Sorting of β1-adrenergic receptors is mediated by pathways that are either dependent on or independent of type I PDZ, protein kinase A (PKA), and SAP97

Abstract

The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A., Naren, A. P., and Bahouth, S. W. (2007) J. Biol. Chem. 282, 5085-5099). We explored the effects of point mutations in the PDZ and in the activity of PKA on recycling of the β1-AR and its binding to the PDZ-binding protein SAP97. These studies indicated that β1-AR recycling was inhibited by PKA inhibitors and by mutations in the PDZ that interfered with SAP97 binding. The trafficking effects of short sequences differing in PDZ and SAP97 binding were examined using chimeric mutant β1-AR. β1-AR chimera containing the type I PDZ of the β2-adrenergic receptor that does not bind to SAP97 failed to recycle except when serine 312 was mutated to aspartic acid. β1-AR chimera with type I PDZ sequences from the C-tails of aquaporin-2 or GluR1 recycled in a SAP97- and PKA-dependent manner. Non-PDZ β1-AR chimera derived from μ-opioid, dopamine 1, or GluR2 receptors promoted rapid recycling of chimeric β1-AR in a SAP97- and PKA-independent manner. Moreover, the nature of the residue at position -3 in the PDZ regulated whether the β1-AR was internalized alone or in complex with SAP97. These results indicate that divergent pathways were involved in trafficking the β1-AR and provide a roadmap for its trafficking via type I PDZs versus non-PDZs.

Keywords: Adrenergic Receptor; Confocal Microscopy; G Protein-coupled Receptor (GPCR); Protein Kinase A (PKA); Trafficking.

FIGURE 1.

Characterization of the residues in the PDZ domain of the β₁-AR that are involved in recycling and in binding to SAP97. A, HEK-293 cells expressing the FLAG-tagged WT β₁-AR (a–f′) and β₁-AR constructs in which the entire PDZ was inactivated (β₁-ARΔPDZ; g–l′) or single point mutations in the PDZ (m–x1′) were plated on poly-l-lysine-covered glass slides. Cells were prelabeled for 1 h with Cy3-anti-FLAG antibody and fixed (a, g, m, s, a1, g1, m1, and s1). The rest of the slides were exposed to 10 μm isoproterenol for 30 min (b, h, n, t, b1, h1, n1, and t1) and then acid-washed and fixed (c, i, o, u, c1, i1, o1, and u1). The rest of the slides were subjected to recycling conditions for the indicated time period and then fixed. Slides incubated with 100 μm alprenolol for 1 h were exposed to acid wash and then fixed (f′, l′, r′, x′, f1′, l1′, r1′, and x1′). The distribution of fluorescent pixels was obtained using confocal microscopy, and the colors shown are pseudo-colors. Scale bars, 5 μm. B, HEK-293 cells were transiently transfected with FLAG-tagged β₁-AR and rat SAP97 cDNAs for 2 days. The cells were lysed, and about 4% of the crude cell lysate (input) was subjected to Western blotting (IB) with anti-SAP97 IgG to verify that equal amounts of SAP97 were loaded onto the resin. Then equal amounts of the remaining cell lysate proteins were incubated with anti-FLAG IgG resin, followed by washing and eluting the resin with Laemmli sample buffer. The eluates were divided into two portions. One portion representing 4% of the eluate by volume was probed with anti-FLAG antibody to verify that equal amounts of the β₁-AR were bound and eluted from the affinity resin. The rest of the FLAG IPs, representing about 90% of resin eluates, were probed with the anti-SAP97 antibody (n = 4). A/W, acid wash.

FIGURE 2.

Identification of the residues at position 477 in the β₁-AR that are involved in recycling and binding to SAP97. A, HEK-293 cells expressing the point mutations of the valine residue at position 477 in the β₁-AR were plated on poly-l-lysine-covered glass slides. Cells were subjected to internalization and recycling as described in the legend to Fig. 1A. Scale bars, 5 μm. B, HEK-293 cells were transiently transfected with FLAG-tagged β₁-AR and SAP97 cDNAs for 2 days. Cell lysates were prepared, and 4% of input lysate proteins were probed by Western blotting (IB) for the expression of SAP97 (lanes 1–6). Then equal amounts of the rest of the lysates were incubated with anti-FLAG IgG resin, eluted, and subjected to Western blotting for the co-IP of SAP97 (lanes 7–12). A/W, acid wash.

FIGURE 3.

Effect substituting the β₂-AR-derived C-tail sequences on recycling of the β₁-AR. A, HEK-293 cells stably expressing the WT β₂-AR or β₁/β₂-AR chimera in whom the last 4 or 10 amino acids of the β₁-AR were mutagenized into the corresponding sequences of the human β₂-AR were used. The recycling of these β₁/β₂-adrenergic receptor chimeras was determined as described in the legend to Fig. 1A. B, HEK-293 cells were transiently transfected with FLAG-tagged β₁-AR, β₂-AR, or β₁-ARΔPDZ or with β₁/β₂-AR chimera and SAP97 cDNAs for 2 days. Equal amounts of cell lysate proteins were incubated with anti-FLAG IgG resin, eluted, and subjected to Western blotting for the co-IP of SAP97. To normalize for input protein levels, ∼4% of each cell lysate were subjected to Western blotting (IB) and probed with the anti-SAP97 antibody. A/W, acid wash.

FIGURE 4.

Effect of SAP97 and H-89/mPKI on recycling of WT β₁-AR or WT β₂-AR. A, HEK-293 cells stably expressing the FLAG-tagged WT β₁-AR and either the scrambled shRNA-EGFP (a–f′) or hSAP97 shRNA-EGFP (g–l′) were used. Similarly, cells stably expressing the FLAG-tagged WT β₂-AR with either the scrambled shRNA-EGFP (m–r′) or hSAP97 shRNA-EGFP (s–x′) were used. These four types of cells were subjected to internalization and recycling as described in the legend to Fig. 1A. The distribution of fluorescent pixels was obtained using confocal microscopy, and the colors shown are pseudo-colors. Scale bars, 5 μm. B, HEK-293 cells stably expressing the WT β₂-AR or WT β₁-AR were preincubated for 30 min with 3 μm H-89 or the myristoylated peptide inhibitor of PKA (mPKI) and then subjected to internalization and recycling as described in the legend to Fig. 1A. Scale bars, 5 μm. C, pixels inside a 300-nm boundary in the isoproterenol/acid-washed images of the indicated β₁-AR construct were set arbitrarily to 100% to indicate 100% internalization. The ratios in alprenolol-treated cells (at 15, 30, and 60 min) were calculated and expressed as the percentage for each time period. The ratios from 15–20 independent images for each condition were calculated and expressed as the mean ± S.E. for each time period. The data were plotted as a logarithmic y axis to calculate the t_0.5 for recycling, as described under “Experimental Procedures.” The mean and S.E. for each time period were compared among the four different groups by one-way analysis of variance with Newman-Keuls post-tests. Statistical results are expressed as NS to indicate no significant difference or *, **, and *** to indicate p < 0.05, p < 0.01, and p < 0.001, respectively. A/W, acid wash.

FIGURE 5.

Effect of the serine 312 to aspartic acid mutation on recycling of β₁/β₂-AR(10) chimera. A, HEK-293 cells were transiently transfected with the FLAG-tagged (S312D) β₁-AR mutant in which the last 10 amino acids were mutagenized to the corresponding sequence in the β₂-AR ((S312D) β₁/β₂-AR(10)) with either the scrambled shRNA-EGFP (a–f′) or hSAP97 shRNA-EGFP (g–l′). In addition, cells that expressed ((S312D) β₁/β₂-AR(10)) were preincubated for 30 min with 3 μm H-89 (m–r′) or mPKI (s–x′) prior to subjecting these cells to internalization and recycling as described in the legend to Fig. 1A. The distribution of fluorescent pixels was obtained using confocal microscopy, and the colors shown are pseudo-colors. Scale bars, 5 μm. B, the recycling data were plotted and analyzed as described in the legend to Fig. 4C, and the means ± S.E. were expressed as NS to indicate no significant difference or *, **, and *** to indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

FIGURE 6.

Effect of additional substitutions in the C-tail of the β₁-AR on recycling and binding to SAP97. A, the last 10 amino acids of the β₁-AR were mutagenized into the sequences described in Table 1. HEK-293 cells stably expressing equivalent levels of these FLAG-tagged β₁-adrenergic receptor chimeras were preincubated for 30 min in buffer or with 3 μm H-89, and their internalization and recycling were determined as described in the legend to Fig. 1A. B, HEK-293 cells stably expressing the β₁-AR chimeras described in A were transiently transfected with SAP97 cDNA for 2 days. The cells were lysed, and about 4% of the crude cell lysate (input) was subjected to Western blotting (IB) and probed with anti-SAP97 IgG to verify that equal amounts of SAP97 were loaded onto the resin. Equal amounts of the remaining cell lysate proteins were incubated with anti-FLAG IgG resin, followed by washing and eluting the resin with Laemmli sample buffer. The eluates were divided into portions. One portion representing 4% of the eluate by volume was probed with anti-FLAG antibody to verify that equal amounts of the β₁-AR were bound and eluted from the affinity resin. The rest, representing about 95% of resin eluates, were probed for the co-IP of SAP97 (n = 4). A/W, acid wash.

FIGURE 7.

Effect of the polarity of the amino acid at position −3 in the type 1 PDZ on the association of SAP97 with internal β₁-AR constructs. Shown is confocal microscopy imaging of SAP97-GFP (green) and FLAG β₁-AR labeled with Cy3-conjugated anti-FLAG M2 IgG (red) in cells expressing either the WT β₁-AR (A) or cells expressing the β₁-AR GluR1(10) (B) prior to (0 min) and 30 min after (ISO 30 min) adding 10 μm isoproterenol. Thereafter, the isoproterenol-exposed cells were washed and incubated for 60 min at 37 °C (ALP 60 min) with 100 μm alprenolol and imaged. The image is representative of 20 cells.

FIGURE 8.

Effect of selected substitutions in the C-tail of the β₁-AR on agonist-induced degradation of mutant receptor chimeras. A, HEK-293 cells expressing the indicated FLAG-tagged receptors were exposed to 10 μm isoproterenol for 0, 3, or 6 h. Total cell extracts were probed by Western blotting with anti-FLAG IgG to compare the levels of β₁-AR chimeras and with anti-β-actin to equalize the amounts of loaded protein lysates. B, band and intensities of anti-FLAG and anti-β-actin were quantified by chemiluminescent imaging, and anti-FLAG intensities were normalized for each construct. Bars, mean receptor levels (relative to 0 h of agonist exposure) were determined from n = 4 independent experiments per construct. Error bars, S.E.

FIGURE 9.

Roadmap for divergent trafficking pathways of chimeric β₁-AR. A, organization of membranous β₁-AR mutants and chimeras. Left, WT or β₁-AR-chimeras with a type I PDZ that bind to SAP97 (blue PDZ) assemble as a complex composed of SAP97-AKAP79-PKA. Activation of these GPCRs increases cyclic AMP, which promotes the dissociation of the catalytic subunit of PKA from PDZ-bound PKA. The catalytic subunit of PKA phosphorylates Ser³¹² in the third intracellular loop to imprint the recycling signal onto the β₁-AR (red P). Middle, β₁-AR-chimeras with an inactive PDZ or with a type I PDZ that did bind SAP97 (red PDZ), did not assemble a SAP97-anchored complex. Right, β₁-AR-chimeras with a type II PDZ or non-PDZ recycling sequences do not assemble a complex (black PDZ). Activation of all of these receptors, however, increased cyclic AMP and promoted the binding of β-arrestin to the GRK-phosphorylated GPCR. In the left panel, we hypothesize that acidic amino acids at P = −3 in WT or β₁-AR mutants would interact with β-arrestin to promote the dissociation of the complex anchored at the PDZ prior to the internalization of the various GPCRs into early endosomes, whereas PDZs with neutral residues at P = −3 are internalized with their PDZ-binding protein (±PBP). B, trafficking roadmap of internalized β₁-AR mutants and chimeras. Left, trafficking roadmap of β₁-AR mutants with a phospho-Ser³¹². These GPCRs traffic by a process termed “sequence-dependent trafficking,” in which they are sorted from early endosomes into membrane extensions (blue arrow) that coalesce into “recycling endosomes”. Middle, β₁-AR with type I PDZs, but without a phospho-Ser³¹², were retained within the “body” of early endosomes. These early endosomes eventually matured into late endosomes/lysosomes, in which the retained “cargo” is eventually degraded. Right, internalized β₁-AR with recycling non-PDZs traffic out of early endosomes into “recycling endosomes” by a process termed “bulk or sequence-independent recycling.” β₁-AR-chimeras that traffic to recycling endosomes recycle back and fuse with the cell membrane (green arrows).