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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 ₁-Adrenergic Receptor Generates a Receptosome Involved in Receptor Recycling and Networking^*

Lidia A. Gardner, Anjaparavanda P. Naren¹, and Suleiman W. Bahouth²

From the 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 ₁-adrenergic receptor (₁-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the ₁-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the ₁-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 ₁-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 ₁-AR. The PDZ and its scaffold were required for efficient recycling of the ₁-AR and for PKA-mediated phosphorylation of the ₁-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 ₁-AR PDZ mutant. Thus, trafficking signals transmitted from the PDZ-associated scaffold in the carboxyl terminus of the ₁-AR to Ser³¹² in the 3rd intracellular loop (3rd IC) were paramount in setting the trafficking itinerary of the ₁-AR. The data presented here show that a novel ₁-adrenergic receptosome is organized at the ₁-AR PDZ to generate a scaffold essential for trafficking and networking of the ₁-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 - and -adrenergic receptors. Among these receptors is the ₁-AR, which is coupled to the G_s-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 ₁-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 ₁-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 ₁-AR (10, 11). The ESKV tetrapeptide conforms to a type I (PSD-95/DLG/ZO1) PDZ ligand (i.e. X(S/T)X, where X at positions -1 and -3 is any amino acid, and 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 ₁-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 ₁-AR (11).

These results indicate that two distinct motifs are involved in recycling of the ₁-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 ₁-AR in HEK-293 and other cell lines (1). AKAP79 promoted the targeting of PKA to the ₁-AR by binding to the carboxyl-terminal 53 amino acids (between residues 425 and 477) of the ₁-AR (1). Here we report that the binding domain of AKAP79 to the ₁-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 ₁-AR. By scaffolding PKA to the ₁-AR, SAP97 facilitates PKA-mediated phosphorylation of Ser³¹², which is critical for trafficking of the internalized ₁-AR to membranes. These results indicate that a novel ₁-adrenergic receptosome is involved in recycling and resensitization of the ₁-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 ₁-AR—To allow rapid assessment of cell surface expression of the ₁-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 ₁-AR or the S312D mutant in the mammalian expression vector pcDNA3.1 (Invitrogen) (14). To generate the ₁-AR(425-441) and ₁-AR(425-463) constructs, the full-length ₁-AR cDNA was cut with SmaI, and the resulting 1.3-kb cDNA encoding ₁-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 ₁-AR-(1-424). The WT ₁-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 ₁-ARPDZ and the S312D-₁-ARPDZ, respectively. Sequences of all the epitope-tagged ₁-AR constructs were verified by automated dideoxy sequencing.

Construction of Fluorescently Tagged ₁-AR, SAP97, AKAP79, and RII Subunit—Amino-terminal fusions of the WT₁-AR and AKAP79 to CFP and YFP were described (1). CFP- or YFP-₁-ARPDZ 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 ₁-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 K_D and the B_max values for ICYP binding by parametric fitting of the data by using the Prism 4 software (GraphPad Corp.).

View this table: [in this window] [in a new window]   TABLE 1 Ligand binding properties of [125I] iodocyanopindolol to wild-type and mutated 1-AR stably expressed in HEK-293 cells Binding of ICYP to 0.5 µg of membranes derived from HEK-293 cells expressing the various 1-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).

Acid Strip Confocal Recycling Microscopy Protocol—HEK-293 cells expressing the FLAG- or Myc-tagged WT ₁-AR or ₁-ARPDZ 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 ₁-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 ₁-AR (1, 15, 16). Cultures were then incubated with culture medium supplemented with 100 µM of the -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 ₁-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, _ex = 514 nm, _em = 530LP; Cy3, _ex = 543 nm, _em = 560 BP) using the LSM-510 multitracking configuration.

FRET Microscopy—Double stable cell lines expressing AKAP79-CFP and ₁-AR-YFP or SAP97-CFP and ₁-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 ₁-AR, SAP97, AKAP79, or the RII -subunit of PKA were performed as follows. Cells stably expressing the indicated FLAG-tagged or Myc-tagged ₁-AR were lysed in radioimmune precipitation assay buffer (1), and the insoluble cellular debris was removed by centrifugation at 14,000 x g_av 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 subunit of PKA by Western blotting.

Pulldown Assays—The human ₁-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 ₂-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 g_av centrifugation of cell lysates, GST or₁-AR_c-tail or₂-AR_c-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% -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-₁-AR (WT and 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 ₁-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 ₁-AR construct.

Adenylyl Cyclase Assays for -AR Desensitization and Resensitization—HEK-293 cells stably expressing the various ₁-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 K_act ± S.E. for each ₁-AR was calculated using the Prism 4 program, and statistical comparisons were analyzed using Prism 4 and Instat programs (GraphPad Corp.).

View larger version (66K): [in this window] [in a new window]   FIGURE 1. Characterization of the site of AKAP79 binding to the 1-AR and its role in 1-AR recycling. A, co-immunoprecipitation of various Myc-tagged 1-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 1-AR (images a-e) or FLAG-1-ARPDZ, in which the last four amino acids in the carboxyl terminus of the 1-AR were mutated to alanine (images f-j), were cultured on glass bottom slides. Recycling of the WT 1-AR and the 1-ARPDZ in response to 10 µM isoproterenol (n = 3) was conducted as described under "Experimental Procedures." Each scale bar represents 5 µm.

Biotinylation Assay of ₁-AR Recycling with Cleavable Biotin—Cells expressing the WT ₁-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 g_av for 20 min at 2 °C. The membrane pellet was dissolved in lysis buffer supplemented with detergents and recentrifuged at 100,000 x g_av 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 ₁-AR.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Characterization of AKAP79 Binding to the ₁-AR—Myc-tagged ₁-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 ₁-AR that bound AKAP79 (Fig. 1A). Deletion of the amino acids between 425 and 441 in the carboxyl-terminal tail of the ₁-AR had little effect on the immunoprecipitation of AKAP79 by this ₁-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 ₁-AR partially overlapped with this sequence. Mutagenic inactivation () of each residue in the type 1 PDZ sequence (ESKV) between amino acids 474 and 477 to alanine (₁-ARPDZ) completely inhibited the interaction between the ₁-AR and AKAP79, confirming that the AKAP79 interacting site overlapped with the type 1 PDZ sequence. The interactions between AKAP79 and the ₁-AR are involved in recycling of the agonist-internalized ₁-AR back into the cell membrane in a process termed "resensitization" (1, 11). This process is involved in trafficking of the agonist-internalized ₁-AR back to the cell membrane (Fig. 1B, images a-e). Inhibition of the binding of AKAP79 to the ₁-AR by inactivating the PDZ prevented the recycling of the agonist-internalized ₁-ARPDZ (Fig. 1B, images f-j). In other experiments we determined that the binding parameters of [¹²⁵I]ICYP to the WT ₁-AR or to the ₁-ARPDZ were comparable (Table 1). The effect of mutagenesis of the PDZ on receptor coupling efficacy to G_s 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 ₁-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 ₁-AR or the ₁-ARPDZ, respectively (Fig. 2A). The EC₅₀ value for the accumulation of cyclic AMP in response to isoproterenol was 7 ± 1.5 nM for the WT ₁-AR and 9 ± 2nM for the WT ₁-ARPDZ (Fig. 2A; p > 0.05). Basal levels of adenylyl cyclase activities in membranes prepared from cells expressing the WT ₁-AR were 17 ± 3 pmol/min/mg, whereas in cells expressing the ₁-ARPDZ, 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 ₁-AR or the ₁-ARPDZ 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 PDZ mutation had no effect on the coupling efficacy of the ₁-AR to G_s.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Effect of point mutants in the PDZ or in Ser312 of the 1-AR and of the WT 1-AR-CFP and -YFP chimera on cyclic AMP accumulation or adenylyl cyclase activation in response to isoproterenol. A, cells stably expressing the levels of 1-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 1-AR or its point mutants, and chimera were determined. The EC50 values for isoproterenol in activating adenylyl cyclase for each 1-AR construct are reported in Table 1.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Identification of SAP97 as an interacting partner at the carboxyl-terminal type 1 PDZ of the 1-AR. A and B, 1-AR does not interact with AKAP79 in far Western blotting assays. FLAG-WT1-AR and FLAG-1-AR 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-1-AR but not with FLAG-1-ARPDZ in far Western assays. E, purified GST-1-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 1-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, ex = 514 nm, em = 530LP; Cy3, ex = 543 nm, em = 560 BP) using LSM-510 multitracking configuration. IB, immunoblot; CBB, Coomassie Brilliant Blue; Iso, isoproterenol.

Co-immunoprecipitations between SAP97 and a variety of ₁-AR constructs were used to determine whether SAP97 binds to the ₁-AR in a PDZ-dependent manner. FLAG-WT ₁-AR or its PDZ mutant was co-transfected into HEK-293 cells with Myc-SAP97 (Fig. 4, panel A, a-c). Precipitates of the WT ₁-AR, co-immunoprecipitated SAP97, and reciprocal immunoprecipitations of SAP97 precipitated the WT ₁-AR (Fig. 4, a and b), but precipitates prepared from cells expressing the ₁-ARPDZ failed to co-immunoprecipitate SAP97 (Fig. 4, panel A, c). Moreover, in precipitates of the WT ₁-AR, we detected co-immunoprecipitations of AKAP79 and the RII- 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 ₁-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 ₁-AR and AKAP79/PKA, consequently knockdown of SAP97 should destabilize the binding between the ₁-AR and AKAP79. Toward this, cells expressing 1.4 pmol/mg Myc-₁-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, ₁-AR precipitates co-immunoprecipitated the ₁-AR, AKAP79, and SAP97 (Fig. 4, h-j). In cells expressing SAP97 siRNA, precipitates of the ₁-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 ₁-AR to co-immunoprecipitate AKAP79 and SAP97, it indicates that SAP97 is likely to serve as a bridging molecule between the ₁-AR and the AKAP79-PKA complex.

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Role of SAP97 in the targeting of AKAP79-PKA complex to the 1-AR microdomain; analysis by co-immunoprecipitation. a-c, co-immunoprecipitation of WT 1-AR and SAP97 from HEK-293 cells. HEK-293 cells stably expressing 1.05 ± 0.12 pmol/mg FLAG-tagged WT 1-AR or 1.2 ± 0.1 pmol/mg FLAG-tagged 1-ARPDZ 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 1-AR and SAP97 as indicated. SAP97 co-immunoprecipitated with WT 1-AR, but not with 1-ARPDZ. d and e, co-immunoprecipitation of 1-AR and AKAP79-PKA complexes from HEK-293 cells. Lysates from HEK-293 cells transfected with Myc-SAP97 and FLAG WT 1-AR were immunoprecipitated with FLAG antibodies. AKAP79 and the RII-subunit of PKA co-immunoprecipitated with the WT 1-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-1-AR in HEK cells (k-m), but the interaction between AKAP79 and the 1-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.

Next, WT ₁-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 ₁-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 ₁-AR and its interaction with SAP97, FRET interaction efficiencies between the ₁-ARPDZ-CFP and SAP97-YFP were analyzed by FRET microscopy (Fig. 5D). Data from several experiments did not reveal FRET interactions between the ₁-ARPDZ and SAP97, confirming that the interaction between SAP97 and the ₁-AR was mediated through the PDZ. Similarly, AKAP79 interacted with the full-length WT ₁-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 subunit of PKA and AKAP79 and between RII and the WT ₁-AR (1). These data complement the immunoprecipitation results in Fig. 4 that have shown binding between the SAP97-AKAP79-PKA complex and the WT ₁-AR.

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

View larger version (65K): [in this window] [in a new window]   FIGURE 5. Acceptor photobleaching analysis of FRET interactions between SAP97, AKAP79, and the1-AR. A and B, cells co-expressing the WT 1-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 1-AR and SAP97 and between the WT 1-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 1-AR, AKAP79, and SAP97 have occurred with relatively high efficiency. D and E, however, in cells co-expressing the 1-ARPDZ-CFP with either SAP97-YFP or AKAP79-YFP no FRET interactions were observed, indicating that mutagenesis of the PDZ domain of the 1-AR abrogated the interactions between the 1-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.

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Sensitized emission FRET microscopy between SAP97, AKAP79, and the WT 1-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) ([bound]/[total d] x [total a]) that was described under "Experimental Procedures." The equation indicates the proportional () 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.

View larger version (64K): [in this window] [in a new window]   FIGURE 7. Effect of the PDZ or SAP97 siRNA on isoproterenol-mediated phosphorylation of the 1-AR. A, isoproterenol (10 µM) treatment of cells expressing the WT 1-AR resulted in a 5.7-fold increase in phosphorylation (lanes 1 and 2), whereas the 1-ARPDZ showed reduced phosphorylation in response to isoproterenol (lanes 3 and 4). siRNA-mediated knockdown of SAP97 also reduced the phosphorylation of the WT1-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 1-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 1-AR or 1-ARPDZ 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 1-AR (lanes 1 and 2) or the 1-ARPDZ (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 1-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.

View larger version (47K): [in this window] [in a new window]   FIGURE 8. Roles of the ESKV sequence in the carboxyl-terminal tail of the 1-AR and Ser312 in the 3rd IC in regulating the recycling and resensitization of the 1-AR in response to isoproterenol. A, recycling of the 1-ARPDZ in response to 10 µM isoproterenol (Iso). HEK-293 cells stably expressing the 1-ARPDZ 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 1-ARPDZ in which the serine residue at position 312 was mutated to aspartic acid (S312D 1-ARPDZ, images q-y). Recycling of the 1-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 1-AR were conducted as described under "Experimental Procedures." Each scale bar represents 5 µm. B, summary of the results of the experiment in