize by co-immunoprecipitations the sequence in the beta1-AR that bound AKAP79 (Fig. 1A). Deletion of the amino acids between 425 and 441 in the carboxyl-terminal tail of the beta1-AR had little effect on the immunoprecipitation of AKAP79 by this beta1-AR mutant. Deletion of the sequence between 425 and 463 significantly reduced the immunoprecipitation of AKAP79, indicating that the binding site between AKA79 and the beta1-AR partially overlapped with this sequence. Mutagenic inactivation ({Delta}) of each residue in the type 1 PDZ sequence (ESKV) between amino acids 474 and 477 to alanine (beta1-AR{Delta}PDZ) completely inhibited the interaction between the beta1-AR and AKAP79, confirming that the AKAP79 interacting site overlapped with the type 1 PDZ sequence. The interactions between AKAP79 and the beta1-AR are involved in recycling of the agonist-internalized beta1-AR back into the cell membrane in a process termed "resensitization" (1, 11). This process is involved in trafficking of the agonist-internalized beta1-AR back to the cell membrane (Fig. 1B, images a-e). Inhibition of the binding of AKAP79 to the beta1-AR by inactivating the PDZ prevented the recycling of the agonist-internalized beta1-AR{Delta}PDZ (Fig. 1B, images f-j). In other experiments we determined that the binding parameters of [125I]ICYP to the WT beta1-AR or to the beta1-AR{Delta}PDZ were comparable (Table 1). The effect of mutagenesis of the PDZ on receptor coupling efficacy to Gs was assessed by measuring basal and isoproterenol-stimulated increases in cyclic AMP accumulation in whole cells and in adenylyl cyclase activities in membranes prepared from cells expressing comparable densities of each beta1-AR construct (Fig. 2). Basal levels of cyclic AMP in cells stably expressing the empty pcDNA3.1 vector were 8 ± 3 and 14 ± 2 pmol/mg of protein in cells expressing the WT beta1-AR or the beta1-AR{Delta}PDZ, respectively (Fig. 2A). The EC50 value for the accumulation of cyclic AMP in response to isoproterenol was 7 ± 1.5 nM for the WT beta1-AR and 9 ± 2nM for the WT beta1-AR{Delta}PDZ (Fig. 2A; p > 0.05). Basal levels of adenylyl cyclase activities in membranes prepared from cells expressing the WT beta1-AR were 17 ± 3 pmol/min/mg, whereas in cells expressing the beta1-AR{Delta}PDZ, these levels were 14 ± 3 pmol/min/mg (p > 0.05). The EC50 value for the activation of adenylyl cyclase in membranes expressing either the WT beta1-AR or the beta1-AR{Delta}PDZ were comparable at 0.1 ± 0.02 µM, and maximal activation of adenylyl cyclase was 82 pmol/min/mg protein by either of these receptors (Fig. 2B). Thus, the {Delta}PDZ mutation had no effect on the coupling efficacy of the beta1-AR to Gs.


Figure 2
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FIGURE 2.
Effect of point mutants in the PDZ or in Ser312 of the beta1-AR and of the WT beta1-AR-CFP and -YFP chimera on cyclic AMP accumulation or adenylyl cyclase activation in response to isoproterenol. A, cells stably expressing the levels of beta1-AR, shown in Table 1, were stimulated with 0, 1, 10, 50, and 100 nM of isoproterenol for 5 min at 37 °C, followed by determining the levels of cyclic AMP as described under "Experimental Procedures." B and C, isoproterenol-mediated activation of adenylyl cyclase in membranes prepared from cells expressing the WT beta1-AR or its point mutants, and chimera were determined. The EC50 values for isoproterenol in activating adenylyl cyclase for each beta1-AR construct are reported in Table 1.

 
To characterize the mechanism by which AKAP79 binds to the beta1-AR, full-length WT beta1-AR or beta1-AR{Delta}PDZ was hybridized to immobilized AKAP79 (Fig. 3, A and B). In these far Western assays, no direct binding of AKAP79 to either beta1-AR construct was observed, indicating that these proteins did not interact directly. An alternative mechanism that can account for indirect binding of AKAP79 to the beta1-AR is through their mutual association with MAGUK proteins (22). MAGUK proteins related to the PSD/SAP family (PSD-95/SAP90, SAP97/hdlg, Chasyn-110/PSD-93, and SAP102) share a common domain organization consisting of three PDZ domains in their amino-terminal half that bind to type 1 PDZs and Src homology 3 and guanylate kinase-like domains at their carboxyl terminus that bind to AKAP79 and other proteins (22-24). MAGUK proteins related to PSD-95 and MAGI-II have been shown to bind to the beta1-AR PDZ (25, 26). Therefore, HEK-293 cells were probed for the expression of these proteins, but none of them was detected in this cell line. Consequently, we explored whether other MAGUK-related proteins were expressed in HEK-293 cells by probing cell extracts with a pan-PDZ antibody (Upstate%20Biotechnology">Upstate Biotechnology, Inc.), which identified a prominent immunoreactive species with an apparent molecular mass of 110-116 kDa (data not shown). This protein was confirmed as human SAP97 by Western blotting with a monoclonal antibody to human SAP97 (Fig. 3C) and with a polyclonal anti-SAP97 antibody (data not shown). Far Western assays between the beta1-AR and SAP97 showed direct binding between SAP97 and the full-length beta1-AR, which was abrogated when the PDZ domain in the beta1-AR was mutated (Fig. 3D). Pulldown assays between SAP97 and glutathione S-transferase (GST) fusions of full-length carboxyl termini of the beta1-AR and the beta2-AR indicated that SAP97 preferentially associated with the carboxyl terminus of the human beta1-AR (Fig. 3E).


Figure 3
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FIGURE 3.
Identification of SAP97 as an interacting partner at the carboxyl-terminal type 1 PDZ of the beta1-AR. A and B, beta1-AR does not interact with AKAP79 in far Western blotting assays. FLAG-WTbeta1-AR and FLAG-beta1-AR {Delta}PDZ (~50 ng) were immobilized onto nitrocellulose filters. The filters were hybridized with 100 ng/ml FLAG-AKAP79, washed, and then probed with the anti-AKAP79 monoclonal antibody. C, HEK-293 cell lysates express SAP97. D, immobilized Myc-SAP97 (~50 ng) interacts with FLAG-beta1-AR but not with FLAG-beta1-AR{Delta}PDZ in far Western assays. E, purified GST-beta1-AR-carboxyl-terminal tail (between amino acids 425-477, left panel) binds preferentially to SAP97 in pulldown assays that were conducted as described under "Experimental Procedures." F, Myc-SAP97 was transiently transfected into a HEK-293 cell line stably expressing the WT beta1-AR-YFP. After 2 days, the cells were exposed to ascorbic acid (No Iso) or to 10 µM isoproterenol for 30 min, acid-washed, and treated with 100 µM alprenolol for 0, 10, 20, 30, and 45 min. At the end of each time period, the cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 in PBS, stained with Cy-3 conjugated to anti-Myc 9E-10 monoclonal antibody, and visualized by dual confocal microscopy (YFP, {lambda}ex = 514 nm, {lambda}em = 530LP; Cy3, {lambda}ex = 543 nm, {lambda}em = 560 BP) using LSM-510 multitracking configuration. IB, immunoblot; CBB, Coomassie Brilliant Blue; Iso, isoproterenol.

 
To further confirm that the beta1-AR could bind to SAP97, we determined whether they were co-localized. Myc-SAP97 was transiently transfected into a HEK-293 cell line stably expressing 1.1 ± 0.2 pmol/mg protein of YFP tagged to the carboxyl terminus of the WT beta1-AR, and their distribution was determined by dual-labeling confocal microscopy (Fig. 3F). In control cells, Cy-3-labeled SAP97 (Fig. 3F, red) was co-localized with YFP-labeled beta1-AR (yellow) at the cell surface of HEK-293 cells (Fig. 3F, image a). In addition, SAP97 was distributed into other intracellular compartments (24). Exposing these cells to isoproterenol caused the internalization of the beta1-AR without altering the cellular distribution of SAP97 (Fig. 3F, image b). After the removal of isoproterenol, the internalized beta1-AR trafficked back into the cell membrane and was co-localized with SAP79 (Fig. 3F, images c-f).

Co-immunoprecipitations between SAP97 and a variety of beta1-AR constructs were used to determine whether SAP97 binds to the beta1-AR in a PDZ-dependent manner. FLAG-WT beta1-AR or its {Delta}PDZ mutant was co-transfected into HEK-293 cells with Myc-SAP97 (Fig. 4, panel A, a-c). Precipitates of the WT beta1-AR, co-immunoprecipitated SAP97, and reciprocal immunoprecipitations of SAP97 precipitated the WT beta1-AR (Fig. 4, a and b), but precipitates prepared from cells expressing the beta1-AR{Delta}PDZ failed to co-immunoprecipitate SAP97 (Fig. 4, panel A, c). Moreover, in precipitates of the WT beta1-AR, we detected co-immunoprecipitations of AKAP79 and the RII-{alpha} subunit of PKA (Fig. 4, panel A, d and e). In cells co-expressing FLAG-AKAP79 and Myc-SAP97 (Fig. 4, panel A, f and g), precipitates of AKAP79 co-immunoprecipitated SAP97 and vice versa, indicating that SAP97 and AKAP79 interacted with each other under these conditions (17, 22). These data show that a quaternary complex composed of SAP97, AKAP79, and PKA was assembled at the type 1 PDZ in the carboxyl-terminal tail of the beta1-AR.

The organization of this scaffold was further studied using siRNAs to SAP97 (Fig. 4, panel B). We predicted that if SAP97 was a bridging molecule between the beta1-AR and AKAP79/PKA, consequently knockdown of SAP97 should destabilize the binding between the beta1-AR and AKAP79. Toward this, cells expressing 1.4 pmol/mg Myc-beta1-AR and FLAG-AKAP79 were transfected with SAP97 siRNA or its scrambled control (Fig. 4, panel B, h-m). In cells expressing the scrambled siRNA, beta1-AR precipitates co-immunoprecipitated the beta1-AR, AKAP79, and SAP97 (Fig. 4, h-j). In cells expressing SAP97 siRNA, precipitates of the beta1-AR failed to co-immunoprecipitate AKAP79 or SAP97 (Fig. 4, k-m). In addition, the expression of SAP97 was not detected either in the lysates or in the immunoprecipitates prepared from these cells, indicating that the siRNA effectively knocked down SAP97 levels (Fig. 4m). Therefore, because the SAP97 siRNA abolished the ability of the beta1-AR to co-immunoprecipitate AKAP79 and SAP97, it indicates that SAP97 is likely to serve as a bridging molecule between the beta1-AR and the AKAP79-PKA complex.


Figure 4
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FIGURE 4.
Role of SAP97 in the targeting of AKAP79-PKA complex to the beta1-AR microdomain; analysis by co-immunoprecipitation. a-c, co-immunoprecipitation of WT beta1-AR and SAP97 from HEK-293 cells. HEK-293 cells stably expressing 1.05 ± 0.12 pmol/mg FLAG-tagged WT beta1-AR or 1.2 ± 0.1 pmol/mg FLAG-tagged beta1-AR{Delta}PDZ were transiently transfected with Myc-SAP97 as indicated. Lysates from these cells were immunoprecipitated (IP) with anti-Myc or anti-FLAG epitope antibodies. Precipitates were immunoblotted (IB) for beta1-AR and SAP97 as indicated. SAP97 co-immunoprecipitated with WT beta1-AR, but not with beta1-AR{Delta}PDZ. d and e, co-immunoprecipitation of beta1-AR and AKAP79-PKA complexes from HEK-293 cells. Lysates from HEK-293 cells transfected with Myc-SAP97 and FLAG WT beta1-AR were immunoprecipitated with FLAG antibodies. AKAP79 and the RII{alpha}-subunit of PKA co-immunoprecipitated with the WT beta1-AR. f and g, co-immunoprecipitation of AKAP79 and SAP97 from HEK-293 cells. Lysates from HEK-293 cells transfected with Myc-SAP97 and FLAG AKAP79 were immunoprecipitated with FLAG or Myc antibodies. AKAP79 co-immunoprecipitated with SAP97. h-m, knockdown of SAP97 expression using siRNA eliminates the binding of FLAG-AKAP79 to Myc-beta1-AR in HEK cells (k-m), but the interaction between AKAP79 and the beta1-AR is not eliminated in cells expressing the scrambled siRNA (h-j). In all experiments, lysates represented 5% of the total extract, whereas immunoprecipitations represent 30% of the total volume.

 
Characterization of the Association between the beta1-AR, SAP97, and AKAP79 by FRET Microscopy—Fluorescence confocal and FRET microscopy provide data relevant to the cellular distribution and proximity of the proteins under study (1, 17-19). To determine whether SAP97 and the beta1-AR interacted with one another, WT beta1-AR-CFP or -YFP and SAP97-YFP or -CFP were generated. The binding and Gs-coupling parameters of beta1-AR-CFP and beta1-AR-YFP were determined by radioligand binding and adenylyl cyclase assays (Table 1 and Fig. 2C). These chimera bound ICYP with affinities (KD) comparable with that of the WT beta1-AR (Table 1). beta1-AR-CFP and beta1-AR-YFP chimera displayed basal activities of adenylyl cyclase that were comparable with that of the WT beta1-AR (Fig. 2C). Moreover, isoproterenol generated a graded escalation in the activity of adenylyl cyclase in chimeric receptors that culminated in an ~6-fold increase in its activity, with EC50 values that were comparable with those of the WT beta1-AR (Table 1).

Next, WT beta1-AR-CFP and SAP97-YFP were cotransfected into HEK-293 cells, and their interaction was assessed by acceptor photobleaching FRET microscopy (Fig. 5A). The imaging data indicated that the interactions between the WT beta1-AR and SAP97 were strong and displayed FRET efficiencies of 24 ± 5.0% (Table 2). To assess the influence of the PDZ site on the distribution of the beta1-AR and its interaction with SAP97, FRET interaction efficiencies between the beta1-AR{Delta}PDZ-CFP and SAP97-YFP were analyzed by FRET microscopy (Fig. 5D). Data from several experiments did not reveal FRET interactions between the beta1-AR{Delta}PDZ and SAP97, confirming that the interaction between SAP97 and the beta1-AR was mediated through the PDZ. Similarly, AKAP79 interacted with the full-length WT beta1-AR with a moderate FRET efficiency of 12 ± 1.1% (Fig. 5B), and this interaction was abolished when the PDZ site was mutated (Fig. 5E). Finally, SAP97 and AKAP79 interacted with a FRET efficiency of 19-21%, indicative of high affinity interactions (Fig. 5C and Table 2). Previously we have shown by FRET microscopy strong interactions between the RII{alpha} subunit of PKA and AKAP79 and between RII{alpha} and the WT beta1-AR (1). These data complement the immunoprecipitation results in Fig. 4 that have shown binding between the SAP97-AKAP79-PKA complex and the WT beta1-AR.


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TABLE 2
FRET efficiencies in (%) as recorded by the acceptor photobleaching method in fixed cells

FRET efficiency was calculated using area averages for donor (D) before and after bleaching. FRET = (D after - D before) / D after. Donor (D) and acceptor (A) threshold values were subtracted from all pixels before FRET calculation. The mean values of FRET efficiencies calculated for at least 10 specific regions of interest in each FRET pair from seven experiments were collected and analyzed with FRET tool software for LSM510 version 3.2.

 
Additionally, we used FRETN microscopy to determine the efficiency of molecular interactions between the WT beta1-AR and the SAP97-AKAP79 complex in live cells (Fig. 6). This method generates high resolution, real time images of sensitized emission FRET using image subtraction, which allows comparison of FRET from multiple cells for a given acceptor-donor pair (18, 19). FRETN showed strong association between the WT beta1-AR and SAP97 (FRETN efficiency of 23 ± 5%). Furthermore, strong interactions between SAP97 and AKAP79 (FRETN efficiency = 21 ± 2%) and between the WT beta1-AR and AKAP79 (FRETN efficiency = 12 ± 1.1%) were observed (Fig. 6, B and C). Finally, in agreement with Nakagawa et al. (20), we confirmed the existence of SAP97 dimers because SAP97-CFP interacted with SAP97-YFP with a high FRET efficiency of 19.7 ± 3% (Fig. 6D).

Effect of the beta1-AR PDZ and SAP97 on Agonist-mediated Phosphorylation of the beta1-AR—To study the role of the beta1-AR PDZ and its associated scaffold in signaling by the beta1-AR, we determined whether inactivation of the beta1-AR PDZ or knockdown of SAP97 produced comparable effects on beta-agonist-mediated phosphorylation of the beta1-AR. Cell lines stably expressing comparable levels of FLAG-tagged WT beta1-AR (1.05 ± 0.12 pmol/mg) and of FLAG-tagged beta1-AR{Delta}PDZ (1.2 ± 0.1 pmol/mg) were used. Isoproterenol-mediated phosphorylation of the WT beta1-AR or beta1-AR{Delta}PDZ increased total phosphorylation of the WT beta1-AR by 6-fold and that of the beta1-AR{Delta}PDZ by 2-fold (Fig. 7A). These experiments were also performed in cells expressing the FLAG-tagged WT beta1-AR with scrambled or SAP97 siRNAs. Isoproterenol increased total phosphorylation of the WT beta1-AR by 5.7-fold in control or in cells expressing the scrambled siRNA (Fig. 7A, compare lanes 1 to 2 and 5 to 6). However, in cells expressing the beta1-AR{Delta}PDZ or in those co-expressing the WT beta1-AR and SAP97 siRNA, isoproterenol increased the phosphorylation of the beta1-AR by ~2.5-fold (Fig. 7A, compare lanes 3 to 4 and 7 to 8). Thus, inactivation of the beta1-AR PDZ or knockdown of SAP97 was roughly equivalent in inhibiting the phosphorylation of the beta1-AR by isoproterenol.


Figure 5
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FIGURE 5.
Acceptor photobleaching analysis of FRET interactions between SAP97, AKAP79, and thebeta1-AR. A and B, cells co-expressing the WT beta1-AR-CFP with either SAP97-YFP or AKAP79-YFP were photobleached, and FRET was recorded by examining the loss of quenching of CFP during acceptor (YFP) photobleaching. FRET was observed between the WT beta1-AR and SAP97 and between the WT beta1-AR and AKAP79, with a FRET efficiency of 24±5 and 12 ± 1.1%, respectively. C, FRET signals between AKAP79 and SAP97 were also recorded with FRET efficiency between 19 and 21%. These data verify that interactions between WT beta1-AR, AKAP79, and SAP97 have occurred with relatively high efficiency. D and E, however, in cells co-expressing the beta1-AR{Delta}PDZ-CFP with either SAP97-YFP or AKAP79-YFP no FRET interactions were observed, indicating that mutagenesis of the PDZ domain of the beta1-AR abrogated the interactions between the beta1-AR and either SAP97 or AKAP79. The color ruler shows the relationship between the pseudo-FRET color and the corresponding FRET efficiency reported in Table 2.

 
The two major kinases that are involved in agonist-mediated phosphorylation of the beta1-AR are the GRK and PKA (27). Thus, the underlying cause for reduced phosphorylation of the beta1-AR{Delta}PDZ might be due either to inhibition of GRK, PKA, or both. The preferred substrates for phosphorylation by PKA are serine/threonine residues that are preceded by RX- or RRX-(where X is any amino acid and R is arginine). This organization is found solely around Ser312 in the 3rd IC, which corresponds to RRPS312 (11). The preferred substrates for phosphorylation by GRK are serine/threonine residues that are preceded by an acidic amino acid (28, 29). This organization is found around four serine residues that reside exclusively in the carboxyl terminus of the beta1-AR (27). To identify the kinase affected by knockdown of SAP97 or by the inactivation of the PDZ, the phosphorylation of the carboxyl terminus versus that of the 3rd IC should be independently determined. Therefore, the phosphorylated beta1-AR was cleaved with cyanogen bromide that cleaves the full-length beta1-AR into a 10-kDa 32P-labeled peptide that encompasses the 3rd IC and into a 15-kDa 32P-labeled peptide that encompasses the carboxyl terminus of the beta1-AR (1). Previously we have shown that isoproterenol increased the phosphorylation of the 10- and 15-kDa peptides by ~6-fold (1). However, the counts/min derived from cyanogen bromide cleavage of an equivalent amount of receptor protein from cells pre-exposed to ascorbic acid were insufficient to accurately estimate the incorporation of 32P into these peptides under basal conditions (1). Therefore, the 32P-labeled beta1-ARs on nitrocellulose filters were digested with cyanogen bromide, and the amounts of 32P incorporated into the resulting peptides were determined. Then an equal number 32P counts/min derived from isoproterenol- and ascorbic acid-treated cells were subjected to electrophoresis on 16% acrylamide/Tricine gels (Fig. 7B). In the samples derived from cells pre-exposed to ascorbic acid, we observed that the 10- and 15-kDa peptides were phosphorylated at a ratio of ~1:3 (Fig. 7B, lane 1). Isoproterenol increased the total phosphorylation of each peptide by ~6-fold (1) but did not alter their phosphorylation ratios, indicating that both the 10- and 15-kDa peptides were substrates for isoproterenol-mediated phosphorylation (Fig. 7B, lane 2). Cleavage of 32P-beta1-AR{Delta}PDZ from control or isoproterenol-treated cells, followed by loading the same number of 32P counts/min as in Fig. 7B, lanes 1 and 2, generated the 15-kDa phosphopeptide only (Fig. 7B, lanes 3 and 4). Thus, mutagenesis of the PDZ abrogated basal and isoproterenol mediated phosphorylation of the 10-kDa peptide.


Figure 6
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FIGURE 6.
Sensitized emission FRET microscopy between SAP97, AKAP79, and the WT beta1-AR. A-D, live cells co-expressing the indicated YFP- and CFP-tagged constructs were imaged at room temperature in glass-bottom Petri dishes. FRETN is presented in pseudo-color. Normalized FRETN values were calculated using LSM510 Macro 1.5 FRET software using the following equation: FRETN = (FRET1/Dfd x Afa) {infty} ([bound]/[total d] x [total a]) that was described under "Experimental Procedures." The equation indicates the proportional ({infty}) relationship between FRETN and the concentrations of the interacting and noninteracting species. In the equation [bound] represents the concentration of interacting pairs of donor-labeled species and acceptor-labeled species. The values for [total d] and [total a] represent the total concentrations (interacting and noninteracting) of the donor- and acceptor-labeled species, respectively. FRET1 is proportional to the FRET signal from the specimen. Dfd is the donor signal that would take place if no FRET occurred and is therefore proportional to the total concentration of the donor. Afa is the acceptor signal that would take place if no FRET occurred and is therefore proportional to the total concentration of the acceptor. FRETN values for the various constructs are presented in Table 2.

 


Figure 7
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FIGURE 7.
Effect of the PDZ or SAP97 siRNA on isoproterenol-mediated phosphorylation of the beta1-AR. A, isoproterenol (10 µM) treatment of cells expressing the WT beta1-AR resulted in a 5.7-fold increase in phosphorylation (lanes 1 and 2), whereas the beta1-AR{Delta}PDZ showed reduced phosphorylation in response to isoproterenol (lanes 3 and 4). siRNA-mediated knockdown of SAP97 also reduced the phosphorylation of the WTbeta1-AR (lanes 7 and 8) when compared with the effect of scrambled siRNA on this parameter (lanes 5 and 6). Electronic counting of the counts/min of 32P incorporated into the beta1-AR was as follows: lane 1, 135; lane 2, 768; lane 3, 138; lane 4, 330; lane 5, 153; lane 6, 780; lane 7, 137; and lane 8, 326. B, phosphorylated WT beta1-AR or beta1-AR{Delta}PDZ was cleaved with cyanogen bromide, lyophilized, and then redissolved in Tricine sample buffer, and 32P incorporated into their peptides was determined as described under "Experimental Procedures." Equal counts/min (1,200 ± 50) of 32P from control-treated cells (ascorbic acid) or isoproterenol-treated cells, harboring WT beta1-AR (lanes 1 and 2) or the beta1-AR{Delta}PDZ (lanes 3 and 4), were subjected to electrophoresis on 16% acrylamide gels in Tricine cathode buffer. These experiments were repeated in cells expressing the WT beta1-AR with scrambled (Scr, lanes 5 and 6) or SAP97 siRNAs (lanes 7 and 8). Electronic counting of 32P incorporated in lane 2 indicated that the % of the counts/min in the 10-versus the 15-kDa band was 34% (190-571 cpm, respectively). In lanes 3 and 4, ~653 cpm were counted in each lane that was exclusively located in the 15-kDa band. In lanes 5 and 6, the % counts/min in the 10-versus the 15-kDa band was 30% (202-623 cpm, respectively). In lanes 7 and 8, ~693 cpm were counted in each lane that was exclusively located in the 15-kDa band.

 
The next series of experiments was conducted in cells expressing the WT beta1-AR with scrambled or SAP97 siRNA (Fig. 7, lanes 5-8). We have shown that siRNA-mediated knockdown of AKAP79 inhibited the phosphorylation of the 10-kDa peptide in response to isoproterenol (1). However, we were not able to estimate the effect of the AKAP79 siRNA on the peptides derived from ascorbic acid-treated cells. Thus, the 32P-labeled WT beta1-AR from control or isoproterenol-treated samples was cleaved, and equal amounts of 32P counts/min as in Fig. 7B, lanes 1-4, were subjected to electrophoresis. The 32P-WT beta1-AR derived from control or isoproterenol-treated cells that expressed the scrambled siRNA generated the 10- and 15-kDa phosphopeptides at a ratio of ~1:3 (Fig. 7B, lanes 5 and 6). However, the 32P-labeled WT beta1-AR derived from control or isoproterenol-treated cells that expressed the SAP97 siRNA generated the 15-kDa phosphopeptide only (Fig. 7B, lanes 7 and 8). Therefore, inactivation of the beta1-AR PDZ or knockdown of SAP97 both inhibited the phosphorylation of the 10-kDa peptide derived from the 3rd IC that contains the putative PKA-Ser312 substrate. These results buttress our claim that inactivation of the PDZ inhibited the targeting of the AKAP79-PKA complex to the beta1-AR and inhibited PKA-mediated phosphorylation of Ser312 in the 3rd IC.


Figure 8
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FIGURE 8.
Roles of the ESKV sequence in the carboxyl-terminal tail of the beta1-AR and Ser312 in the 3rd IC in regulating the recycling and resensitization of the beta1-AR in response to isoproterenol. A, recycling of the beta1-AR{Delta}PDZ in response to 10 µM isoproterenol (Iso). HEK-293 cells stably expressing the beta1-AR{Delta}PDZ were mock-transfected (pcDNA, images a-h) or transfected with an expression vector for the catalytic subunit of PKA (images i-p). In addition, HEK-293 cells were transiently transfected with the point mutant of beta1-AR{Delta}PDZ in which the serine residue at position 312 was mutated to aspartic acid (S312D beta1-AR{Delta}PDZ, images q-y). Recycling of the beta1-AR in response to 10 µM isoproterenol (n = 3) for 30 min followed by an acid wash (A/W) to remove the antibody bound to extracellular beta1-AR were conducted as described under "Experimental Procedures." Each scale bar represents 5 µm. B, summary of the results of the experiment in A are presented as line graphs of n = 3. The isoproterenol-internalized beta1-AR{Delta}PDZ did not recycle (images d-h), whereas the beta1-AR{Delta}PDZ with cPKA and the S312D beta1-AR{Delta}PDZ recycling was rapid with a t0.5 of 25 ± 5 and 18 ± 4 min, respectively. C, comparison of adenylyl cyclase activities in response to short term isoproterenol (short term desensitization) and in response to isoproterenol followed by antagonist (desensitization followed by resensitization) treatments in HEK-293 cells expressing WT, {Delta}PDZ, S312D, and S312D{Delta}PDZ constructs of the beta1-AR. These experiments were replicated (n = 3) each in triplicate.

 
Cross-talk between the beta1-AR PDZ Scaffold and Ser312—Two distinct domains in the beta1-AR, namely Ser312 in the 3rd IC and the PDZ in the carboxyl-terminal tail, participate in imparting a recycling signal to the beta1-AR. If, as suggested earlier, one of the functions of the PDZ domain is to target PKA to the beta1-AR, then we hypothesized that overexpression of PKA might overcome the effect of inactivating the PDZ on recycling of the beta1-AR. To address this question, the recycling experiment for beta1-AR{Delta}PDZ was conducted under conditions of increased PKA activation (Fig. 8A). HEK-293 cells expressing 1.2 ± 0.1 pmol/mg of FLAG-beta1-AR{Delta}PDZ were transfected with the empty vector or with the vector expressing cPKA. In cells expressing the empty vector, the agonist-internalized beta1-AR{Delta}PDZ did not recycle (Fig. 8A, images d-g). In cells expressing cPKA, the beta1-AR{Delta}PDZ recycled back to the cell surface within 45 min from the removal of isoproterenol (Fig. 8A, images l-o). A boundary was drawn around the inner circumference of the cells in Fig. 8A in order to determine the distribution of pixels between membranous and intracellular compartments. The density of the pixels residing inside the boundary versus those residing outside the boundary was used as an index for internalized and membranous beta1-AR, respectively. The pixel data were plotted as a function of time after the removal of isoproterenol in order to calculate the recycling kinetics of the beta1-AR (Fig. 8B). The data indicate that in cells expressing the beta1-AR{Delta}PDZ with the empty pcDNA vector, the internalized beta1-AR{Delta}PDZ did not recycle (Fig. 8A, images d-g). However, in cells expressing the beta1-AR{Delta}PDZ and cPKA, the agonist-internalized beta1AR{Delta}PDZ recycled with a t0.5 = 20 ± 5 min, indicating that super induction of cPKA overcame the effect of the PDZ mutation on recycling of the beta1-AR. If the recycled beta1-AR{Delta}PDZ was inserted properly into the cell membrane, then Cy3-conjugated anti-FLAG IgG bound to the amino-terminal FLAG epitope should be oriented extracellularly. In this case, a second acid wash would strip Cy3-IgG from the recycled receptor population. In agreement with these assumptions, we observed reduced cell fluorescence in the recycled beta1-AR{Delta}PDZ in cPKA expressing cells, but not in pcDNA expressing cells (Fig. 8A, compare image p with h). It should be emphasized however, that a 30-min pretreatment with forskolin, which activates all the isoforms of adenylyl cyclase and markedly activates PKA, did not restore the recycling phenotype to beta1-AR{Delta}PDZ (data not shown), suggesting that chronic activation of PKA might be necessary to restore the recycling of the beta1-AR{Delta}PDZ.

If one of the functions of the beta1-AR PDZ is to facilitate the phosphorylation of Ser312 by targeting PKA to the beta1-AR, then it is logical to hypothesize that replacement of Ser312 with the phosphoserine mimic aspartic acid would restore the recycling of the beta1-AR{Delta}PDZ. This hypothesis was tested directly by generating a beta1-AR{Delta}PDZ construct in which the serine at position 312 was mutated to aspartic acid (S312D{Delta}PDZ). Indeed the agonist-internalized S312D{Delta}PDZ recycled efficiently (Fig. 8A, images t-w), with kinetics comparable with those of the WT-beta1-AR (t0.5 15 ± 4 min) (Fig. 8B). Because recycling of the agonist-desensitized and internalized GPCR is a priori for its resensitization, we determined whether beta1-AR-mediated activation of adenylyl cyclase was functionally resensitized in those beta1-AR{Delta}PDZ constructs that were capable of recycling (Fig. 8C). Rapid desensitization of adenylyl cyclase in membranes expressing all the four beta1-AR constructs described in Fig. 8C was observed after 10 min of exposing the cells to isoproterenol, indicating that mutagenesis of either the PDZ or Ser312 alone or in combination did not affect short term desensitization of the receptor. The resensitization assay involves the desensitization of the beta1-AR by exposing cells to isoproterenol for 3 h, followed by incubating the cells with 100 µM of the beta-antagonist alprenolol to induce the recycling of the beta1-AR and subsequent resensitization of its adenylyl cyclase activity (1, 11). In this assay, we observed the resensitization of adenylyl cyclase activity in the WT beta1-AR, but the activity of adenylyl cyclase of the beta1-AR{Delta}PDZ was significantly desensitized (Fig. 8C). Functional resensitization of adenylyl cyclase activity of the beta1-AR{Delta}PDZ was restored in the context of S312D{Delta}PDZ construct, indicating that the modification of Ser312 to its phosphoserine mimic "aspartic acid" resuscitated the recycling and resensitization of beta1-AR{Delta}PDZ.

Characterization of the Role of SAP97 in Recycling of the Human beta1-AR—Thus far, we have shown that cross-talk between the beta1-AR PDZ domain and Ser312 was involved in regulating the recycling and resensitization of the beta1-AR in HEK-293 cells. To determine the role of SAP97 in this phenomenon, the effects of SAP97 knockdown and overexpression on recycling of the WT beta1-AR were assessed (Figs. 9 and 10). In cells stably expressing 1.1 pmol/mg protein of WT beta1-AR-YFP, knockdown of SAP97 inhibited the recycling of the WT beta1-AR as determined by the confocal recycling assay (Fig. 9A, images i-l). However, knockdown of SAP97 had variable effects on agonist-induced internalization of the beta1-AR as well, whereas in some cells SAP97 had no effect on internalization, and in others it reduced the internalization by ~35% (compare internal pixels in Fig. 9A, images h versus b). Therefore the effect of knockdown of SAP97 on trafficking of the beta1-AR was determined by surface biotinylation because in this assay the internalization and recycling data are derived from the entire cell population rather than from few imaged cells (1). HEK-293 cells stably expressing FLAG-tagged WT beta1-AR with scrambled or SAP97 siRNA were surface-biotinylated with cleavable biotin followed by quenching of excess biotin with glycine. The amount of biotin incorporated into the beta1-AR under this condition indexed total cellular beta1-AR biotinylation (Fig. 9B, lanes 1 and 6). The cells were then exposed to isoproterenol for 30 min, followed by cleavage of the remaining cell surface biotin (Fig. 9B, lanes 2 and 7). The amount of biotin recovered in this step indexed the amount of biotinylated beta1-AR that was internalized in response to isoproterenol, whereas the ratio of internal to total biotin indexed the percentile of total receptors that were internalized. Isoproterenol induced the internalization of ~60% of total beta1-AR in control cells, whereas the internalization of the beta1-AR in SAP97 siRNA-treated cells was reduced by ~20 (n = 4). To initiate recycling, isoproterenol was replaced with the beta-antagonist alprenolol, and the cells were warmed to 37 °C for an additional 15, 30, or 60 min (Fig. 9B, lanes 3-5 and 8-10). After each time period, the cells were cooled to 4 °C, and biotin was cleaved for the second time to ensure cleavage of any newly appearing "recycled" surface biotin. Thus, the loss of biotin from the second cleavage step indexed the recycling of the beta1-AR. The data indicate that by 60 min, more than 90% of the biotin was lost from the beta1-AR in cells expressing the scrambled siRNA, reflecting membrane recycling and subsequent biotin cleavage (Fig. 9B, lanes 3-5). In contrast, the internalized (biotinylated) beta1-AR in cells expressing SAP97 siRNA was not changed even after 1 h from the removal of isoproterenol, reflecting their internal distribution (Fig. 9b, compare lanes 9 and 10 with lanes 4 and 5). Next, we quantified the amount of biotin remaining as a function of time after the removal of isoproterenol and determined that the beta1-AR recycled with a t0.5 of 25 ± 5 min (Fig. 9C).

In follow-up experiments, the effect of overexpression of Myc-SAP97 or Myc-PSD-95 in cells stably expressing 1.1 ± 0.2 pmol/mg protein of WT beta1-AR-CFP on isoproterenol-mediated beta1-AR internalization and recycling was determined by confocal microscopy (Fig. 10A). The rationale for analyzing the effect of PSD-95 is that PSD-95 binds to the beta1-AR PDZ with high affinity and interferes with the internalization of the receptor, but its effect on recycling is unknown (26). However, because HEK-293 cells do not express PSD-95 (Fig. 10B), PSD-95 was overexpressed along with SAP97. Overexpression of SAP97 did not affect the rate or magnitude of isoproterenol-mediated internalization of the beta1-AR (t0.5 = 5 min ± 1 min). On the other hand, overexpression of PSD-95 markedly inhibited the magnitude (-50%) and rate of isoproterenol-mediated internalization of the WT beta1-AR (Fig. 10, A, images u-z, and C). Overexpression of SAP97 did not affect the rate or magnitude of beta1-AR recycling, but PSD-95 reduced both the rate and the magnitude of beta1-AR recycling (Fig. 10C). Thus, these MAGUK proteins exerted different effects on internalization, recycling, and resensitization of the beta1-AR. Finally, knockdown of AKAP79 or SAP97 in conjunction with FRET microscopy was conducted to determine the organization of the scaffolding complex that binds to the beta1-AR PDZ (Fig. 11). Knockdown of AKAP79 did not prevent the association between the WT beta1-AR-YFP and SAP97-CFP as assessed by acceptor photobleaching FRET microscopy (Fig. 11A). Knockdown of SAP97, however, abolished the interaction between WT beta1-AR and AKAP79, indicating that SAP97 served as bridging molecule between the beta1-AR and the AKAP79-PKA complex (Fig. 11, B and C).


Figure 9
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FIGURE 9.
Effect of down-regulating the expression of SAP97 on recycling of the WT beta1-AR as determined by confocal and cell-surface biotinylation recycling assays. A, recycling assay of WT beta1-AR-YFP in HEK-293 cells where SAP97 expression was unaffected (scrambled (Scr) siRNA, upper panel) or knocked down using the SAP97 siRNA approach (lower panel). B, recycling of surface-biotinylated WT beta1-AR in cells expressing the scrambled (lanes 1-5) or the SAP97 siRNA (lanes 6-10). C, rate of biotinylated beta1-AR recycling in cells in which the expression of SAP97 was knocked down versus those with control SAP97 levels. Internalized WT beta1-AR did not recycle in HEK-293 cells in which SAP97 was knocked down, whereas it recycled with t0.5 of 25 ± 5 min in scrambled siRNA controls.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type 1 PDZ domains in the tails of GPCR such as the beta1-or the beta2-AR are necessary for efficient recycling of these receptors (10, 30-32). The type 1 PDZ in the beta2-AR (DSLL) interacts with the PDZ-binding domain in the Na+/H+ exchanger regulatory factor ((NHERF) also known as ezrin-radixin-moesin (ERM)-binding phosphoprotein-50 (EBP50)), and this interaction is required for recycling of the beta2-AR (31). Similarly, inactivation of the ESKV sequence of the beta1-AR by a variety of point mutations generated beta1-AR mutants that, although efficiently internalized in response to isoproterenol, were not recycled (10, 32). The type 1 PDZ in the human beta1-AR has attracted wide attention because it interacted with numerous proteins related to the MAGUK family, and these interactions exerted different effects on its functions. The association of the beta1-AR with the MAGUK protein PSD-95, for example, inhibited the internalization of the beta1-AR but facilitated its interaction with N-methyl-D-aspartate (NMDA) receptors (26). Another family of PDZ-interacting proteins was the MAGI-related proteins, which increased the magnitude of agonist-induced internalization of the beta1-AR (25, 33). Two additional PDZ-binding proteins were shown to interact with the type 1-PDZ in the carboxyl-terminal tail of the beta1-AR. The first was a protein called the beta1-AR-binding partner, which is involved in regulating beta1-AR-mediated activation of extracellular signal-regulated kinases 1/2 (34). The other was the cystic fibrosis transmembrane conductance regulator-associated ligand, which is involved in surface expression of the beta1-AR (35).

These MAGUK proteins, however, are static entities because they lack ATP-binding and catalytic core motifs and are thought to function as adaptor proteins (23). Therefore, in addition to identifying SAP97 as a novel MAGUK protein that interacts with the beta1-AR PDZ, we have confirmed that an AKAP79-PKA complex binds to SAP97. This novel organization generates a dynamic MAGUK-beta1-AR complex with ATP binding and catalytic core motifs to create a novel beta1-AR signalosome that broadens the range of functions attributed to this receptor.


Figure 10
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FIGURE 10.
Effect of overexpression of SAP97 or PSD95 on isoproterenol-mediated internalization and recycling of the WT-beta1-AR. A, cells stably expressing 1.1 pmol/mg protein of WT beta1-AR-YFP were transfected with the empty pcDNA 3.1 vector (upper panel), Myc-SAP97 (middle panel), or Myc-PSD95 (lower panel) to determine the effects of MAGUK proteins on the internalization and recycling of the WT beta1-AR. B, blots of HEK-293 cell extracts (5 µg in each lane) prepared from mock (pcDNA 3.1), Myc-SAP97, or Myc-PSD95 transfected cells. IB, immunoblot. C, quantification of WT beta1-AR internalization and recycling in control HEK-293 cells or in cells overexpressing SAP97 or PSD95. The effect of isoproterenol on the distribution of YFP pixels outside versus those inside a 300-nm boundary in images a-f, k-p, and u-z, was determined. Because the number of pixels inside the boundary progressively increased after the addition of isoproterenol, this was reflected as a reduction in the percentile of membranous receptors in the + isoproterenol images. After the initiation of recycling (-isoproterenol), there was a progressive decline in the pixels inside the 300-nm boundary and a corresponding increase in the pixels outside this boundary in images g-j, q-t, and a'-d'. The data are derived from n = 2 experiments each utilizing 5-7 cell images per time point.

 
In addition to its assigned role in recycling of the agonist-internalized beta1-AR, a central function of this beta1-adrenergic receptosome is to ensure potency, fidelity, and reversibility of beta1-AR signaling. Potency and fidelity of signaling are ensured by high affinity connections between the PDZ and the various members of the scaffold. These interconnections ensure that cyclic AMP, generated by agonist-mediated activation of the receptor-Gs-adenylyl cyclase axis, activates the PKA bound to the PDZ domain of the beta1-AR (Fig. 11C). Activated PKA phosphorylates Ser312 in the 3rd IC of the beta1-AR as well as any acceptor protein bound to the MAGUK or AKAP members of the scaffold (Fig. 11C). The cyclic AMP signal is quickly terminated by phosphodiesterases bound to AKAP79 or somewhere else in this scaffold (36). In addition to activating the Gs-adenylyl cyclase axis, agonist binding to the beta1-AR causes conformational changes in the receptor that facilitate GRK-mediated phosphorylation of acidotrophic residues such as Ser475 at position -2 in the middle of the beta1-AR PDZ (28, 29). These modifications promote physical separation between the scaffold and the beta1-AR PDZ and facilitate the internalization of the receptor (1) (Fig. 3F). Thus, functional synergism between homologous desensitization through GRK and homologous resensitization through PKA are capable of generating several cycles of receptor activation, desensitization, and recovery, which potentially can maintain the signaling output from cells with low density of beta1-AR.

In the model described above, we proposed that one of the functions of the beta1-AR PDZ was to target PKA to the beta1-AR to facilitate the phosphorylation of Ser312. This is a novel idea because it hypothesizes that a major function of the PDZ-associated scaffold is the transmission of signaling information. This suggestion is novel because the hypothesis of Gage et al. (10), which states "that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCR," does not take into account the involvement of other downstream elements in GPCR recycling. Thus, we proceeded to test our hypothesis that "PDZ domain-mediated protein interactions transmit recycling signal(s) that promote rapid recycling of the GPCR." At first we confirmed the involvement of the beta1-AR PDZ, SAP97, in addition to AKAP79 (1) in phosphorylating Ser312, thereby providing a vivid example of cross-talk between a domain in the extreme carboxyl terminus and another in the 3rd IC of the GPCR (Figs. 7 and 11C). If the phosphorylation of Ser312 lies downstream from the PDZ in setting the trafficking itinerary for the beta1-AR, then the PDZ-generated recycling signal, which is phospho-Ser312, could be replicated either by super-induction of PKA or by mutagenesis of Ser312 in the 3rd IC of the beta1-AR to the phosphoserine mimic aspartic acid (S312D). Our data in Fig. 8 showed that the beta1-AR{Delta}PDZ would recycle under conditions where the catalytic activity of PKA was chronically elevated or when its putative target Ser312 in the beta1-AR{Delta}PDZ mutant was replaced by its phosphoserine mimic aspartic acid. These findings imply that Ser312 is downstream from the PDZ and apparently occupies a more dominant position than the type 1 PDZ in setting the trafficking itinerary of the beta1-AR. We arrived at this conclusion because our model extended beyond the binary MAGUK-PDZ model into a quaternary model that incorporates an AKAP, which is involved in targeting PKA to the beta1-AR microdomain (Fig. 11C).


Figure 11
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FIGURE 11.
Characterization of the WT-beta1-AR, SAP97, and AKAP79 scaffold by acceptor photobleaching FRET microscopy. A, FRET was obtained in HEK-293 cells expressing WT beta1-AR-YFP and SAP97-CFP when AKAP79 was knocked down with siRNA. B, FRET was not obtained in HEK-293 cells expressing WT beta1-AR-YFP and AKAP79-CFP when SAP97 was knocked down with siRNA. C, organization of the beta1-AR receptosome. Based upon our data, we hypothesize that the beta1-AR is in a macromolecular complex with SAP97, AKAP79, and the PKA tetramer. Agonist-induced activation of the beta1-AR generates cyclic AMP, which binds to PKA and releases the cPKA that phosphorylates Ser312 in the 3rd IC of the beta1-AR. In addition, cPKA might phosphorylate other targets that are brought into the vicinity of the beta1-AR microdomain through their association with AKAP79 or SAP97.

 
The role of PKA targeting in setting the trafficking itinerary of GPCR is a nascent field with relatively few reports. The role of PKA in translocating aquaporin-2 from intracellular compartments into cell membranes in response to vasopressin and in recycling of agonist-internalized NMDA receptors is well documented (15, 37). Nevertheless, beyond these few examples, the significance of PKA and its targeting to the PDZ motif in trafficking of the agonist-internalized GPCR remains to be substantiated. Recently, the EBP-50-binding protein ezrin was identified as an AKAP-like protein, which binds to the RI subunit of PKA, instead of the more common RII subunit (38). These data suggest that EBP-50, which binds to the beta2-AR PDZ and is required for the recycling of the beta2-AR, targets an AKAP-like protein to the beta2-AR PDZ. Similarly, aquaporin-2 has a type 1 "G(S/T)KA" PDZ sequence at its extreme carboxyl terminus, and its recycling requires EBP-50 and involves AKAP/PKA-mediated phosphorylation of Ser256 (37). These observations suggest that PDZ-mediated targeting of PKA to the GPCR might be more common than expected and could be involved in the recycling of these and other proteins.

The identification of SAP97 as an organizer of a scaffold composed of AKAP79 and PKA converts the PDZ-binding domain from a static multiprotein-binding complex into a dynamic network with increased number, range, and intensity of signals that are transmitted via the beta1-AR. The PDZ domains of the PSD/SAP family of MAGUK proteins interact with the carboxyl-terminal type 1 PDZ motif found in a variety of membrane and intracellular proteins, including Shaker K+ channels, NMDA receptors, and the beta1-AR, which binds to PDZ-3 domain of SAP97 (33, 39, 40). The Src homology 3 and guanylate kinase domains are involved in the clustering activity of SAP97 because they bind to the GKAP/PAPAP/DAP family of postsynaptic density proteins as well as to many other signaling/scaffolding proteins, including AKAP79 and other PKA-binding proteins (22, 41). SAP97 contains an additional L27 domain in its amino terminus that is involved in dimerization of SAP97 and in binding to other MAGUK proteins such as mLIN-2/CASK and DLG3 (20, 23). These protein-protein interactions can therefore diversify the signaling of the beta1-AR and may explain some of the neuronal and cardiovascular functions attributed to the beta1-AR. For example, myocardial beta1-ARs selectively phosphorylate via PKA many substrates that are involved in regulating myocardial contractility through AKAP-dependent mechanisms (42). In neurons, SAP97 and AKAP79 both interact with the GluR1 {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit, and the interaction between SAP97 and AKAP79 might recruit PKA to ionotropic glutamate receptors (17, 22). Thus it is conceivable that multiplexing between a beta1-AR-MAGUK-AKAP79 complex might scaffold {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, L-type Ca2+ channels, and other signaling molecules to the beta1-AR microdomain to facilitate their phosphorylation by the beta1-AR signaling pathway as has been observed in hippocampal neurons (43). These scaffold-mediated connections between the beta1-AR and voltage-gated channels, for example, might underlie the observed effects of the beta1-AR on synaptic plasticity (44, 45) and in the formation of emotionally charged memories that could result in post-traumatic stress disorders (46-48). The mechanism of beta1-selective beta-blockers in blocking the reconsolidation of traumatic memories, especially those following acts of terror, might be explained by the mélange of proteins that are scaffolded directly or indirectly with the beta1-AR through this novel receptosome (49, 50).


    FOOTNOTES
 
* This work was supported by Grant-in-aid 6071785 from the Southeastern Affiliate of the American Heart Association (to S. W. B.) and by an NIDDK grant from the National Institutes of Health (to A. P. N.). 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 Career investigator of the American Lung Association. Back

2 To whom correspondence should be addressed: Dept. of Pharmacology, the University of Tennessee Health Sciences Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-1503; Fax: 901-448-7206; E-mail: sbahouth{at}utmem.edu.

3 The abbreviations used are: GPCR, G protein-coupled receptors; beta1-AR, beta1-adrenergic receptor; WT, wild type; AKAP, A-kinase anchoring proteins; PDZ, PSD-95/DLG/ZO1; MAGUK, membrane-associated guanylate-kinase; PKA, cyclic AMP-dependent protein kinase; cPKA, catalytic subunit of cyclic AMP-dependent protein kinase; beta1-AR{Delta}PDZ, beta1-AR mutant in which the type 1 PDZ "ESKV" sequence is mutated to alanine; 3rd IC, third intracellular loop; ICYP, [125I]iodocyanopindolol; FRET, fluorescence resonance energy transfer; FRETN, normalized FRET; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NMDA, N-methyl-D-aspartate; BSA, bovine serum albumin. Back


    ACKNOWLEDGMENTS
 
We thank Steven Tavalin for providing SAP97, Carolyn Mathews at the Confocal Microscopy Facility for technical assistance and Danny Morse for the illustration.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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