Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

Abstract

Background: AKAP79/150 scaffolds PKA, PKC, and calcineurin into networks. Results: Knockdown of AKAP79/150 in cardiac myocytes inhibited the recycling of the ␤ 1 -AR and increased ␤ 1 -AR-mediated production of cyclic AMP. Conclusion: AKAP79/150 is involved in trafficking and signaling of myocardial ␤ 1 -AR. Significance: Our results indicate that AKAP79/150 might be cardioprotective via its key role in regulating the signaling intensity of cardiac ␤ 1 -AR.

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

“…As expected, mutagenesis of the type I PDZ amino acids in the ␤ 1 -AR to alanine (␤ 1 -AR⌬PDZ) prevented its recycling (Fig. 1A, j-lЈ), and its intracellular pixels were not stripped by the second acid wash (11,13,14).…”

“…The effect of SAP97 on recycling was dependent on its bipartite binding to the ␤ 1 -AR PDZ and to the protein kinase A anchoring protein 79 (AKAP79)-PKA complex (13). Thus, the interaction between the ␤ 1 -AR and SAP97 created a SAP97-AKAP79-PKA complex that was tethered by SAP97 to the type I PDZ of the ␤ 1 -AR (11,(13)(14).…”

“…Confocal fluorescence microscopy was performed on coded slides, and images were acquired using an Olympus FV1000 confocal microscope (11,(13)(14). To calculate the recycling kinetics, a boundary was drawn around the inner circumference of cells using ImageJ software (National Institutes of Health).…”

“…Co-immunoprecipitation and ␤-AR Degradation AssaysCo-immunoprecipitations between SAP97 and the various FLAG-␤ 1 -AR or ␤ 2 -AR constructs were performed as described previously (11,(13)(14). Briefly, equal amounts of cellular protein lysates were incubated with preimmune IgG-agarose beads (control) or with anti-FLAG IgG-agarose beads for 4 h at 4°C.…”

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

“…To this end, HEK293 cells were nucleofected with an AKAP5-specific shRNA vector to knock down the protein, as described previously (25), or with scrambled shRNA. AKAP5 shRNA completely prevented GPR30 inhibition of FSK-and PGE2-stimulated cAMP production 48 and 96 h post-nucleofection, respectively, whereas GPR30 inhibition still occurred with scrambled shRNA (Fig.…”