The angiotensin Ⅱ type 1 receptor and receptor－associated proteins

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.     

2-Aminoimidazoles inhibitors of TGF-β receptor 1

The 4-(5-fluoro-6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-1H-imidazol-2-ylamine 3 is a potent and selective inhibitor of TGF-βR1. Substitution of the amino group of 3 typically led to a slight decrease in the affinity for the receptor and in TGF-β-inducted PAI-luciferase reporter activity. However, 2-acetamidoimidazoles were identified as attractive candidates for further optimization as a result of their significant activity combined to their superior pharmacokinetic profile.     

Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking

Very little is understood about the trafficking of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) to the plasma membrane. Rab guanosine triphosphatases (GTPases) are known to participate in the trafficking of various GPCRs via a direct interaction during the endocytic pathway, but whether this occurs in the anterograde pathway is unknown. We evaluated the potential interaction of Rab1, a GTPase known to regulate β2-adrenergic receptor (β2AR) trafficking, and its effect on export from the ER. Our results show that GTP-bound Rab1 interacts with the F(x)(6)LL motif of β2AR. Receptors lacking the interaction motif fail to traffic properly, suggesting that a direct interaction with Rab1 is required for β2AR anterograde trafficking.     

The angiotensin Ⅱ type 1 receptor and receptor-associated proteins

INTRODUCTIONThe renin-angiotensin system (RAS) is consid-ered to be the major regulator of blood pressure)electrolyte balance and renal, neuronal as well as en-docrine functions related to cardiovascular control.The RAS is the key factor in most cases essential hy-pertension, as indicated by successes in treatment ofhypertensive patients with various angiotensin I con-verting enzyme (ACE) inhibitors and receptor block-ers. Renin was a central subject of intense investigation because of…     

Functional selectivity of adenosine A1 receptor ligands?

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this ‘biased’ form of signaling. We screened over 800 compounds targeting the class of adenosine A₁ receptors using a β-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [³H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of β-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A₁ receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated.     

Nogo receptor 3, a paralog of Nogo-66 receptor 1 （NgR1）, may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system (CNS).Nogo-66 (a 66-residue domain of Nogo-A) expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 (NgR1).A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered.However,detailed composition of the complex and its signaling mechanisms remain to be fully elucidated.In this study,we show that Nogo receptor 3 (NgR3),a paralog of NgR1,is a binding protein for NgR1.The interaction is highly specific because other members of the reticulin family,to which Nogo-A belongs,do not bind to NgR3.Neither does NgR3 show any binding activity with Nogo receptor 2 (NgR2),another NgR1 paralog.Majority of NgR3 domains are required for its binding to NgR1.Moreover,a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture.These in vitro results,together with previously reported overlapping expression profile between NgR1 and NgR3,suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.     

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

IL-13 receptor α1 differentially regulates aeroallergen-induced lung responses

IL-13 and IL-4 are hallmark cytokines of Th2-associated diseases including asthma. Recent studies revealed that IL-13Rα1 regulates asthma pathogenesis by mediating both IL-4- and IL-13-mediated responses. Nonetheless, the relative contribution of each cytokine in response to aeroallergen challenge and the degree of functional dichotomy between IL-4 and IL-13 in asthma remains unclear. Consistent with prior publications, we demonstrate that IL-13Rα1 regulates aeroallergen-induced airway resistance and mucus production but not IgE and Th2 cytokine production. We demonstrate that aeroallergen-induced eosinophil recruitment and chemokine production were largely dependent on IL-13Rα1 after Aspergillus but not house dust mite (HDM) challenges. Notably, Aspergillus-challenged mice displayed increased IL-13Rα1-dependent accumulation of dendritic cell subsets into lung-draining lymph nodes in comparison with HDM-challenged mice. Comparison of IL-4 and IL-13 levels in the different experimental models revealed increased IL-4/IL-13 ratios after HDM challenge, likely explaining the IL-13Rα1-independent eosinophilia and chemokine production. Consistently, eosinophil adoptive transfer experiments revealed near ablation of lung eosinophilia in response to Aspergillus in Il13ra1(-/-) mice, suggesting that Aspergillus-induced lung eosinophil recruitment is regulated by IL-13-induced chemokine production rather than altered IL-13 signaling in eosinophils. Furthermore, the near complete protection observed in Il13ra1(-/-) mice in response to Aspergillus challenge was dependent on mucosal sensitization, as alum/Aspergillus-sensitized mice that were rechallenged with Aspergillus developed IL-13Rα1-independent eosinophilia although other asthma parameters remained IL-13Rα1 dependent. These results establish that IL-13Rα1 is required for aeroallergen-induced airway resistance and that allergen-induced chemokine production and consequent eosinophilia is dictated by the balance between IL-4 and IL-13 production in situ.     

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.     

Cardiac and neuroprotection regulated by α1-adrenergic receptor subtypes

Sympathetic nervous system regulation by the α₁-adrenergic receptor (AR) subtypes (α_1A, α_1B, α_1D) is complex, whereby chronic activity can be either detrimental or protective for both heart and brain function. This review will summarize the evidence that this dual regulation can be mediated through the different α₁-AR subtypes in the context of cardiac hypertrophy, heart failure, apoptosis, ischemic preconditioning, neurogenesis, locomotion, neurodegeneration, cognition, neuroplasticity, depression, anxiety, epilepsy, and mental illness.     

Influence of interleukin-12 receptor β1 polymorphisms on tuberculosis

Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A-->G, 684C-->T, 1094T-->C, and 1132G-->C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity.     

Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.     

Association of ��2-adrenergic receptor and insulin receptor substrate-1 polymorphisms with obesity in a Northern Indian population

BACKGROUND:

Imbalance in hormonal levels, regulated by host genetic factors, are known to be a major cause of obesity. Therefore, we aimed to evaluate association of genetic polymorphisms of β₂-adrenergic receptor (β₂-AR) and insulin receptor substrate-1 (IRS-1) with hormonal levels in northern Indian obese.

METHODS:

A total of 111 obese and 89 age matched non-obese subjects were studied after taking detailed clinical profile. Hormonal assays in serum/plasma for different hormones were done using IRMA and RIA kits. Genetic analysis of β₂-AR (-47 and -20, T to C) and IRS-1 (Arg972Gly) was done using PCR-RFLP.

STATISTICAL ANALYSIS:

Statistical analysis was performed by SPSS (version 11.5) software. All continuous variables were expressed as mean ± SD and tested by ANOVA test. Comparisons of categorical variables were assessed using X² tests or Fisher''s exact test. P-value <0.05 was considered as significant.

RESULTS:

Analysis showed that obese subjects had significantly higher value of blood pressure (systolic), WHR, leptin insulin and glucagon and lower value of GH. In β₂-AR (-47) T/C and IRS-1 Gly972Arg gene polymorphisms we did not found significant differences in genotype or allele frequencies. Moreover, none of the studied hormonal or metabolic parameters showed any association with the gene polymorphisms.

CONCLUSIONS:

Study reveals no significant association of β₂-AR (-47 and -20, T to C) and IRS-1 Gly 972 Arg polymorphisms with obesity in northern Indians.