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Lee E Limbird - One of the best experts on this subject based on the ideXlab platform.
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genetic evidence for involvement of multiple effector systems in alpha 2a Adrenergic Receptor inhibition of stimulus secretion coupling
Molecular Pharmacology, 1996Co-Authors: P P Lakhlani, David M Lovinger, Lee E LimbirdAbstract:The alpha 2A-Adrenergic Receptor (alpha 2AAR), via its interaction with the pertussis toxin-sensitive Gi/G(o) class of G proteins, modulates multiple effector systems, including inhibition of adenylyl cyclase and Ca2+ channels and activation of K+ channels. Mutation of a membrane-embedded aspartate residue, highly conserved among G protein-coupled Receptors, in the alpha 2AAR to asparagine (D79N alpha 2AAR) results in selective uncoupling of the Receptor to K+ currents but retention of inhibition of cAMP production and of voltage-sensitive Ca2+ currents when expressed in AtT20 anterior pituitary cells in culture. It is known that attenuation of cAMP synthesis alone cannot account for alpha 2AAR suppression of stimulus-secretion coupling; thus, the D79N alpha 2AAR provides a unique tool with which to assess the relative contribution of K+ current activation and Ca2+ current suppression in mediating the cellular responses of alpha 2AAR. The wild-type alpha 2AAR suppresses basal and secretagogue-evoked adrenocorticotropic hormone (ACTH) release in a manner indistinguishable from response to the endogenous somatostatin Receptor. In contrast, the D79N alpha 2AAR does not attenuate basal ACTH release and is only partially effective in suppressing ACTH secretion evoked by the secretagogue isoproterenol. Regulation of ACTH release evoked by 8-bromo-cAMP, which bypasses Receptor regulation of cAMP synthesis, suggests that attenuation of cAMP production, although not sufficient for inhibition of ACTH secretion, nevertheless participates in a functionally relevant manner. Taken together, the present findings indicate that alpha 2AAR-mediated suppression of neuropeptide secretion requires concomitant regulation of K+ and Ca2+ currents in parallel with attenuation of cAMP production.
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mutation of an aspartate residue highly conserved among g protein coupled Receptors results in nonreciprocal disruption of alpha 2 Adrenergic Receptor g protein interactions a negative charge at amino acid residue 79 forecasts alpha 2a Adrenergic rec
Journal of Biological Chemistry, 1994Co-Authors: B P Ceresa, Lee E LimbirdAbstract:We have mutated the aspartate residue in the putative second transmembrane spanning domain of the alpha 2A-Adrenergic Receptor (alpha 2AAR) to the non-negatively charged asparagine (D79N) and glutamine (D79Q) and the negatively charged glutamate (D79E) residue in an effort to better characterize the role of this residue, highly conserved among G-protein-coupled Receptors, in Na+ regulation of ligand binding and in Receptor G-protein coupling. Allosteric modulation of Receptor-ligand interactions by Na+ is retained by the D79E alpha 2AAR but lost upon mutation to the uncharged D79N and D79Q residues. Loss of allosteric effects of Na+ is paralleled by a complete loss of retrograde information transfer from G-proteins to alpha 2AAR in AtT20 cells, measured via the sensitivity of radiolabeled agonist binding to Gpp(NH)p. In contrast to the complete elimination of retrograde signaling via the D79N and D79Q alpha 2AAR, anterograde information transfer from Receptor to G-protein is modified in a more subtle quantitative way, since agonist-stimulated GTPase activity via D79N and D79Q alpha 2AAR, although apparently attenuated compared to wild type and D79E alpha 2AAR, is no less than the GTPase activity elicited by endogenous somatostatin Receptors in AtT20 cells. These data indicate that a negative charge at amino acid residue 79 forecasts sensitivity to allosteric regulation by monovalent cations and its mutation to non-negatively charged residues elicits a nonparallel modulation of Receptor-->G-protein versus G-protein-->Receptor communication between alpha 2AAR and pertussis toxin-sensitive GTP-binding proteins.
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unique structural features important for stabilization versus polarization of the alpha 2a Adrenergic Receptor on the basolateral membrane of madin darby canine kidney cells
Journal of Biological Chemistry, 1994Co-Authors: Jeffrey R Keefer, Matthew E Kennedy, Lee E LimbirdAbstract:The alpha 2A-Adrenergic Receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters Receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases Receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.
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the alpha 2a Adrenergic Receptor is targeted directly to the basolateral membrane domain of madin darby canine kidney cells independent of coupling to pertussis toxin sensitive gtp binding proteins
Journal of Biological Chemistry, 1993Co-Authors: Jeffrey R Keefer, Lee E LimbirdAbstract:The alpha 2-Adrenergic Receptor (alpha 2-AR) is a member of the seven transmembrane-spanning G-protein-coupled Receptor superfamily. In the kidney, the alpha 2-AR is most abundant in the epithelial cells of the proximal tubule where it is important in enhancing Na+ reabsorption via the modulation of Na+/H+ exchange. Radioligand binding and physiological studies suggest that the alpha 2-AR residues primarily on the basolateral surface of these proximal tubule cells in vivo. To investigate the mechanisms underlying alpha 2-AR polarization in epithelial cells, we permanently expressed wild-type and an epitope-tagged version of the alpha 2A-AR in Madin-Darby canine kidney (MDCK) cells. Using a steady-state surface biotinylation assay, we observe that 80-90% of the alpha 2A-AR in MDCK cell clones is located on the basolateral membrane domain. Immunolocalization studies confirm the biotinylation results and demonstrate that the alpha 2A-AR is actually confined primarily to the lateral domain of the basolateral surface. Metabolic labeling experiments suggest that basolateral polarization of the alpha 2A-AR is achieved by direct targeting of the Receptor to the basolateral domain. Targeting of the alpha 2A-AR to the basolateral surface is not perturbed by pertussis toxin-treatment of MDCK cells, suggesting that coupling of the alpha 2A-AR to GTP-binding proteins is not important for Receptor polarization.
Jeffrey R Keefer - One of the best experts on this subject based on the ideXlab platform.
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unique structural features important for stabilization versus polarization of the alpha 2a Adrenergic Receptor on the basolateral membrane of madin darby canine kidney cells
Journal of Biological Chemistry, 1994Co-Authors: Jeffrey R Keefer, Matthew E Kennedy, Lee E LimbirdAbstract:The alpha 2A-Adrenergic Receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters Receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases Receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.
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the alpha 2a Adrenergic Receptor is targeted directly to the basolateral membrane domain of madin darby canine kidney cells independent of coupling to pertussis toxin sensitive gtp binding proteins
Journal of Biological Chemistry, 1993Co-Authors: Jeffrey R Keefer, Lee E LimbirdAbstract:The alpha 2-Adrenergic Receptor (alpha 2-AR) is a member of the seven transmembrane-spanning G-protein-coupled Receptor superfamily. In the kidney, the alpha 2-AR is most abundant in the epithelial cells of the proximal tubule where it is important in enhancing Na+ reabsorption via the modulation of Na+/H+ exchange. Radioligand binding and physiological studies suggest that the alpha 2-AR residues primarily on the basolateral surface of these proximal tubule cells in vivo. To investigate the mechanisms underlying alpha 2-AR polarization in epithelial cells, we permanently expressed wild-type and an epitope-tagged version of the alpha 2A-AR in Madin-Darby canine kidney (MDCK) cells. Using a steady-state surface biotinylation assay, we observe that 80-90% of the alpha 2A-AR in MDCK cell clones is located on the basolateral membrane domain. Immunolocalization studies confirm the biotinylation results and demonstrate that the alpha 2A-AR is actually confined primarily to the lateral domain of the basolateral surface. Metabolic labeling experiments suggest that basolateral polarization of the alpha 2A-AR is achieved by direct targeting of the Receptor to the basolateral domain. Targeting of the alpha 2A-AR to the basolateral surface is not perturbed by pertussis toxin-treatment of MDCK cells, suggesting that coupling of the alpha 2A-AR to GTP-binding proteins is not important for Receptor polarization.
Dan Xue - One of the best experts on this subject based on the ideXlab platform.
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dexmedetomidine an alpha 2a Adrenergic Receptor agonist mitigates experimental autoimmune encephalomyelitis by desensitization of cxcr7 in microglia
Biochemistry, 2018Co-Authors: Yi Huang, Dan XueAbstract:The autoimmune disease multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is characterized by an ascending paralysis that is characterized by extensive infiltration of the central nervous system by inflammatory cells. Although several studies to some extent uncover the cellular mechanisms of microglia that govern EAE pathogenesis, the molecular mechanisms that orchestrate the movement of microglia remain unknown, and potential novel therapeutic strategies are still required. In this study, we report that dexmedetomidine, an alpha 2a Adrenergic Receptor agonist, attenuates the clinical severity of EAE with less infiltration of microglia. During EAE, dexmedetomidine inhibits SDF-1- and I-TAC-induced chemotaxis of microglia mediated by CXCR7 but not CXCR4 or CXCR3. Most importantly, the alpha 2a Adrenergic Receptor is essential in dexmedetomidine-induced CXCR7 desensitization in microglia. Further experiments confirmed that CXCR7 desensitization required atypical protein kinase C ζ activation, while conventional and novel protein kinase C isoforms were not involved. Altogether, our data elucidate the mechanism of dexmedetomidine-induced CXCR7 desensitization in microglia and amelioration in EAE, which might lead to a better understanding of the therapeutic effects of dexmedetomidine as well as its implications for CXCR7 desensitization in autoimmune disease.
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Dexmedetomidine, an Alpha 2a Adrenergic Receptor Agonist, Mitigates Experimental Autoimmune Encephalomyelitis by Desensitization of CXCR7 in Microglia
2018Co-Authors: Yi Huang, Dan XueAbstract:The autoimmune disease multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is characterized by an ascending paralysis that is characterized by extensive infiltration of the central nervous system by inflammatory cells. Although several studies to some extent uncover the cellular mechanisms of microglia that govern EAE pathogenesis, the molecular mechanisms that orchestrate the movement of microglia remain unknown, and potential novel therapeutic strategies are still required. In this study, we report that dexmedetomidine, an alpha 2a Adrenergic Receptor agonist, attenuates the clinical severity of EAE with less infiltration of microglia. During EAE, dexmedetomidine inhibits SDF-1- and I-TAC-induced chemotaxis of microglia mediated by CXCR7 but not CXCR4 or CXCR3. Most importantly, the alpha 2a Adrenergic Receptor is essential in dexmedetomidine-induced CXCR7 desensitization in microglia. Further experiments confirmed that CXCR7 desensitization required atypical protein kinase C ζ activation, while conventional and novel protein kinase C isoforms were not involved. Altogether, our data elucidate the mechanism of dexmedetomidine-induced CXCR7 desensitization in microglia and amelioration in EAE, which might lead to a better understanding of the therapeutic effects of dexmedetomidine as well as its implications for CXCR7 desensitization in autoimmune disease
Matthew E Kennedy - One of the best experts on this subject based on the ideXlab platform.
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unique structural features important for stabilization versus polarization of the alpha 2a Adrenergic Receptor on the basolateral membrane of madin darby canine kidney cells
Journal of Biological Chemistry, 1994Co-Authors: Jeffrey R Keefer, Matthew E Kennedy, Lee E LimbirdAbstract:The alpha 2A-Adrenergic Receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters Receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases Receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.
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mutations of the alpha 2a Adrenergic Receptor that eliminate detectable palmitoylation do not perturb Receptor g protein coupling
Journal of Biological Chemistry, 1993Co-Authors: Matthew E Kennedy, L E LimbirdAbstract:Abstract The alpha 2A-Adrenergic Receptor (alpha 2AAR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled Receptor superfamily, the primary structure of the alpha 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-palmitoylation. However, mutation of Cys-442 does not alter Adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta 2-Adrenergic Receptor attenuates coupling to Gs (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to Gt (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha 2AAR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing Receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of Receptor-agonist interactions to guanine nucleotides in competition for [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-Adrenergic Receptor-Gs and rhodopsin-Gt coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different Receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as Receptor expression, turnover, and localization.
L E Limbird - One of the best experts on this subject based on the ideXlab platform.
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coupling of the alpha 2a Adrenergic Receptor to multiple g proteins a simple approach for estimating Receptor g protein coupling efficiency in a transient expression system
Journal of Biological Chemistry, 1994Co-Authors: O Chabre, Bruce R Conklin, S Brandon, Henry R Bourne, L E LimbirdAbstract:It is now widely appreciated that G-protein-coupled cell-surface Receptors can modulate distinct signal transduction pathways via coupling to different GTP-binding proteins. In the present study, we have used a transient co-expression approach to study the coupling of a single alpha 2-Adrenergic Receptor (alpha 2AAR) population to three different G protein subtypes (Gi, Gq, and Gs) acting on two different cellular effectors in HEK 293 cells. In all cases, the affinity of the Receptor for the alpha 2A-Adrenergic agonist, UK14304, is unchanged (KD approximately equal to 670 nM). However, there is a dramatic difference in the EC50 of UK14304 in eliciting inhibition of endogenous adenylyl cyclase via endogenous Gi (0.09 nM) versus activation of phospholipase C via co-transfected Gq (50 nM) or stimulation of endogenous adenylyl cyclase via co-transfected Gs (70 nM) in HEK 293 cells. These findings are consistent with the interpretations that the alpha 2AAR preferentially interacts with Gi rather than Gs or Gq. When the alpha 2AAR was mutated at Asp79, a residue highly conserved among G-protein-coupled Receptors, the mutant D79N alpha 2AAR lost the ability to couple to Gq and Gs and, although it was able to couple to inhibition of cyclase via pertussis toxin-sensitive pathways (Gi), it did so with a lower potency than observed for the wild-type alpha 2AAR (EC50 = 7.2 nM). The most straightforward interpretation of these data is that the D79N mutation in the alpha 2AAR reduces the efficiency of coupling of the alpha 2AAR to all G-proteins, thus eliminating signal transduction through those pathways less efficiently coupled to the alpha 2AAR. Since the transient expression assays described permit manipulation of the structure of both the Receptor or the G-protein, the present strategies could be exploited to delineate the complementary domains specifying the affinity and/or efficacy of Receptor coupling to distinct GTP-binding proteins.
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mutations of the alpha 2a Adrenergic Receptor that eliminate detectable palmitoylation do not perturb Receptor g protein coupling
Journal of Biological Chemistry, 1993Co-Authors: Matthew E Kennedy, L E LimbirdAbstract:Abstract The alpha 2A-Adrenergic Receptor (alpha 2AAR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled Receptor superfamily, the primary structure of the alpha 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-palmitoylation. However, mutation of Cys-442 does not alter Adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta 2-Adrenergic Receptor attenuates coupling to Gs (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to Gt (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha 2AAR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing Receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of Receptor-agonist interactions to guanine nucleotides in competition for [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-Adrenergic Receptor-Gs and rhodopsin-Gt coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different Receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as Receptor expression, turnover, and localization.