Protein kinase A regulates AKAP250 (gravin) scaffold binding to the  2-adrenergic receptor

Abstract

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 b 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. Keywords: b-adrenergic receptor/AKAP/gravin/protein kinase A/protein kinase C/scaffold IntroductionRecent advances in our understanding of the workings of cell signaling have highlighted the roles of molecules, often referred to as`scaffolds', in organizing the interactions of molecules in signaling pathways. In Saccharomyces cerevisiae, for example, STE5 is an essential component of the pheromone-mediated mitogen-activated protein kinase (MAPK) pathway. The STE5 scaffold recruits MAPK cascade members STE11, STE7 and FUS3 to generate speci®city for the pheromone pathway (Kim et al., 1998). In vertebrates, A-kinase-anchoring proteins (AKAPs) constitute a diverse family of such scaffold molecules, organizing not only protein kinases, but also protein phosphatases and other molecules, into multivalent signaling complexes (Pawson and Scott, 1997;Colledge and Scott, 1999;Bauman and Scott, 2002). AKAPs bind the RII subunits of the cAMP-dependent kinase (PKA), providing discrete localization of PKA within the cell.The muscle-speci®c AKAP, termed`mAKAP', coordinates multivalent signaling complexes in both the sarcoplasmic reticulum and perinuclear membrane of muscle cells . Another family of AKAPs are derived from a single gene, producing the splice products AKAP350, AKAP450, CG-NAP and Yotiao that are involved in coordinating multivaltent signaling complexes with N-methyl-D-aspartate (NMDA) receptors, protein phosphatases PP1 and calcineurin, as well as PKA Bauman and Scott, 2002). Gravin (also known as AKAP250 and AKAP12) is another prominent member of the AKAP family, a family that includes >50 members. As scaffold molecules, the AKAPs display binding sites for several well known protein kinases and phosphatases. Gravin, for example, binds the RII subunit of PKA, protein kinase C (PKC), protein phosphatase 2B (PP2B) (Nauert et al., 1997;Shih et al., 1999;Fan et al.,...

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…Mutation of the binding site for the RII subunit of PKA was shown recently to abolish the ability of PKA to phosphorylate only those AKAP molecules lacking the RII-binding site [50]. When performed in A431 cells expressing both the wild-type and RII-deficient AKAPs, in contrast, the RII-deficient AKAPs are no longer phosphorylated in response to agonist, demonstrating a new dimension in our understanding of spatial constraints on signalling complexes.…”

“…The two C-terminal motifs (TEG-EGVSTWESFKRLVTPRKKSKSKLEEK and EPGKEESW-VSIKKFIPGRRKKRPD, where the bold residues show identity between AKAP79 and AKAP250) are found also in AKAP79, suggesting that the region comprising residues 753-827 is essential for AKAP250 binding to β 2 ARs. The first N-terminal AKAP motif of AKAP250 is lacking in AKAP79, and by itself fails to confer β 2 AR-binding capacity to the AKAP [50]. Deletion of AKAP79 sequence 108-427 (containing the AKAP motifs) does appear to abolish β 2 AR binding [47].…”

“…In silico analysis of AKAP250 revealed three 'AKAP motifs' at residue positions 603-633, 752-782 and 797-827. Deletion/mutagenesis of these AKAP motifs led to loss of β 2 AR-binding capability of AKAP250 [50]. The two C-terminal motifs (TEG-EGVSTWESFKRLVTPRKKSKSKLEEK and EPGKEESW-VSIKKFIPGRRKKRPD, where the bold residues show identity between AKAP79 and AKAP250) are found also in AKAP79, suggesting that the region comprising residues 753-827 is essential for AKAP250 binding to β 2 ARs.…”

“…This same domain appears to provide AKAP79 binding to NMDA (N-methyl-Daspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) GluR 1 receptors via the MAGUK (membrane-associated guanylate kinase) proteins PSD-95 (post synaptic density protein-95) and SAP97 (synapse-associated protein 97) respectively [49]. That the interactions between the β 2 AR and the scaffold are dynamic in character and can be regulated by phosphorylation ( Figure 5) was discovered only recently [50]. Although the full composition of the AKAP-based β 2 AR signalling complexes remains to be established, two critical elements have been targeted for an exhaustive analysis of dynamic phosphorylation: the AKAP250 scaffold and the receptor itself.…”

“…When performed in A431 cells expressing both the wild-type and RII-deficient AKAPs, in contrast, the RII-deficient AKAPs are no longer phosphorylated in response to agonist, demonstrating a new dimension in our understanding of spatial constraints on signalling complexes. One would have presumed initially that, with a mixture of wild-type and mutant AKAPs being expressed in the same cells, the wildtype scaffold would donate the PKA catalytic activity and still phosphorylate the RII-deficient AKAPs, but this was not the case [50].…”