cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.

Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.

Dual role of the beta(2)-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta(2)-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta(2)-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta(2)-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.

beta-arrestin binding to the beta(2)-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta(2)-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats

Rodrigues SF, Tran ED, Fortes ZB, Schmid-Schonbein GW. Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H25-H35, 2010. First published April 9, 2010; doi:10.1152/ajpheart.00620.2009.-We recently observed the enhanced serine and matrix metalloproteinase (MMP) activity in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) rat and the cleavage of membrane receptors in the SHR by MMPs. We demonstrate in vivo that MMP-7 and MMP-9 injection leads to a vasoconstrictor response in microvessels of rats that is blocked by a specific MMP inhibitor (GM-6001, 1 mu M). Multiple pathways may be responsible. Since the beta(2)-adrenergic receptor (beta(2)-AR) is susceptible to the action of endogenous MMPs, we hypothesize that MMPs in the plasma of SHRs are able to cleave the extracellular domain of the beta(2)-AR. SHR arterioles respond in an attenuated fashion to beta(2)-AR agonists and antagonists. Aorta and heart muscle of control Wistar rats were exposed for 24 h (37 C) to fresh plasma of male Wistar and WKY rats and SHRs with and without doxycycline (30 mu M) and EDTA (10 mM) to reduce MMP activity. The density of extracellular and intracellular domains of beta(2)-AR was determined by immunohistochemistry. The density of the extracellular domain of beta(2)-AR is reduced in aortic endothelial cells and cardiac microvessels of SHRs compared with that of WKY or Wistar rats. Treatment of the aorta and the heart of control Wistar rats with plasma from SHRs, but not from WKY rats, reduced the number of extracellular domains, but not intracellular domains, of beta(2)-AR in aortic endothelial cells and cardiac microvessels. MMP inhibitors (EDTA and doxycycline) prevented the cleavage of the extracellular domain. Thus MMPs may contribute to the reduced density of the extracellular domain of beta(2)-AR in blood vessels and to the increased arteriolar tone of SHRs compared with normotensive rats.

Prognostic significance of beta-2 adrenergic receptor in oral squamous cell carcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Short-Term Effectiveness of Non-Surgical Treatment in Reducing Levels of Interleukin-1 beta and Proteases in Gingival Crevicular Fluid From Patients With Type 2 Diabetes Mellitus and Chronic Periodontitis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Human beta-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

Objective: Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human beta defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. Methods: Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after nonsurgical periodontal treatment. Salivary HBD-2, and GCF TNF-alpha levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. Results: P. gingivalis was significantly (p < 0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p < 0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-alpha, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p < 0.001). Conclusions: Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression. (C) 2012 Elsevier Ltd. All rights reserved.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

Virulent Salmonella enterica infections can be exacerbated by concomitant infection of the host with a live attenuated S. enterica vaccine via Toll-like receptor 4-dependent interleukin-10 production with the involvement of both TRIF and MyD88.

During systemic disease in mice, Salmonella enterica grows intracellularly within discrete foci of infection in the spleen and liver. In concomitant infections, foci containing different S. enterica strains are spatially separated. We have investigated whether functional interactions between bacterial populations within the same host can occur despite the known spatial separation of the foci and independence of growth of salmonellae residing in different foci. In this study we have demonstrated that bacterial numbers of virulent S. enterica serovar Typhimurium C5 strain in mouse tissues can be increased by the presence of the attenuated aroA S. Typhimurium SL3261 vaccine strain in the same tissue. Disease exacerbation does not require simultaneous coinjection of the attenuated bacteria. SL3261 can be administered up to 48 hr after or 24 hr before the administration of C5 and still determine higher tissue numbers of the virulent bacteria. This indicates that intravenous administration of a S. enterica vaccine strain could potentially exacerbate an established infection with wild-type bacteria. These data also suggest that the severity of an infection with a virulent S. enterica strain can be increased by the prior administration of a live attenuated vaccine strain if infection occurs within 48 hr of vaccination. Exacerbation of the growth of C5 requires Toll-like receptor 4-dependent interleukin-10 production with the involvement of both Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta and myeloid differentiation factor 88.

Cloning of the cDNA for the human beta 1-adrenergic receptor.

Screening of a human placenta lambda gt11 library has led to the isolation of the cDNA for the human beta 1-adrenergic receptor (beta 1AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human beta 2-adrenergic receptor (beta 2AR). The 2.4-kilobase cDNA for the human beta 1AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian beta AR but only 54% homologous with the human beta 2AR. This suggests that the avian gene encoding beta AR and the human gene encoding beta 1AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with beta 1AR binding. This pattern is quite distinct from the pattern obtained when the beta 2AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical beta 1AR specificity. This contrasts with the typical beta 2 subtype specificity observed when the human beta 2AR cDNA is expressed in this system. Mammalian beta 1AR and beta 2AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.