Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

γ-aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result following receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact on anxiety, stress and depression, and for therapeutic drugs such as general anaesthetics. To understand the basis for modulating GABAAR function, high-resolution structures are required. Here we present the first atomic structures of a GABAAR chimera at 2.8Å resolution, including those bound with potentiating and inhibitory neurosteroids. These define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable neurosteroids to be exploited for therapeutic drug design to regulate GABAARs in neurological disorders.

A-kinase anchoring protein 79 (AKAP79) is a human anchoring protein that organizes cAMP-dependent protein kinase (PKA), Ca 2þ ∕calmodulin (CaM)-dependent protein phosphatase (PP2B), and protein kinase C (PKC) for phosphoregulation of synaptic signaling. Quantitative biochemical analyses of selected AKAP79 complexes have determined the quaternary structure of these signaling complexes. We show that AKAP79 dimerizes, and we demonstrate that, upon addition of a lysine-reactive cross-linker, parallel homomeric dimers are stabilized through K328-K328 and K333-K333 cross-links. An assembly of greater complexity comprising AKAP79, PP2B, a type II regulatory subunit fragment (RII 1-45) of PKA, and CaM was reconstituted in vitro. Using native MS, we determined the molecular mass of this complex as 466 kDa. This indicates that dimeric AKAP79 coordinates two RII 1-45 homodimers, four PP2B heterodimers, and two CaM molecules. Binding of Ca 2þ ∕CaM to AKAP79 stabilizes the complex by generating a second interface for PP2B. This leads to activation of the anchored phosphatases. Our architectural model reveals how dimeric AKAP79 concentrates pockets of second messenger responsive enzyme activities at the plasma membrane. F ollowing its discovery, cyclic AMP (cAMP) was thought to be freely diffusible within the cell (1). Subsequently, it was demonstrated that utilization of this second messenger is more sophisticated. For example, treatment of cardiac myocytes with norepinephrine, but not prostaglandin E1, stimulates contraction and glycogen metabolism, despite the fact that both hormones elevate cAMP to activate cAMP-dependent protein kinase (PKA) (2). It is now recognized that intracellular cAMP levels fluctuate in cellular microdomains where cAMP effector proteins such as PKA, Epac guanine nucleotide exchange factors, and cyclic nucleotide-gated ion channels reside (3-5). Likewise, other second messengers such as Ca 2þ and certain phospholipids operate in cellular microdomains where they interface with their own effector proteins (6).A-kinase anchoring proteins (AKAPs) organize responses to these second messengers. AKAP79 is a prototypical AKAP, exemplifying the three properties that are characteristic of the family: (i) an amphipathic α-helix (residues 387-406) that binds to the D∕D domain (residues 1-45) of PKA RII subunits (7); (ii) a subcellular localization signal, in its case, three tandem membrane-binding basic regions (MBBRs) that bind to PIP 2 (8); and (iii) the ability to interact with multiple signaling molecules. AKAP79 targets PKA, the Ca 2þ -dependent Ser/Thr protein phosphatase PP2B (calcineurin) (9), and protein kinase C (PKC) (10) to substrates including transmembrane receptors and ion channels. A more comprehensive list of AKAP79 binding partners is shown in Fig. S1. Ancillary protein-lipid and proteinprotein interactions serve to localize AKAP79 with particular membrane substrates (8). For example, a leucine zipper-like motif in AKAP79 associates with the cytoplasmic tail of L-type Ca 2þ channels, enabling...