Severe photochemical pollution formation associated with strong HONO emissions from dew and guttation evaporation.

The unknown daytime source of HONO has been extensively investigated due to unexplained atmospheric oxidation capacity and current modelling bias, especially during cold seasons. In this study, abrupt morning increases in atmospheric HONO at a rural site in the North China Plain (NCP) were observed almost on daily basis, which were closely linked to simultaneous rises in atmospheric water vapor content and NH3 concentrations. Dew and guttation water formation was frequently observed on wheat leaves, from which water samples were taken and chemically analyzed for the first time. Results confirmed that such natural processes likely governed the daily nighttime deposition and daytime release of HONO and NH3, which have not been considered in the numerous HONO budget studies investigating its large missing daytime source in the NCP. The dissolved HONO and NH3 in leaf surface water droplets reached 1.4 and 23 mg L-1 during the morning on average, resulting in averaged atmospheric HONO and NH3 increases of 0.89 ± 0.61 and 43.7 ± 29.3 ppb during morning hours, with relative increases of 186 ± 212 % and 233 ± 252 %, respectively. The high atmospheric oxidation capacity contained within HONO was stored in near surface liquid water (such as dew, guttation and soil surface water) during nighttime, which prevented its atmospheric dispersion after sunset and protected it from photodissociation during early morning hours. HONO was released in a blast during later hours with stronger solar radiation, which triggered and then accelerated daytime photochemistry through the rapid photolysis of HONO and subsequent OH production, especially under high RH conditions, forming severe secondary gaseous and particulate pollution. Results of this study demonstrate that global ecosystems might play significant roles in atmospheric photochemistry through nighttime dew formation and guttation processes.

Exploring HONO formation and its role in driving secondary pollutants formation during winter in the North China Plain.

Daytime HONO photolysis is an important source of atmospheric hydroxyl radicals (OH). Knowledge of HONO formation chemistry under typical haze conditions, however, is still limited. In the Multiphase chemistry experiment in Fogs and Aerosols in the North China Plain in 2018, we investigated the wintertime HONO formation and its atmospheric implications at a rural site Gucheng. Three different episodes based on atmospheric aerosol loading levels were classified: clean periods (CPs), moderately polluted periods (MPPs) and severely polluted periods (SPPs). Correlation analysis revealed that HONO formation via heterogeneous conversion of NO2 was more efficient on aerosol surfaces than on ground, highlighting the important role of aerosols in promoting HONO formation. Daytime HONO budget analysis indicated a large missing source (with an average production rate of 0.66 ± 0.26, 0.97 ± 0.47 and 1.45 ± 0.55 ppbV/hr for CPs, MPPs and SPPs, respectively), which strongly correlated with photo-enhanced reactions (NO2 heterogeneous reaction and particulate nitrate photolysis). Average OH formation derived from HONO photolysis reached up to (0.92 ± 0.71), (1.75 ± 1.26) and (1.82 ± 1.47) ppbV/hr in CPs, MPPs and SPPs respectively, much higher than that from O3 photolysis (i.e., (0.004 ± 0.004), (0.006 ± 0.007) and (0.0035 ± 0.0034) ppbV/hr). Such high OH production rates could markedly regulate the atmospheric oxidation capacity and hence promote the formation of secondary aerosols and pollutants.

Multiphase chemistry experiment in Fogs and Aerosols in the North China Plain (McFAN): integrated analysis and intensive winter campaign 2018.

Fine-particle pollution associated with winter haze threatens the health of more than 400 million people in the North China Plain. The Multiphase chemistry experiment in Fogs and Aerosols in the North China Plain (McFAN) investigated the physicochemical mechanisms leading to haze formation with a focus on the contributions of multiphase processes in aerosols and fogs. We integrated observations on multiple platforms with regional and box model simulations to identify and characterize the key oxidation processes producing sulfate, nitrate and secondary organic aerosols. An outdoor twin-chamber system was deployed to conduct kinetic experiments under real atmospheric conditions in comparison to literature kinetic data from laboratory studies. The experiments were spanning multiple years since 2017 and an intensive field campaign was performed in the winter of 2018. The location of the site minimizes fast transition between clean and polluted air masses, and regimes representative for the North China Plain were observed at the measurement location in Gucheng near Beijing. The consecutive multi-year experiments document recent trends of PM2.5 pollution and corresponding changes of aerosol physical and chemical properties, enabling in-depth investigations of established and newly proposed chemical mechanisms of haze formation. This study is mainly focusing on the data obtained from the winter campaign 2018. To investigate multiphase chemistry, the results are presented and discussed by means of three characteristic cases: low humidity, high humidity and fog. We find a strong relative humidity dependence of aerosol chemical compositions, suggesting an important role of multiphase chemistry. Compared with the low humidity period, both PM1 and PM2.5 show higher mass fraction of secondary inorganic aerosols (SIA, mainly as nitrate, sulfate and ammonium) and secondary organic aerosols (SOA) during high humidity and fog episodes. The changes in aerosol composition further influence aerosol physical properties, e.g., with higher aerosol hygroscopicity parameter κ and single scattering albedo SSA under high humidity and fog cases. The campaign-averaged aerosol pH is 5.1 ± 0.9, of which the variation is mainly driven by the aerosol water content (AWC) concentrations. Overall, the McFAN experiment provides new evidence of the key role of multiphase reactions in regulating aerosol chemical composition and physical properties in polluted regions.

Dust-Dominated Coarse Particles as a Medium for Rapid Secondary Organic and Inorganic Aerosol Formation in Highly Polluted Air.

Secondary aerosol (SA) frequently drives severe haze formation on the North China Plain. However, previous studies mostly focused on submicron SA formation, thus our understanding of SA formation on supermicron particles remains poor. In this study, PM2.5 chemical composition and PM10 number size distribution measurements revealed that the SA formation occurred in very distinct size ranges. In particular, SA formation on dust-dominated supermicron particles was surprisingly high and increased with relative humidity (RH). SA formed on supermicron aerosols reached comparable levels with that on submicron particles during evolutionary stages of haze episodes. These results suggested that dust particles served as a medium for rapid secondary organic and inorganic aerosol formation under favorable photochemical and RH conditions in a highly polluted environment. Further analysis indicated that SA formation pathways differed among distinct size ranges. Overall, our study highlights the importance of dust in SA formation during non-dust storm periods and the urgent need to perform size-resolved aerosol chemical and physical property measurements in future SA formation investigations that are extended to the coarse mode because the large amount of SA formed thereon might have significant impacts on ice nucleation, radiative forcing, and human health.

High Concentrations of Atmospheric Isocyanic Acid (HNCO) Produced from Secondary Sources in China.

Isocyanic acid (HNCO) is a potentially toxic atmospheric pollutant, whose atmospheric concentrations are hypothesized to be linked to adverse health effects. An earlier model study estimated that concentrations of isocyanic acid in China are highest around the world. However, measurements of isocyanic acid in ambient air have not been available in China. Two field campaigns were conducted to measure isocyanic acid in ambient air using a high-resolution time-of-flight chemical ionization mass spectrometer (ToF-CIMS) in two different environments in China. The ranges of mixing ratios of isocyanic acid are from below the detection limit (18 pptv) to 2.8 ppbv (5 min average) with the average value of 0.46 ppbv at an urban site of Guangzhou in the Pearl River Delta (PRD) region in fall and from 0.02 to 2.2 ppbv with the average value of 0.37 ppbv at a rural site in the North China Plain (NCP) during wintertime, respectively. These concentrations are significantly higher than previous measurements in North America. The diurnal variations of isocyanic acid are very similar to secondary pollutants (e.g., ozone, formic acid, and nitric acid) in PRD, indicating that isocyanic acid is mainly produced by secondary formation. Both primary emissions and secondary formation account for isocyanic acid in the NCP. The lifetime of isocyanic acid in a lower atmosphere was estimated to be less than 1 day due to the high apparent loss rate caused by deposition at night in PRD. Based on the steady state analysis of isocyanic acid during the daytime, we show that amides are unlikely enough to explain the formation of isocyanic acid in Guangzhou, calling for additional precursors for isocyanic acid. Our measurements of isocyanic acid in two environments of China provide important constraints on the concentrations, sources, and sinks of this pollutant in the atmosphere.

Hygroscopicity of amino acids and their effect on the water uptake of ammonium sulfate in the mixed aerosol particles.

Amino acids are important water-soluble nitrogen-containing compounds in atmospheric aerosols. They can be involved in cloud formation due to their hygroscopicity and have significant influences on the hygroscopicity of inorganic compounds, which have not yet been well characterized. In this work, the hygroscopic properties of three amino acids, including aspartic acid, glutamine, and serine, as well as their mixtures with ammonium sulfate (AS) were investigated using a hygroscopicity tandem differential mobility analyzer (HTDMA) system. The gradual water uptake of aspartic acid, glutamine and serine particles indicates that they exist as liquid phase at low RH. When mixing either aspartic acid or glutamine with AS by mass ratio of 1:3, we observed a clear phase transition but with a lower deliquescence relative humidity (DRH) with respect to that of pure AS. This suggests the crystallization of AS in the presence of each of these two amino acids. However, as the mass fractions of these two amino acids increased in the mixed particles, the deliquescence transition process was not obvious. In contrast, the crystallization of AS was efficiently hampered even at low content (i.e., 25% by mass) of serine in the mixed particles. The Zdanovskii-Stokes-Robinson (ZSR) method in general underestimated the hygroscopic growth of any mixtures at RH below 79% (prior to AS deliquescence), suggesting both amino acid and the partially dissolved AS contributed the overall hygroscopicity at RH in this range. Relatively good agreements were reached between the measurements and model predictions using the Extended Aerosol Inorganic Model (E-AIM) assuming solid state AS in the mixed particles for 1:3 aspartic acid-AS and glutamine-AS systems. However, the model failed to simulate the water uptake behaviors of any other systems. It demonstrates that the interactions between components within the aerosols have a significant effect on the phase state of the mixed particles.

Photochemical Aqueous-Phase Reactions Induce Rapid Daytime Formation of Oxygenated Organic Aerosol on the North China Plain.

Secondary organic aerosol (SOA) constitutes a large fraction of organic aerosol worldwide, however, the formation mechanisms in polluted environments remain poorly understood. Here we observed fast daytime growth of oxygenated organic aerosol (OOA) (with formation rates up to 10 µg m-3 h-1) during low relative humidity (RH, daytime average 38 ± 19%), high RH (53 ± 19%), and fog periods (77 ± 13%, fog occurring during nighttime with RH reaching 100%). Evidence showed that photochemical aqueous-phase SOA (aqSOA) formation dominantly contributed to daytime OOA formation during the periods with nighttime fog, while both photochemical aqSOA and gas-phase SOA (gasSOA) formation were important during other periods with the former contributing more under high RH and the latter under low RH conditions, respectively. Compared to daytime photochemical aqSOA production, dark aqSOA formation was only observed during the fog period and contributed negligibly to the increase in OOA concentrations due to fog scavenging processes. The rapid daytime aging, as indicated by the rapid decrease in m,p-xylene/ethylbenzene ratios, promoted the daytime formation of precursors for aqSOA formation, e.g., carbonyls such as methylglyoxal. Photooxidants related to aqSOA formation such as OH radical and H2O2 also bear fast daytime growth features even under low solar radiative conditions. The simultaneous increases in ultraviolet radiation, photooxidant, and aqSOA precursor levels worked together to promote the daytime photochemical aqSOA formation. We also found that biomass burning emissions can promote photochemical aqSOA formation by adding to the levels of aqueous-phase photooxidants and aqSOA precursors. Therefore, future mitigation of air pollution in a polluted environment would benefit from stricter control on biomass burning especially under high RH conditions.

Predicting cloud condensation nuclei number concentration based on conventional measurements of aerosol properties in the North China Plain.

Cloud condensation nuclei (CCN) play an important role in the formation and evolution of cloud droplets. However, the dataset of global CCN number concentration (NCCN) is still scarce due to the lack of direct CCN measurements, hindering an accurate evaluation of its climate effects. Alternative approaches to determine NCCN have thus been proposed to calculate NCCN based on measurements of other aerosol properties, such as particle number size distribution, bulk aerosol chemical composition and aerosol optical properties. To better understand the interaction between haze pollution and climate, we performed direct CCN measurements in the winter of 2018 at the Gucheng site, a typical polluted suburban site in North China Plain (NCP). The results show that the average CCN concentrations were 3.81 × 103 cm-3, 5.35 × 103 cm-3, 9.74 × 103 cm-3, 1.27 × 104 cm-3, 1.44 × 104 cm-3 at measured supersaturation levels of 0.114%, 0.148%, 0.273%, 0.492% and 0.864%, respectively. Based on these observational data, we have further investigated two methods of calculating NCCN from: (1) bulk aerosol chemical composition and particle number size distribution; (2) bulk aerosol chemical composition and aerosol optical properties. Our results showed that both methods could well reproduce the observed concentration (R2 > 0.88) and variability of NCCN with a 9% to 23% difference in the mean value. Further error analysis shows that the estimated NCCN tends to be underestimated by about 20% during the daytime while overestimated by <10% at night compared with the measured NCCN. These results provide quantitative instructions for the NCCN prediction based on conventional aerosol measurements in the NCP.

Method to retrieve the nocturnal aerosol optical depth with a CCD laser aerosol detective system.

Aerosol optical depth (AOD) is a crucial parameter in describing the atmospheric pollution and analyzing the influences of aerosol on the radiative equilibrium. Currently, no method can precisely and continuously measure the nocturnal AOD. In this study, a novel method was developed to retrieve the nocturnal AOD based on a remote sensing instrument called the charge-coupled device-laser aerosol detective system (CCD-LADS). CCD-LADS consists of a CCD camera, a continuous laser, a fisheye lens, and related filters. The AOD can be calculated by integrating the aerosol extinction coefficient profile retrieved from CCD-LADS measurements. The retrieved AOD was validated with AERONET and MODIS data sets. The comparison shows good agreement.

Unification of de novo and acquired ibrutinib resistance in mantle cell lymphoma.

The novel Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-ß1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies.

Concurrence of B-lymphoblastic leukemia and myeloproliferative neoplasm with copy neutral loss of heterozygosity at chromosome 1p harboring a MPL W515S mutation.

B-lymphoblastic leukemia (B-ALL) is a neoplasm of precursors committed to B-cell lineage, whereas myeloproliferative neoplasm (MPN) is a clonal proliferation derived from myeloid stem cells. Concurrent B-ALL with MPN is uncommon except in the presence of abnormalities of the PDGFRA, PDGFRB, or FGFR1 genes or the BCR-ABL1 fusion gene. Herein, we describe a rare concurrence, B-ALL with MPN without the aforementioned genetic aberrations, in a 64-year-old male patient. The patient was initially diagnosed with B-ALL with normal karyotype and responded well to aggressive chemotherapy but had sustained leukocytosis and splenomegaly. The posttreatment restaging bone marrow was free of B-ALL but remained hypercellular with myeloid predominance. Using a single nucleotide polymorphism microarray study, we identified a copy neutral loss of heterozygosity at the terminus of 1p in the bone marrow samples taken at diagnosis and again at remission, 49% and 100%, respectively. Several additional genetic abnormalities were present in the initial marrow sample but not in the remission marrow samples. Retrospective molecular studies detected a MPL W515S homozygous mutation in both the initial and remission marrows for B-ALL, at 30-40% and 80% dosage effect, respectively. In summary, we present a case of concurrent B-ALL and MPN and demonstrate a stepwise cytogenetic and molecular approach to the final diagnosis.

c-MYC-miRNA circuitry: a central regulator of aggressive B-cell malignancies.

MYC (c-Myc) deregulation has been frequently associated with aggressive lymphomas and adverse clinical outcome in B-cell malignancies. MYC has been implicated in controlling the expression of miRNAs, and MYC-regulated miRNAs affect virtually all aspects of the hallmarks of MYC-driven lymphomas. Increasing evidence has indicated that there is significant cross-talk between MYC and miRNAs, with MYC regulating expression of a number of miRNAs, resulting in widespread repression of miRNA and, at the same time, MYC being subjected to regulation by miRNAs, leading to sustained MYC activity and the corresponding MYC downstream pathways. Thus, these combined effects of MYC overexpression and downregulation of miRNAs play a central regulatory role in the MYC oncogenic pathways and MYC-driven lymphomagenesis. Here, we provide biological insight on the function of MYC-regulated miRNAs, the mechanisms of MYC-induced miRNA repression, and the complicated feedback circuitry underlying lymphoma progression, as well as potential therapeutic targets in aggressive B-cell lymphomas.

AKAR2-AKAP12 fusion protein "biosenses" dynamic phosphorylation and localization of a GPCR-based scaffold.

BACKGROUND: The cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation. AKAP12 is a scaffold that docks PKA, G-protein-coupled receptors, cell membrane negatively-charged phospholipids, and catalyzes receptor resensitization and recycling. In the current work, the AKAR2 biosensor was fused to the N-terminus of AKAP12 to evaluate its ability to function and report on dynamic phosphorylation of the AKAP12 scaffold. RESULTS: AKAR2-AKAP12 can be expressed in mammalian cells, is fully functional, and reveals the spatial-temporal activation of AKAP12 undergoing phosphorylation by PKA in response to beta-adrenergic activation in human epidermoid carcinoma A431 cells. CONCLUSION: The dynamic phosphorylation of AKAP12 "biosensed" by AKAR2-AKAP12 reveals the scaffold in association with the cell membrane, undergoing rapid phosphorylation by PKA. The perinuclear, cytoplasmic accumulation of phosphorylated scaffold reflects the phosphorylated, PKA-activated form of AKAP12, which catalyzes the resensitization and recycling of desensitized, internalized G-protein-coupled receptors.

G-protein-coupled receptor-associated A-kinase anchoring proteins AKAP5 and AKAP12: differential signaling to MAPK and GPCR recycling.

BACKGROUND: A-kinase Anchoring Protein AKAP5 and AKAP12 both dock to the beta2-adrenergic receptor, the former constitutively, the latter dynamically in response to activation of the receptor with agonist. RESULTS: In the current work we analyze the ability of each AKAP to contribute to two downstream signaling events, the activation of mitogen-activate protein kinase and the resensitization/recycling of the internalized, desensitized beta2-adrenergic receptor to the cell membrane. Although both AKAP share a large number of docking partners in common (e.g., beta2-adrenergic receptor, protein kinases A and C, protein phosphatase-2B, and negatively-charged membrane phospholipids), AKAP5 and AKAP12 are shown to segregate with respect to activation of Erk1,2 and to resensitization/recycling of beta2-adrenergic receptor. A431 cells were found to highly express AKAP12, but little of AKAP5. HEK293 cells, in contrast, were found to highly express AKAP5, but little of AKAP12. Suppression of the expression of AKAP5 in either A431 cells or HEK293 cells leads to loss of the ability of the beta2-adrenergic receptor to activate Erk1,2. Suppression of the expression of AKAP12 in either cell line leads to loss of the ability of these cells to resensitize the beta2-adrenergic receptor. CONCLUSION: Knock-down experiments of endogenous AKAP 5 and AKAP12 in two cell lines used commonly to study beta2-adrenergic receptor signaling clearly discriminate between the activation of mitogen-activated protein kinase (a downstream read-out solely mediated by AKAP5) and receptor recycling (a downstream read-out solely mediated by AKAP12).

Lysophosphatidic acid regulates trafficking of beta2-adrenergic receptors: the Galpha13/p115RhoGEF/JNK pathway stimulates receptor internalization.

Lysophosphatidic acid is an important lipid ligand regulating many aspects of cell function, including proliferation and migration. Operating via heterotrimeric G proteins to downstream effectors, lysophosphatidic acid was shown to regulate the function and trafficking of the G protein-coupled beta(2)-adrenergic receptor. C3 exotoxin, expression of dominant negative RhoA, and inhibition of c-Jun N-terminal kinase blocked the ability of lysophosphatidic acid to sequester the beta(2)-adrenergic receptor, whereas expression of constitutively active Galpha(13), p115RhoGEF, or RhoA mimicked lysophosphatidic acid (LPA) action, stimulating the internalization of the Galpha(s)-coupled beta(2)-adrenergic receptor. This study revealed a novel cross-talk exerted from the LPA/Galpha(13)/p115RhoGEF/RhoA pathway to the beta(2)-adrenergic receptor/Galpha(s)/adenylyl cyclase pathway, attenuating the ability of beta-adrenergic agonists to act following stimulation of cells by LPA as may occur during beta-adrenergic therapy of an inflammatory response.

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.

Regulation of AKAP-membrane interactions by calcium.

The AKAP gravin is a scaffold for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of beta2-adrenergic receptors. Gravin binds to the receptor through well characterized protein-protein interactions. These interactions are facilitated approximately 1000-fold when gravin is anchored to the cytoplasmic leaflet of the plasma membrane. Although the N-terminal region (approximately 550 residues) is highly negatively charged and probably natively unfolded, it could anchor gravin to the inner leaflet through hydrophobic insertion of its N-terminal myristate and electrostatic binding of three short positively charged domains (PCDs). Loss of the site of N-myristoylation was found to affect neither AKAP macroscopic localization nor AKAP function. Synthetic peptides corresponding to PCD1-3 bound in vitro to unilamellar phospholipid vesicles with high affinity, a binding reversed by calmodulin in the presence of Ca2+. In vivo gravin localization is regulated by intracellular Ca2+, a function mapping to the N terminus of the protein harboring PCD1, PCD2, and PCD3. Mutation of any two PCDs eliminates membrane association of the non-myristoylated gravin, the sensitivity to Ca2+/calmodulin, and the ability of this scaffold to catalyze receptor resensitization and recycling.

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.

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

Cell signalling mediated via GPCRs (G-protein-coupled receptors) is a major paradigm in biology, involving the assembly of receptors, G-proteins, effectors and downstream elements into complexes that approach in design 'solid-state' signalling devices. Scaffold molecules, such as the AKAPs (A-kinase anchoring proteins), were discovered more than a decade ago and represent dynamic platforms, enabling multivalent signalling. AKAP79 and AKAP250 were the first to be shown to bind to membrane-embedded GPCRs, orchestrating the interactions of various protein kinases (including tyrosine kinases), protein phosphatases (e.g. calcineurin) and cytoskeletal elements with at least one member of the superfamily of GPCRs, the prototypical beta2-adrenergic receptor. In this review, the multivalent interactions of AKAP250 with the cell membrane, receptor, cytoskeleton and constituent components are detailed, providing a working model for AKAP-based GPCR signalling complexes. Dynamic regulation of the AKAP-receptor complex is mediated by ordered protein phosphorylation.

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.