Assembly of a β2‐adrenergic receptor—GluR1 signalling complex for localized cAMP signalling

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the β₂‐adrenergic receptor (β₂AR), the trimeric G_s protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA‐type glutamate receptor subunit GluR1, which is linked to the β₂AR through stargazin and PSD‐95 and their homologues. Only GluR1 associated with the β₂AR is phosphorylated by PKA on β₂AR stimulation. Peptides that interfere with the β₂AR–GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic β₂AR–cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.     

Effects of the β‐agonist,isoprenaline, on the down‐regulation,functional responsiveness and trafficking of β2‐adrenergic receptors with N‐terminal polymorphisms

The β₂‐AR (β₂‐adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N‐terminal polymorphisms of β₂‐AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down‐regulation of β₂‐AR variants following β‐agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β₂‐AR (designated β₂‐AR‐RE, β₂‐AR‐GE, β₂‐AR‐RQ and β₂‐AR‐GQ) were studied using site‐directed mutagenesis and recombinant expression in HEK‐293 cells (human embryonic kidney cells). Ligand‐binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg¹⁶ (β₂‐AR‐RE and β₂‐AR‐RQ) underwent increased down‐regulation compared with isoforms with Gly¹⁶ (β₂‐AR‐GE and β₂‐AR‐GQ). Consistent with these differences in down‐regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β₂‐AR‐RE relative to β₂‐AR‐GE. Confocal microscopy revealed that the receptor isoforms had similar co‐localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co‐localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co‐localization of β₂‐AR‐RE with the lysosomal marker LAMP1 (lysosome‐associated membrane protein 1) compared with that of β₂‐AR‐GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β‐agonist involves differences in the efficiency with which agonist‐activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.     

Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

The role of β‐adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere‐derived cells

The effects of β adrenergic receptors (β‐ARs) and p38 mitogen‐activated protein kinases (MAPK) pathways on cardiosphere‐derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β‐ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1‐AR blocker (Met group), β2‐AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non‐selective β‐AR blocker (PRO group), and β‐AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST‐1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β‐AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1‐AR as well as c‐Kit and β2‐AR, respectively. These results suggested that β‐ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.     

Insights into β2‐adrenergic receptor binding from structures of the N‐terminal lobe of ARRDC3

ARRDC3 is one of six known human α‐arrestins, and has been implicated in the downregulation of the β2‐adrenergic receptor (β2AR). ARRDC3 consists of a two‐lobed arrestin fold and a C‐terminal tail containing two PPYX motifs. In the current model for receptor downregulation by ARRDC3, the arrestin fold portion is thought to bind the receptor, while the PPXY motifs recruit ubiquitin ligases of the NEDD4 family. Here we report the crystal structures of the N‐terminal lobe of human ARRDC3 in two conformations, at 1.73 and 2.8 Å resolution, respectively. The structures reveal a large electropositive region that is capable of binding phosphate ions of crystallization. Residues within the basic patch were shown to be important for binding to β2AR, similar to the situation with β‐arrestins. This highlights potential parallels in receptor recognition between α‐ and β‐arrestins.     

Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin

Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α_2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α₂ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α_2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α_2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α_2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α_2AAR endocytosis and that α_2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α_2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α_2AARs. Importantly, we show that α₂AR agonist-mediated α_2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α₂ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α_2AARs and accelerating α_2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α_2AARs.     

Arrestin domain‐containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the β2‐adrenergic receptor

Prolonged stimulation of the β2‐adrenergic receptor (β2AR) leads to receptor ubiquitination and downregulation. Using a genome‐wide RNA interference screen, we identified arrestin domain‐containing 3 (ARRDC3) as a gene required for β2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co‐localizes with activated β2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and β2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)‐mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for β2AR ubiquitination.     

Syntheses and β‐adrenergic binding affinities of (R)‐ and (S)‐fluoronaphthyloxypropanolamines

The β‐adrenergic receptors mediate several physiological processes including heart rate (β₁), bronchodilation (β₂), and lipolysis (β₃). Therefore, selectivity is important for a possible therapeutic agent acting via these receptors. Aryloxypropanolamines are β‐receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring in this class could lead to significant biological effects because of the unique chemical characteristics of fluorine. Because the target compound has a chiral center, we set out to synthesize the two enantiomers so that effects of stereochemistry on biological activity could be evaluated. Syntheses of the enantiomers were performed starting with commercially available fluoronaphthalene and subsequent use of the chiral synthon (2R)‐ or (2S)‐glycidyl 3‐nitrobenzenesulfonate, depending on the desired enantiomer. High‐pressure liquid chromatography (HPLC) methods were used to characterize %ee. Each enantiomer was synthesized. They exhibited nanomolar binding activities on β‐adrenergic receptors. The (S)‐enantiomer was found to be up to 310 times more potent than the (R). It was also found to be about five‐fold more selective for β₂‐ than for β₁‐receptors. The current report demonstrates the importance of stereochemistry for the fluoroaromatic β‐receptor ligands. Chirality 11:144–148, 1999. © 1999 Wiley‐Liss, Inc.     

Cardiac β‐adrenergic receptor activation mediates distinct and cell type‐dependent changes in the expression and distribution of connexin 43

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β₂‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β₂‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β₂‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.     

Binding of protein phosphatase 2A to the L-type calcium channel Cav1.2 next to Ser1928, its main PKA site, is critical for Ser1928 dephosphorylation

The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.     

Surface L‐type Ca2+ channel expression levels are increased in aged hippocampus

Age‐related increase in L‐type Ca²⁺ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca²⁺ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age‐related cognitive deficits. Here, using specific antibodies against Ca_v1.2 and Ca_v1.3 subunits of LTCCs, we systematically re‐examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Ca_v1.2 and Ca_v1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Ca_v1.2 in the CA1 and CA3 regions and of Ca_v1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Ca_v1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Ca_v1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age‐related cognitive deficits cannot be attributed to a global change in L‐type channel expression nor to the level of phosphorylation of Ca_v1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age‐related increase in L‐type Ca²⁺ channel activity in CA1 pyramidal neurons.     

Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon β2‐adrenergic receptor activation

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the “second wave” of signaling from endosomes. Here, we set out to determine whether G_s signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin‐coated structures (CCSs) or caveolae upon activation by β₂‐adrenergic receptors (β₂AR). Activated β₂AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after β₂AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gα_s and Gγ₂ exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon β₂AR stimulation, no redistribution to CCSs, and weak activation‐independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated G_s signaling cascade members and confirm that β₂AR and arrestin 3 accumulate in CCSs, while Gα_s, Gγ₂ and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.     

Amyloid beta peptide-(1-42) induces internalization and degradation of beta2 adrenergic receptors in prefrontal cortical neurons

Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β(2) adrenergic receptor (β(2)AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β(2)AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β(2)AR in fibroblasts and endogenous β(2)AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β(2)AR into lysosome, and prolonged Aβ treatment causes β(2)AR degradation. The Aβ-induced β(2)AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β(2)AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β(2)AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β(2)AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β(2)AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β(2)AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities.     

Gender differences in β‐adrenoceptor system in cardiac hypertrophy due to arteriovenous fistula

This study was undertaken to determine alterations in the β‐adrenoceptor (β‐AR) signaling system in male and female rats at 4 weeks after the induction of arteriovenous (AV) fistula or shunt. AV shunt produced a greater degree of cardiac hypertrophy and larger increase in cardiac output in male than in female animals. Increases in plasma levels of norepinephrine and epinephrine (EPI) due to AV shunt were also higher in male than females. While no difference in the β₁‐AR affinity was seen in males and females, AV shunt induced increase in β₁‐AR density in female rats was higher than that in males. Furthermore, no changes in basal adenylyl cyclase (AC) V/VI mRNA levels were seen; however, the increase in EPI‐stimulated AC activities was greater in AV shunt females than in males. AV shunt decreased myocardial β₁‐AR mRNA level in male rats and increased β₂‐AR mRNA level in female hearts; an increase in G_i‐protein mRNA was detected only in male hearts. Although GRK2 gene expression was increased in both sexes, an increase in GRK3 mRNA was seen only in AV shunt female rats. β‐arrestin1 mRNA was elevated in females whereas β‐arrestin 2 gene expression was increased in both male and female AV shunt rats. While these data demonstrate gender associated differences in various components of the β‐AR system in cardiac hypertrophy due to AV shunt, only higher levels of plasma catecholamines may account for the greater increase in cardiac output and higher degree of cardiac hypertrophy in males. J. Cell. Physiol. 226: 181–186, 2010. © 2010 Wiley‐Liss, Inc.     

β‐adrenergic blockade attenuates cardiac dysfunction and myofibrillar remodelling in congestive heart failure

Although β‐adrenoceptor (β‐AR) blockade is an important mode of therapy for congestive heart failure (CHF), subcellular mechanisms associated with its beneficial effects are not clear. Three weeks after inducing myocardial infarction (MI), rats were treated daily with or without 20 and 75 mg/kg atenolol, a selective β₁‐AR antagonist, or propranolol, a non‐selective β‐AR antagonist, for 5 weeks. Sham operated rats served as controls. All animals were assessed haemodynamically and echocardiographically and the left ventricle (LV) was processed for the determination of myofibrillar ATPase activity, α‐ and β‐myosin heavy chain (MHC) isoforms and gene expression as well as cardiac troponin I (cTnI) phosphorylation. Both atenolol and propranolol at 20 and 75 mg/kg doses attenuated cardiac hypertrophy and lung congestion in addition to increasing LV ejection fraction and LV systolic pressure as well as decreasing heart rate, LV end‐diastolic pressure and LV diameters in the infarcted animals. Treatment of infarcted animals with these agents also attenuated the MI‐induced depression in myofibrillar Ca²⁺‐stimulated ATPase activity and phosphorylated cTnI protein content. The MI‐induced decrease in α‐MHC and increase in β‐MHC protein content were attenuated by both atenolol and propranolol at low and high doses; however, only high dose of propranolol was effective in mitigating changes in the gene expression for α‐MHC and β‐MHC. Our results suggest that improvement of cardiac function by β‐AR blockade in CHF may be associated with attenuation of myofibrillar remodelling.     

RalA GTPase Tethers Insulin Granules to L‐ and R‐Type Calcium Channels Through Binding α2δ‐1 Subunit

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage‐gated calcium (Ca_v) channels. RalA knockdown (KD) in INS‐1 cells and primary rat β‐cells resulted in a reduction in Ca²⁺ currents arising specifically from L‐(Ca_v1.2 and Ca_v1.3) and R‐type (Ca_v2.3) Ca²⁺ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca²⁺ currents. RalA co‐immunoprecipitated with the Ca_vα₂δ‐1 auxiliary subunit known to bind the three Ca_vs. Moreover, the functional molecular interactions between Ca_vα₂δ‐1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α₂δ‐1 to insulin granules without affecting the localization of the other Ca_v subunits. Furthermore, we confirmed that RalA and α₂δ‐1 functionally interact since RalA KD‐induced inhibition of Ca_v currents could not be recovered by RalA when α₂δ‐1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α₂δ‐1 on insulin granules to tether these granules to PM Ca²⁺ channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.     

Somatostatin receptor-2 negatively regulates β-adrenergic receptor mediated Ca dependent signaling pathways in H9c2 cells

In the present study, we report that somatostatin receptor 2 (SSTR2) plays a crucial role in modulation of β₁AR and β₂AR mediated signaling pathways that are associated with increased intracellular Ca^2 + and cardiac complications. In H9c2 cells, SSTR2 colocalizes with β₁AR or β₂AR in receptor specific manner. SSTR2 selective agonist inhibits isoproterenol and formoterol stimulated cAMP formation and PKA phosphorylation in concentration dependent manner. In the presence of SSTR2 agonist, the expression of PKCα and PKCβ was comparable to the basal condition, however SSTR2 agonist inhibits isoproterenol or formoterol induced PKCα and PKCβ expression, respectively. Furthermore, the activation of SSTR2 not only inhibits calcineurin expression and its activity, but also blocks NFAT dephosphorylation and its nuclear translocation. SSTR2 selective agonist abrogates isoproterenol mediated increase in cell size and protein content (an index of hypertrophy). Taken together, the results described here provide direct evidence in support of cardiac protective role of SSTR2 via modulation of Ca^2 + associated signaling pathways attributed to cardiac hypertrophy.     

A macromolecular trafficking complex composed of β2-adrenergic receptors,A-Kinase Anchoring Proteins and L-type calcium channels

Abstract

Sympathetic modulation of cardiac L-type calcium channels is an important mechanism for regulating heart rate and cardiac contractility. At the molecular level, activation of β-adrenergic receptors (βAR) increases calcium influx into cardiac myocytes by activating protein kinase A (PKA), leading to subsequent phosphorylation of L-type calcium channels. In the case of the β₂AR, this process is facilitated by the presence of A-Kinase Anchoring Proteins (AKAPs) that serve as scaffolding proteins for the L-type calcium channel and the β₂AR complex. Our work has shown that, in addition to facilitating PKA phosphorylation of the channel, AKAPs also promote an increase in the Cav1.2 channel surface expression. Here we review the molecular mechanisms of β₂AR/AKAP/L-type channel interactions and trafficking.     

β‐Adrenergic‐induced CD40 overexpression on gingival fibroblasts: Role of PGE2

CD40, a member of the tumour necrosis factor‐α receptor family, is constitutively expressed by cells of haematopoietic and non‐haematopoietic origin, including fibroblasts. Signalling through this receptor molecule regulates inflammatory mediator secretion by many cell types. The work has been performed in healthy subjects and the authors studied, by cellular culture, flow cytometric analysis and ELISA assay, the expression of CD40 and PGE₂ (prostaglandin E₂) generation on gingival fibroblasts stimulated by β‐AR (β‐adrenoceptor) agonists. Herein, the authors demonstrate that β‐AR subtype activation via their own specific agonists markedly increased CD40 expression on human gingival fibroblasts. This effect was prevented by β‐AR subtype‐specific antagonists. In addition, gingival fibroblast β‐AR stimulation resulted in an increase in PGE₂ generation. The inhibition of PLA₂ (phospholipase A₂) and COX‐1 (cyclo‐oxygenase‐1) but not COX‐2 impaired β‐AR increase of PGE₂, an effect that was restored by the addition of low concentrations of PGE₂, suggesting that PGE₂ generation is implicated in the mechanism underlying β‐AR‐agonist‐mediated CD40 overexpression. Our work has revealed an endogenous β‐AR mediator network involving gingival fibroblasts.     

Deletion of the C-terminal phosphorylation sites in the cardiac β-subunit does not affect the basic β-adrenergic response of the heart and the Ca(v)1.2 channel

Phosphorylation of the cardiac β subunit (Ca(v)β(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)β(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Ca(v)β(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α(1C) subunit. The β-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished β-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)β(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.