The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
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oxygen regulated beta 2 Adrenergic Receptor hydroxylation by egln3 and ubiquitylation by pvhl
Science Signaling, 2009Co-Authors: Liang Xie, Robert J Lefkowitz, Kunhong Xiao, Erin J Whalen, Michael T Forrester, Robert S Freeman, Guohua Fong, Steven P Gygi, Jonathan S StamlerAbstract:Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled Receptors (GPCRs) play an essential role in surface Receptor homeostasis, thereby tuning many physiological processes. Although beta-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the beta(2)-Adrenergic Receptor (beta(2)AR), a prototypic GPCR, the molecular cues that mark Receptors for ubiquitylation and the regulation of Receptor degradation by the proteasome remain poorly understood. We show that the von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the beta(2)AR, thereby decreasing Receptor abundance. We further show that the interaction of pVHL with beta(2)AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the beta(2)AR to serve as an endogenous beta(2)AR prolyl hydroxylase. Under hypoxic conditions, Receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating Receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous beta(2)AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.
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β-Arrestin-dependent signaling and trafficking of 7-transmembrane Receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Sudha K Shenoy, William E. Miller, Arun K Shukla, Seungkirl Ahn, Aalok S. Modi, Kunhong Xiao, Magali Berthouze, Keith D. Wilkinson, Robert J LefkowitzAbstract:Beta-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seven-transmembrane Receptors (7TMRs). Agonist-stimulated ubiquitination of beta-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid beta(2)-Adrenergic Receptor (beta(2)AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds beta-arrestin2 and leads to the deubiquitination of beta-arrestins. USP33 and Mdm2 function reciprocally and favor respectively the stability or lability of the Receptor beta-arrestin complex, thus regulating the longevity and subcellular localization of Receptor signalosomes. Receptors such as the beta(2)AR, previously shown to form loose complexes with beta-arrestin ("class A") promote a beta-arrestin conformation conducive for binding to the deubiquitinase, whereas the vasopressin V2R, which forms tight beta-arrestin complexes ("class B"), promotes a distinct beta-arrestin conformation that favors dissociation of the enzyme. Thus, USP33-beta-arrestin interaction is a key regulatory step in 7TMR trafficking and signal transmission from the activated Receptors to downstream effectors.
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protein kinase a mediated phosphorylation of the β2 Adrenergic Receptor regulates its coupling to gs and gi demonstration in a reconstituted system
Journal of Biological Chemistry, 2002Co-Authors: Musa A Zamah, Martha Delahunty, Louis M Luttrell, Robert J LefkowitzAbstract:While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled Receptor, recent studies have indicated that some aspects of beta(2)-Adrenergic Receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type Receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant Receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp Receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by Receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala Receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-Adrenergic Receptor switches its predominant coupling from G(s) to G(i).
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regulation of Receptor fate by ubiquitination of activated beta 2 Adrenergic Receptor and beta arrestin
Science, 2001Co-Authors: Sudha K Shenoy, Patricia Mcdonald, Trudy A Kohout, Robert J LefkowitzAbstract:Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled Receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-Adrenergic Receptors (beta2ARs) led to rapid ubiquitination of both the Receptors and the Receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced Receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited Receptor internalization with marginal effects on Receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the Receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.
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beta arrestin mediated adp ribosylation factor 6 activation and beta 2 Adrenergic Receptor endocytosis
Journal of Biological Chemistry, 2001Co-Authors: Audrey Claing, William E. Miller, Wei Chen, Nicolas Vitale, Joel Moss, Richard T Premont, Robert J LefkowitzAbstract:Abstract β-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled Receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which β-arrestin orchestrates the process of Receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the β2-Adrenergic Receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance Receptor endocytosis. Endogenous β-arrestin is found in complex with ARNO. Upon agonist stimulation of the Receptor, β-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including β-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated Receptor recruits a small G protein necessary for the endocytic process and controls its activation.
Stephen B Liggett - One of the best experts on this subject based on the ideXlab platform.
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an ile to met polymorphism in the catalytic domain of adenylyl cyclase type 9 confers reduced beta 2 Adrenergic Receptor stimulation
Pharmacogenetics, 2003Co-Authors: Kersten M Small, Kari M Brown, Cheryl T Theiss, Carrie A Seman, Scott T Weiss, Stephen B LiggettAbstract:Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled Receptors such as the beta2-Adrenergic Receptor (beta2AR). Genetic variation in the Receptor component of this pathway can alter signal transduction and the response to beta-agonists in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an Ile to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile772) and polymorphic (Met772) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met772 AC9 has lower basal and beta2AR-mediated adenylyl cyclase activities compared to Ile772 AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met772 membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta2AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma.
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complex promoter and coding region beta 2 Adrenergic Receptor haplotypes alter Receptor expression and predict in vivo responsiveness
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Connie M Drysdale, Dennis W Mcgraw, Catharine B Stack, Claiborne J Stephens, Richard S Judson, Krishnan Nandabalan, Kevin M Arnold, Gualberto Ruano, Stephen B LiggettAbstract:The human β2-Adrenergic Receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5′ upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common β2-Adrenergic Receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to β agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. β2-Adrenergic Receptor mRNA levels and Receptor density in cells transfected with the haplotype associated with the greater physiologic response were ≈50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.
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myocardial signaling defects and impaired cardiac function of a human beta 2 Adrenergic Receptor polymorphism expressed in transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jamal Turki, Stuart A Green, John N Lorenz, Elizabeth T Donnelly, Marie T Jacinto, Stephen B LiggettAbstract: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.
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influence of beta 2 Adrenergic Receptor genotypes on signal transduction in human airway smooth muscle cells
American Journal of Respiratory Cell and Molecular Biology, 1995Co-Authors: Stuart A Green, J Turki, P Bejarano, Ian P Hall, Stephen B LiggettAbstract:The phenotypic relevance of allelic variation in the structure of the beta 2-Adrenergic Receptor (beta 2AR) expressed in lung cells is unknown. In particular, altered responsiveness of the beta 2AR expressed on airway smooth muscle, which are responsible for bronchodilation in the treatment of asthma, may be an important factor in the ultimate physiologic response to agonist. To approach this, we established primary cultures of human airway smooth muscle cells obtained at autopsy and developed a method to determine the beta 2AR genotype at the polymorphic loci of codons 16 and 27, using allele-specific polymerase chain reactions. Radioligand binding studies revealed that these cells expressed approximately 70 fmol/mg of Receptor which was exclusively of the beta 2AR subtype. All cell lines obtained (n = 10) exhibited normal agonist binding and Receptor-mediated activation of the adenylyl cyclase second messenger pathway. However, distinct differences were found in the response to long-term agonist exposur...
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influence of beta 2 Adrenergic Receptor genotypes on signal transduction in human airway smooth muscle cells
American Journal of Respiratory Cell and Molecular Biology, 1995Co-Authors: Stuart A Green, J Turki, P Bejarano, Ian P Hall, Stephen B LiggettAbstract:The phenotypic relevance of allelic variation in the structure of the beta 2-Adrenergic Receptor (beta 2AR) expressed in lung cells is unknown. In particular, altered responsiveness of the beta 2AR expressed on airway smooth muscle, which are responsible for bronchodilation in the treatment of asthma, may be an important factor in the ultimate physiologic response to agonist. To approach this, we established primary cultures of human airway smooth muscle cells obtained at autopsy and developed a method to determine the beta 2AR genotype at the polymorphic loci of codons 16 and 27, using allele-specific polymerase chain reactions. Radioligand binding studies revealed that these cells expressed approximately 70 fmol/mg of Receptor which was exclusively of the beta 2AR subtype. All cell lines obtained (n = 10) exhibited normal agonist binding and Receptor-mediated activation of the adenylyl cyclase second messenger pathway. However, distinct differences were found in the response to long-term agonist exposure between the different beta 2AR genotypes. Cells expressing Arg at codon 16 (Arg16) traditionally referred to as wild-type, underwent 77.8 +/- 8.1% downregulations of beta 2AR following prolonged (24-h) exposure to the beta 2AR agonist isoproterenol (10 microM). In contrast, cells expressing Gly16 beta 2AR underwent enhanced agonist-promoted downregulation (95.6 +/- 1.7%, P < 0.05 versus Arg16), whereas cells expressing Glu27 beta 2AR were relatively resistant to such downregulation (29.5 +/- 12.7%, P < 0.01 versus Arg16). For cells expressing Glu27 beta 2AR, this difference resulted in a significant attenuation of agonist-promoted functional desensitization (33 +/- 7 versus 90 +/- 5% desensitization for Arg16, P < 0.001) following preincubation with 1 microM isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)
Virginia M Sanders - One of the best experts on this subject based on the ideXlab platform.
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norepinephrine and beta 2 Adrenergic Receptor stimulation regulate cd4 t and b lymphocyte function in vitro and in vivo
Pharmacological Reviews, 2001Co-Authors: Adam P Kohm, Virginia M SandersAbstract:Historically, norepinephrine and the sympathetic nervous system have been associated with the "fight or flight" response, and they also contribute to the regulation of autonomic activity within the body, such as cardiovascular function. In addition, evidence over the past 30 years suggests that norepinephrine may also regulate the function of immune cells that protect the body against pathogens. The presence of sympathetic nerve fibers and the release of norepinephrine within lymphoid organs represent a mechanism by which signals from the central nervous system may influence immune cell function. The T cell-dependent antibody response is essential to successful host defense against numerous environmental pathogens. It is during this response that CD4+ T and B lymphocytes are activated to produce cytokines and antibody, respectively, leading to immune competence and protection. The goal of this review is to discuss the evidence supporting the release of norepinephrine within lymphoid organs and the expression of the beta2-Adrenergic Receptor by CD4+ T and B lymphocytes. We also discuss the mechanisms by which beta2-Adrenergic Receptor stimulation affects the level of cytokine and antibody produced by these cells both in vitro and in vivo. In cases where conflicting findings have been reported, we discuss potential variables that may have contributed to these conflicting findings. To conclude, we discuss the disease- and health-specific implications of the basic research being done in the area of sympathetic nervous system regulation of T and B lymphocyte function.
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norepinephrine and beta 2 Adrenergic Receptor stimulation regulate cd4 t and b lymphocyte function in vitro and in vivo
Pharmacological Reviews, 2001Co-Authors: Adam P Kohm, Virginia M SandersAbstract:Historically, norepinephrine and the sympathetic nervous system have been associated with the “fight or flight” response, and they also contribute to the regulation of autonomic activity within the body, such as cardiovascular function. In addition, evidence over the past 30 years suggests that norepinephrine may also regulate the function of immune cells that protect the body against pathogens. The presence of sympathetic nerve fibers and the release of norepinephrine within lymphoid organs represent a mechanism by which signals from the central nervous system may influence immune cell function. The T cell-dependent antibody response is essential to successful host defense against numerous environmental pathogens. It is during this response that CD4+ T and B lymphocytes are activated to produce cytokines and antibody, respectively, leading to immune competence and protection. The goal of this review is to discuss the evidence supporting the release of norepinephrine within lymphoid organs and the expression of the β2-Adrenergic Receptor by CD4+ T and B lymphocytes. We also discuss the mechanisms by which β2-Adrenergic Receptor stimulation affects the level of cytokine and antibody produced by these cells both in vitro and in vivo. In cases where conflicting findings have been reported, we discuss potential variables that may have contributed to these conflicting findings. To conclude, we discuss the disease- and health-specific implications of the basic research being done in the area of sympathetic nervous system regulation of T and B lymphocyte function.
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the role of norepinephrine and beta 2 Adrenergic Receptor stimulation in the modulation of th1 th2 and b lymphocyte function
Advances in Experimental Medicine and Biology, 1998Co-Authors: Virginia M SandersAbstract:The most important function of the sympathetic nervous system, which is part of the peripheral autonomic nervous system, is to affect modestadjustments in many different cellular activities in the body to maintain homeostasis. With this function of the sympathetic nervous system in mind, a long-term goal of my research is to achieve a better understanding of the role the sympathetic nervous system plays in maintaining immune homeostasis. An immediate goal is to understand the mechanism by which stimulation of the lymphocyte-associated β2-Adrenergic Receptor (β2AR) by norepinephrine or a pharmacologic agonist modulates lymphocyte function in vitro.By achieving this immediate goal, we plan to use these data to rationally design in vivomodels to study the mechanisms by which the sympathetic nervous system affects immune system function in normal and disease states.
Stuart A Green - One of the best experts on this subject based on the ideXlab platform.
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myocardial signaling defects and impaired cardiac function of a human beta 2 Adrenergic Receptor polymorphism expressed in transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jamal Turki, Stuart A Green, John N Lorenz, Elizabeth T Donnelly, Marie T Jacinto, Stephen B LiggettAbstract: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.
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influence of beta 2 Adrenergic Receptor genotypes on signal transduction in human airway smooth muscle cells
American Journal of Respiratory Cell and Molecular Biology, 1995Co-Authors: Stuart A Green, J Turki, P Bejarano, Ian P Hall, Stephen B LiggettAbstract:The phenotypic relevance of allelic variation in the structure of the beta 2-Adrenergic Receptor (beta 2AR) expressed in lung cells is unknown. In particular, altered responsiveness of the beta 2AR expressed on airway smooth muscle, which are responsible for bronchodilation in the treatment of asthma, may be an important factor in the ultimate physiologic response to agonist. To approach this, we established primary cultures of human airway smooth muscle cells obtained at autopsy and developed a method to determine the beta 2AR genotype at the polymorphic loci of codons 16 and 27, using allele-specific polymerase chain reactions. Radioligand binding studies revealed that these cells expressed approximately 70 fmol/mg of Receptor which was exclusively of the beta 2AR subtype. All cell lines obtained (n = 10) exhibited normal agonist binding and Receptor-mediated activation of the adenylyl cyclase second messenger pathway. However, distinct differences were found in the response to long-term agonist exposur...
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influence of beta 2 Adrenergic Receptor genotypes on signal transduction in human airway smooth muscle cells
American Journal of Respiratory Cell and Molecular Biology, 1995Co-Authors: Stuart A Green, J Turki, P Bejarano, Ian P Hall, Stephen B LiggettAbstract:The phenotypic relevance of allelic variation in the structure of the beta 2-Adrenergic Receptor (beta 2AR) expressed in lung cells is unknown. In particular, altered responsiveness of the beta 2AR expressed on airway smooth muscle, which are responsible for bronchodilation in the treatment of asthma, may be an important factor in the ultimate physiologic response to agonist. To approach this, we established primary cultures of human airway smooth muscle cells obtained at autopsy and developed a method to determine the beta 2AR genotype at the polymorphic loci of codons 16 and 27, using allele-specific polymerase chain reactions. Radioligand binding studies revealed that these cells expressed approximately 70 fmol/mg of Receptor which was exclusively of the beta 2AR subtype. All cell lines obtained (n = 10) exhibited normal agonist binding and Receptor-mediated activation of the adenylyl cyclase second messenger pathway. However, distinct differences were found in the response to long-term agonist exposure between the different beta 2AR genotypes. Cells expressing Arg at codon 16 (Arg16) traditionally referred to as wild-type, underwent 77.8 +/- 8.1% downregulations of beta 2AR following prolonged (24-h) exposure to the beta 2AR agonist isoproterenol (10 microM). In contrast, cells expressing Gly16 beta 2AR underwent enhanced agonist-promoted downregulation (95.6 +/- 1.7%, P < 0.05 versus Arg16), whereas cells expressing Glu27 beta 2AR were relatively resistant to such downregulation (29.5 +/- 12.7%, P < 0.01 versus Arg16). For cells expressing Glu27 beta 2AR, this difference resulted in a significant attenuation of agonist-promoted functional desensitization (33 +/- 7 versus 90 +/- 5% desensitization for Arg16, P < 0.001) following preincubation with 1 microM isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)
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genetic polymorphisms of the beta 2 Adrenergic Receptor in nocturnal and nonnocturnal asthma evidence that gly16 correlates with the nocturnal phenotype
Journal of Clinical Investigation, 1995Co-Authors: Jamal Turki, Stuart A Green, J Pak, Richard J Martin, Stephen B LiggettAbstract:Nocturnal asthma represents a unique subset of patients with asthma who experience worsening symptoms and airflow obstruction at night. The basis for this phenotype of asthma is not known, but beta 2-Adrenergic Receptors (beta 2AR) are known to downregulate overnight in nocturnal asthmatics but not normal subjects or nonnocturnal asthmatics. We have recently delineated three polymorphic loci within the coding block of the beta 2AR which alter amino acids at positions 16, 27, and 164 and impart specific biochemical and pharmacologic phenotypes to the Receptor. In site-directed mutagenesis/recombinant expression studies we have found that glycine at position 16 (Gly16) imparts an accelerated agonist-promoted downregulation of beta 2AR as compared to arginine at this position (Arg16). We hypothesized that Gly16 might be overrepresented in nocturnal asthmatics and thus determined the beta 2AR genotypes of two well-defined asthmatic cohorts: 23 nocturnal asthmatics with 34 +/- 2% nocturnal depression of peak expiratory flow rates, and 22 nonnocturnal asthmatics with virtually no such depression (2.3 +/- 0.8%). The frequency of the Gly16 allele was 80.4% in the nocturnal group as compared to 52.2% in the nonnocturnal group, while the Arg16 allele was present in 19.6 and 47.8%, respectively. This overrepresentation of the Gly16 allele in nocturnal asthma was significant at P = 0.007 with an odds ratio of having nocturnal asthma and the Gly16 polymorphism being 3.8. Comparisons of the two cohorts as to homozygosity for Gly16, homozygosity for Arg16, or heterozygosity were also consistent with segregation of Gly16 with nocturnal asthma. There was no difference in the frequency of polymorphisms at loci 27 (Gln27 or Glu27) and 164 (Thr164 or Ile164) between the two groups. Thus the Gly16 polymorphism of the beta 2AR, which imparts an enhanced downregulation of Receptor number, is overrepresented in nocturnal asthma and appears to be an important genetic factor in the expression of this asthmatic phenotype.
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amino terminal polymorphisms of the human beta 2 Adrenergic Receptor impart distinct agonist promoted regulatory properties
Biochemistry, 1994Co-Authors: Stuart A Green, Jamal Turki, Michael Innis, Stephen B LiggettAbstract:We have recently delineated three naturally occurring polymorphisms of the human beta 2-Adrenergic Receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the Receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these Receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated Receptors displayed normal agonist binding and functional coupling to Gs, resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of Receptor expression. After 24-h exposure to 10 microM isoproterenol, wild-type beta 2AR underwent a 26 +/- 3% reduction in Receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly+Gln27-->Glu also underwent an increased downregulation compared to wild-type beta 2AR (39 +/- 4%). The rates of new Receptor synthesis after irreversible alkylation were not different between these Receptors, nor were the rates of agonist-promoted Receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNA minimally increased during agonist exposure, and wild-type beta 2AR and the other mutated Receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of Receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Adam P Kohm - One of the best experts on this subject based on the ideXlab platform.
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norepinephrine and beta 2 Adrenergic Receptor stimulation regulate cd4 t and b lymphocyte function in vitro and in vivo
Pharmacological Reviews, 2001Co-Authors: Adam P Kohm, Virginia M SandersAbstract:Historically, norepinephrine and the sympathetic nervous system have been associated with the "fight or flight" response, and they also contribute to the regulation of autonomic activity within the body, such as cardiovascular function. In addition, evidence over the past 30 years suggests that norepinephrine may also regulate the function of immune cells that protect the body against pathogens. The presence of sympathetic nerve fibers and the release of norepinephrine within lymphoid organs represent a mechanism by which signals from the central nervous system may influence immune cell function. The T cell-dependent antibody response is essential to successful host defense against numerous environmental pathogens. It is during this response that CD4+ T and B lymphocytes are activated to produce cytokines and antibody, respectively, leading to immune competence and protection. The goal of this review is to discuss the evidence supporting the release of norepinephrine within lymphoid organs and the expression of the beta2-Adrenergic Receptor by CD4+ T and B lymphocytes. We also discuss the mechanisms by which beta2-Adrenergic Receptor stimulation affects the level of cytokine and antibody produced by these cells both in vitro and in vivo. In cases where conflicting findings have been reported, we discuss potential variables that may have contributed to these conflicting findings. To conclude, we discuss the disease- and health-specific implications of the basic research being done in the area of sympathetic nervous system regulation of T and B lymphocyte function.
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norepinephrine and beta 2 Adrenergic Receptor stimulation regulate cd4 t and b lymphocyte function in vitro and in vivo
Pharmacological Reviews, 2001Co-Authors: Adam P Kohm, Virginia M SandersAbstract:Historically, norepinephrine and the sympathetic nervous system have been associated with the “fight or flight” response, and they also contribute to the regulation of autonomic activity within the body, such as cardiovascular function. In addition, evidence over the past 30 years suggests that norepinephrine may also regulate the function of immune cells that protect the body against pathogens. The presence of sympathetic nerve fibers and the release of norepinephrine within lymphoid organs represent a mechanism by which signals from the central nervous system may influence immune cell function. The T cell-dependent antibody response is essential to successful host defense against numerous environmental pathogens. It is during this response that CD4+ T and B lymphocytes are activated to produce cytokines and antibody, respectively, leading to immune competence and protection. The goal of this review is to discuss the evidence supporting the release of norepinephrine within lymphoid organs and the expression of the β2-Adrenergic Receptor by CD4+ T and B lymphocytes. We also discuss the mechanisms by which β2-Adrenergic Receptor stimulation affects the level of cytokine and antibody produced by these cells both in vitro and in vivo. In cases where conflicting findings have been reported, we discuss potential variables that may have contributed to these conflicting findings. To conclude, we discuss the disease- and health-specific implications of the basic research being done in the area of sympathetic nervous system regulation of T and B lymphocyte function.