AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the beta1-adrenergic receptor promotes recycling and functional resensitization of the receptor

Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3'-5' cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized beta(1)-AR (Gardner, L. A., Delos Santos, N. M., Matta, S. G., Whitt, M. A., and Bahouth, S. W. (2004) J. Biol. Chem. 279, 21135-21143). Because PKA is compartmentalized near target substrates by interacting with protein kinase A anchoring proteins (AKAPs), the present study was undertaken to identify the AKAP involved in PKA-mediated phosphorylation of the beta(1)-AR and in its recycling and resensitization. Here, we report that Ht-31 peptide-mediated disruption of PKA/AKAP interactions prevented the recycling and functional resensitization of heterologously expressed beta(1)-AR in HEK-293 cells and endogenously expressed beta(1)-AR in SK-N-MC cells and neonatal rat cortical neurons. Whereas several endogenous AKAPs were identified in HEK-293 cells, small interfering RNA-mediated down-regulation of AKAP79 prevented the recycling of the beta(1)-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the beta(1)-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the beta(1)-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the beta(1)-AR because disruption of PKA/AKAP interactions or small interfering RNA-mediated down-regulation of AKAP79 both inhibited this response. Thus, AKAP79 provides PKA to phosphorylate the beta(1)-AR and thereby dictate the recycling and resensitization itineraries of the beta(1)-AR.     

Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the beta1-adrenergic receptor

A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) beta(1)-AR recycled with a t(0.5) of 14 +/- 3 min. Mutagenesis of Ser(312) to alanine (Ser(312) --> Ala beta(1)-AR) or to the phosphoserine mimic aspartic acid (Ser(312) --> Asp beta(1)-AR) resulted in beta(1)-AR constructs that were pharmacologically indistinguishable from the WT beta(1)-AR. The internalized Ser(312) --> Asp beta(1)-AR recycled efficiently with a t(0.5) of 11 +/- 3 min, whereas the internalized Ser(312) --> Ala beta(1)-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized beta(1)-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT beta(1)-AR. Phosphorylation studies revealed that the beta(1)-AR is partially phosphorylated by PKA and that phosphorylation of the beta(1)-AR by the catalytic subunit of PKA occurs exclusively at Ser(312). Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.     

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

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.     

Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.     

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.     

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

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

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β₁-adrenergic receptor (β₁-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β₁-AR. Recycling of the β₁-AR in AKAP5 knockdown cells was rescued by shRNA-resistant AKAP5. However, truncated mutants of AKAP5 with deletions in the domains involved in membrane targeting or in binding to calcineurin or PKA failed to restore the recycling of the β₁-AR, indicating that full-length AKAP5 was required. Furthermore, recycling of the β₁-AR in rat neonatal cardiac myocytes was dependent on targeting the AKAP5-PKA complex to the C-terminal tail of the β₁-AR. To analyze the role of AKAP5 more directly, recycling of the β₁-AR was determined in ventricular myocytes from AKAP5^−/− mice. In AKAP5^−/− myocytes, the agonist-internalized β₁-AR did not recycle, except when full-length AKAP5 was reintroduced. These data indicate that AKAP5 exerted specific and profound effects on β₁-AR recycling in mammalian cells. Biochemical or real time FRET-based imaging of cyclic AMP revealed that deletion of AKAP5 sensitized the cardiac β₁-AR signaling pathway to isoproterenol. Moreover, isoproterenol-mediated increase in contraction rate, surface area, or expression of β-myosin heavy chains was significantly greater in AKAP5^−/− myocytes than in AKAP5^+/+ myocytes. These results indicate a significant role for the AKAP5 scaffold in signaling and trafficking of the β₁-AR in cardiac myocytes and mammalian cells.     

Src docks to A-kinase anchoring protein gravin, regulating beta2-adrenergic receptor resensitization and recycling

Gravin (AKAP12) is a membrane-associated scaffold that provides docking for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of beta(2)-adrenergic receptors. Gravin binds to the cell membrane in a Ca(2+)-sensitive manner and to receptors through well characterized protein-protein interactions. Although the interaction of serine/threonine, cyclic AMP-dependent protein kinase with protein kinase A-anchoring proteins is well described and involves a kinase regulatory subunit binding domain in the C terminus of these proteins, far less is known about tyrosine kinase docking to members of this family of scaffolds. The non-receptor tyrosine kinase Src regulates resensitization of beta(2)-adrenergic receptors and docks to gravin. Gravin displays nine proline-rich domains distributed throughout the molecule. One class I ligand for Src homology domain 3 docking, found in the N terminus ((10)RXPXXP(15)) of gravin, is shown to bind Src. Binding of Src to gravin activates the intrinsic tyrosine kinase of Src. Mutagenesis/deletion of the class I ligand (P15A,P16A) on the N terminus of gravin abolishes both the docking of Src to gravin as well as the receptor resensitization and recycling catalyzed by gravin. The Src-binding peptide-(1-51) of gravin behaves as a dominant-negative for AKAP gravin regulation of receptor resensitization/recycling. The tyrosine kinase Src plays an essential role in the AKAP gravin-mediated receptor resensitization and recycling, an essential aspect of receptor biology.     

D‐AKAP2:PKA RII:PDZK1 ternary complex structure: Insights from the nucleation of a polyvalent scaffold

A‐kinase anchoring proteins (AKAPs) regulate cAMP‐dependent protein kinase (PKA) signaling in space and time. Dual‐specific AKAP2 (D‐AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D‐AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α‐helix to PKA and a β‐strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D‐AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D‐AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C‐terminus of D‐AKAP2, which contains two binding motifs—the D‐AKAP2_AKB and the PDZ motif—that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D‐AKAP2_AKB binds to the D/D domain of the R‐subunit and the C‐terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D‐AKAP2 would exhibit a differential mode of binding to the two PKA isoforms.     

Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor

A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the beta(2)-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor-scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold-receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin-receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold-receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization.     

Interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) inhibits beta1-adrenergic receptor surface expression

G protein-coupled receptors such as the beta1-adrenergic receptor (beta1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the beta1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The beta1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the beta1AR-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the beta1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the beta1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length beta1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of beta1AR. Interaction with CAL promotes retention of beta1AR within the cell, whereas PSD-95, another beta1AR-associated PDZ domain-containing protein, competitively blocks beta1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel beta1AR-binding partner, modulates beta1AR intracellular trafficking, thereby revealing a new mechanism of regulation for beta1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane.     

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by > 70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by > 70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.     

Structural characterization of the intermolecular interactions of synapse-associated protein-97 with the NR2B subunit of N-methyl-D-aspartate receptors

The synapse-associated protein-97 (SAP97) is important in the proper trafficking and cell surface maintenance of the N-methyl-D-aspartate ionotropic glutamate receptor. The molecular scaffold/receptor interaction is mediated by the association of the C terminus of the NR2B subunit of the N-methyl-D-aspartate receptor with the PDZ domains of SAP97. Here, we characterize the binding of the C terminus of NR2B with the PDZ domains of SAP97 and determine the structure of the PDZ1-NR2B complex employing high-resolution NMR. Based on fluorescence anisotropy, the NR2B subunit binds to the first and second PDZ domains of SAP97, with higher affinity for PDZ2; no appreciable binding to PDZ3 could be measured. The structural features of the NR2B bound to PDZ1 is consistent with the canonical PDZ-binding motif with the glutamic acid at the -3 position of the C terminus (i.e. -E-S-D-V) interacting with the beta2/beta3 loop. Two sites within the loop of PDZ1 were replaced with the corresponding residue from PDZ2, D243G and P245Q. The former mutation, designed to remove a possible Coulombic repulsion between E(-3)(NR2B) and Asp-243 (PDZ1) has only a minimal effect on binding. The P245Q mutation leads to a 2-fold increase in binding affinity of NR2B, approaching that observed for wild-type PDZ2. These results indicate that modification of the beta2/beta3 loop provides an avenue for regulating the ligand specificity of PDZ domains.     

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.     

Identification of an IQGAP1/AKAP79 complex in beta-cells

IQGAP1, is a recently discovered scaffold protein proposed to regulate membrane cytoskeleton events through protein-protein interactions with F-actin, E-cadherin, beta-catenin, and CLIP170. The binding of IQGAP1 to its partners is regulated by calcium/calmodulin (Ca(++)/CaM) and the small molecular weight guanine nucleotide triphosphatases (GTPases), Cdc42, and Rac1. Here we identify a novel IQGAP1 scaffolding function by isolating the cyclic AMP dependent kinase (PKA) with IQGAP1. IQGAP1 was co-purified with PKA using 5'-cyclic AMP (cAMP) affinity chromatography and PKA activity was co-immunoprecipitated with IQGAP1 using an anti-IQGAP1 antibody. The association of IQGAP1 with PKA was shown to occur through a direct interaction between A kinase anchoring protein 79 (AKAP79) and the carboxyl-terminal domain of IQGAP1. This suggests that cAMP/PKA may be coupled with Ca(++)/CaM and GTPases through an IQGAP1/AKAP79 complex.     

G-Protein-coupled receptor-associated A-kinase anchoring proteins: AKAP79 and AKAP250 (gravin)

A-kinase anchoring proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAPs are multivalent and a subset of these scaffold proteins also display the ability to associate with the prototypic member of G-protein-coupled receptors, the beta(2)-adrenergic receptor. Both AKAP79 (also known as AKAP5) and AKAP250 (also known as gravin or AKAP12) have been shown to associate with the beta(2)-adrenergic receptor, but each directs downstream signaling events in decidedly different manners. The primary structures, common and unique protein motifs are of interest. Both proteins display largely natively unfolded primary sequences that provide a necklace on which short, structured regions of sequence are found. Membrane association appears to involve both interactions with the lipid bilayer via docking to a G-protein-coupled receptor as well as interactions of short positively charged domains with the inner leaflet of the cell membrane. Gravin, unlike AKAP79, displays a canonical site at its N-terminus that is subject to N-myristoylation. AKAP79 appears to function in switching signaling pathways of the receptor from adenylylcyclase to activation of the mitogen-activated protein kinase cascade. Gravin, in contrast, is essential for the resensitization and recycling of the receptors following agonist-induced activation, desensitization, and internalization. Each AKAP provides a template that enables space-time continuum features to G-protein-coupled signaling pathways as well as a paradigm for explaining apparent compartmentalization of cell signaling.     

beta 1-adrenergic receptor association with the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 (MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95

The beta1-adrenergic receptor (beta1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining beta1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the beta1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The beta1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the beta1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length beta1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the beta1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the beta1AR. Further cellular experiments revealed that MAGI-2 has no effect on beta1AR oligomerization but does promote association of beta1AR with the cytoplasmic signaling protein beta-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific beta1AR binding partner that modulates beta1AR function and facilitates the physical association of the beta1AR with intracellular proteins involved in signal transduction and synaptic regulation.     

Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.