Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.     

The intermediate filament protein, synemin, is an AKAP in the heart

Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.     

Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.     

Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells

Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.     

Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.     

Isoform-specific targeting of PKA to multivesicular bodies

Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.     

Molecular basis of AKAP specificity for PKA regulatory subunits

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.     

A-KinaseAnchoringProteins: a key to selective activation of cAMP-responsive events?

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family ofA-KinaseAnchoringProteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.     

A-kinase anchor proteins as potential regulators of protein kinase A function in oocytes

In the mammalian oocyte, the cAMP-dependent protein kinase (PKA) has critical functions in the maintenance of meiotic arrest and oocyte maturation. Because PKA is spatially regulated, its localization was examined in developing oocytes. Both regulatory subunits (RI and RII) and the catalytic subunit (C) of PKA were found in oocytes and metaphase II-arrested eggs. In the oocyte, RI and C were predominantly localized in the cortical region, while RII showed a punctate distribution within the cytoplasm. After maturation to metaphase II, RI remained in the cortex and was also localized to the meiotic spindle, while RII was found adjacent to the spindle. C was diffuse within the cytoplasm of the egg but was enriched in the cytoplasm surrounding the metaphase spindle, much like RII. The polarized localization and redistribution of RI, RII, and C suggested that PKA might be tethered by A-kinase anchor proteins (AKAPs), proteins that tether PKA close to its physiological substrates. An AKAP, AKAP140, was identified that was developmentally regulated and phosphorylated in oocytes and eggs. AKAP140 was shown to be a dual-specific AKAP, having the ability to bind both RI and RII. By compartmentalizing PKA, AKAP140 and/or other AKAPs could spatially regulate PKA activity during oocyte development.     

Isoform specific differences in binding of a dual-specificity A-kinase anchoring protein to type I and type II regulatory subunits of PKA

Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMP-dependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIalpha than to RIalpha. The higher affinity for RIIalpha was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIalpha and RIIalpha was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIalpha was independent of solution ionic strength, whereas RIIalpha had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIalpha but not to RIIalpha. These isoform-specific differences provide a foundation for developing isoform-specific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.     

Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.

Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.     

Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.     

Alpha4 integrins are type I cAMP-dependent protein kinase-anchoring proteins

A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.     

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.     

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.     

Related protein-protein interaction modules present drastically different surface topographies despite a conserved helical platform

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.