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Eamonn Kelly - One of the best experts on this subject based on the ideXlab platform.
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analysis of mglur1a constitutive Internalization using a pulse chase enzyme linked immuno sorbant assay elisa
Journal of Biochemical and Biophysical Methods, 2005Co-Authors: Giordano Pula, Stuart J Mundell, Peter J Roberts, Eamonn KellyAbstract:The surface expression of G protein-coupled receptors is regulated by Internalization. For many receptors, a constitutive level of Internalization in the absence of agonist has been reported. The constitutive Internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse–chase ELISA method that allows the investigation of mGluR1a constitutive Internalization. When investigated by pulse–chase ELISA, the constitutive Internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive Internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse–chase labelling procedure. The application of the pulse–chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive Internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive Internalization. Interestingly, only DMII underwent significant constitutive Internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse–chase ELISA appears to be an efficient tool to analyze the constitutive Internalization of different mGluR1 splice variants.
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agonist induced Internalization of metabotropic glutamate receptor 1a structural determinants for protein kinase c and g protein coupled receptor kinase mediated Internalization
Journal of Neurochemistry, 2003Co-Authors: Stuart J Mundell, Giordano Pula, Peter J Roberts, Katy Carswell, Eamonn KellyAbstract:To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced Internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid Internalization whilst Internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a Internalization, had no effect on the Internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent Internalization of mGlu1a, produced negligible Internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced Internalization of mGlu1a and DM-III, whilst Internalization of DM-I and DM-II was not significantly affected. The glutamate-induced Internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319–418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and Internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a Internalization requires the distal C terminus of the receptor (Ser894–Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869–Val893) in the proximal C-terminal tail.
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metabotropic glutamate receptor 1 Internalization induced by muscarinic acetylcholine receptor activation differential dependency of Internalization of splice variants on nonvisual arrestins
Molecular Pharmacology, 2002Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, David Holman, Peter J Roberts, Eamonn KellyAbstract:In this study, we characterized the glutamate- or second-messenger kinase-dependent Internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant Internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid Internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced Internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b Internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M1 muscarinic receptors in human embryonic kidney 293 cells, induced the Internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca2+ calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced Internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor Internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the Internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.
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rapid agonist induced desensitization and Internalization of the a2b adenosine receptor is mediated by a serine residue close to the cooh terminus
Journal of Biological Chemistry, 2001Co-Authors: Annelise Matharu, Jeffrey L Benovic, Stuart J Mundell, Eamonn KellyAbstract:The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and Internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and Internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and Internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and Internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and Internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor Internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor Internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and Internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect Internalization of the receptor to an arrestin- and clathrin-independent pathway.
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agonist induced Internalization of the metabotropic glutamate receptor 1a is arrestin and dynamin dependent
Journal of Neurochemistry, 2001Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, Peter J Roberts, Eamonn KellyAbstract:At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent Internalization to endosomes. Further quantification of receptor Internalization was provided by ELISA experiments which showed rapid agonist-induced Internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a Internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a Internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced Internalization of mGluR1a is an arrestin- and dynamin-dependent process.
Stuart J Mundell - One of the best experts on this subject based on the ideXlab platform.
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analysis of mglur1a constitutive Internalization using a pulse chase enzyme linked immuno sorbant assay elisa
Journal of Biochemical and Biophysical Methods, 2005Co-Authors: Giordano Pula, Stuart J Mundell, Peter J Roberts, Eamonn KellyAbstract:The surface expression of G protein-coupled receptors is regulated by Internalization. For many receptors, a constitutive level of Internalization in the absence of agonist has been reported. The constitutive Internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse–chase ELISA method that allows the investigation of mGluR1a constitutive Internalization. When investigated by pulse–chase ELISA, the constitutive Internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive Internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse–chase labelling procedure. The application of the pulse–chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive Internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive Internalization. Interestingly, only DMII underwent significant constitutive Internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse–chase ELISA appears to be an efficient tool to analyze the constitutive Internalization of different mGluR1 splice variants.
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agonist induced Internalization of metabotropic glutamate receptor 1a structural determinants for protein kinase c and g protein coupled receptor kinase mediated Internalization
Journal of Neurochemistry, 2003Co-Authors: Stuart J Mundell, Giordano Pula, Peter J Roberts, Katy Carswell, Eamonn KellyAbstract:To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced Internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid Internalization whilst Internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a Internalization, had no effect on the Internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent Internalization of mGlu1a, produced negligible Internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced Internalization of mGlu1a and DM-III, whilst Internalization of DM-I and DM-II was not significantly affected. The glutamate-induced Internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319–418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and Internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a Internalization requires the distal C terminus of the receptor (Ser894–Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869–Val893) in the proximal C-terminal tail.
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metabotropic glutamate receptor 1 Internalization induced by muscarinic acetylcholine receptor activation differential dependency of Internalization of splice variants on nonvisual arrestins
Molecular Pharmacology, 2002Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, David Holman, Peter J Roberts, Eamonn KellyAbstract:In this study, we characterized the glutamate- or second-messenger kinase-dependent Internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant Internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid Internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced Internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b Internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M1 muscarinic receptors in human embryonic kidney 293 cells, induced the Internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca2+ calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced Internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor Internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the Internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.
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rapid agonist induced desensitization and Internalization of the a2b adenosine receptor is mediated by a serine residue close to the cooh terminus
Journal of Biological Chemistry, 2001Co-Authors: Annelise Matharu, Jeffrey L Benovic, Stuart J Mundell, Eamonn KellyAbstract:The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and Internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and Internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and Internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and Internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and Internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor Internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor Internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and Internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect Internalization of the receptor to an arrestin- and clathrin-independent pathway.
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agonist induced Internalization of the metabotropic glutamate receptor 1a is arrestin and dynamin dependent
Journal of Neurochemistry, 2001Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, Peter J Roberts, Eamonn KellyAbstract:At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent Internalization to endosomes. Further quantification of receptor Internalization was provided by ELISA experiments which showed rapid agonist-induced Internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a Internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a Internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced Internalization of mGluR1a is an arrestin- and dynamin-dependent process.
Jeffrey L Benovic - One of the best experts on this subject based on the ideXlab platform.
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rapid agonist induced desensitization and Internalization of the a2b adenosine receptor is mediated by a serine residue close to the cooh terminus
Journal of Biological Chemistry, 2001Co-Authors: Annelise Matharu, Jeffrey L Benovic, Stuart J Mundell, Eamonn KellyAbstract:The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and Internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and Internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and Internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and Internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and Internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor Internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor Internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and Internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect Internalization of the receptor to an arrestin- and clathrin-independent pathway.
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role of the differentially spliced carboxyl terminus in thromboxane a2 receptor trafficking identification of a distinct motif for tonic Internalization
Journal of Biological Chemistry, 2001Co-Authors: Jeanluc Parent, Pascale Labrecque, Moulay Driss Rochdi, Jeffrey L BenovicAbstract:Abstract The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced Internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic Internalization. Tonic Internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic Internalization but had no role in agonist-induced Internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic Internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic Internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced Internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic Internalization of a cell surface receptor.
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trafficking of the hiv coreceptor cxcr4 role of arrestins and identification of residues in the c terminal tail that mediate receptor Internalization
Journal of Biological Chemistry, 1999Co-Authors: Michael J Orsini, Jeanluc Parent, Stuart J Mundell, Jeffrey L BenovicAbstract:Abstract The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic Internalization as well as Internalization in response to stimulation with phorbol esters and ligand (SDF-1α). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor Internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1α and phorbol esters (PMA) promoted rapid Internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 Internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 Internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1α. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1α-mediated receptor Internalization. The ability of coexpressed GRK2 and arrestins to promote Internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor Internalization.
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arrestin independent Internalization of the m1 m3 and m4 subtypes of muscarinic cholinergic receptors
Journal of Biological Chemistry, 1998Co-Authors: Katharine B Lee, Jeffrey L Benovic, Robin Palsrylaarsdam, Marlene M HoseyAbstract:Abstract To understand what processes contribute to the agonist-induced Internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented Internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682–23689), the agonist-induced Internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the Internalization of the m1, m3, and m4 mAChR whereas the Internalization of the β2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced Internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the Internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced Internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.
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role of clathrin mediated endocytosis in agonist induced down regulation of the β2 adrenergic receptor
Journal of Biological Chemistry, 1998Co-Authors: Alison W Gagnon, Lorena A Kallal, Jeffrey L BenovicAbstract:Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced Internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced Internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR Internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced Internalization and down-regulation of the beta2AR, suggesting that receptor Internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor Internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.
Leeyuan Liuchen - One of the best experts on this subject based on the ideXlab platform.
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differential regulation of the human κ opioid receptor by agonists etorphine and levorphanol reduced dynorphin a and u50 488h induced Internalization and phosphorylation
Journal of Pharmacology and Experimental Therapeutics, 2003Co-Authors: Jianguo Li, Fengqin Zhang, Leeyuan LiuchenAbstract:We previously observed that ( trans )-3,4-dichloro- N -methyl- N -[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted Internalization and phosphorylation of the FLAG-tagged human κ opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5′- O -(3-[ 35 S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted Internalization of the FLAG-hkor in a time- and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor Internalization, effectively blocked dynorphin A(1-17)- and U50,488H-induced Internalization. Interestingly, the full agonists etorphine and levorphanol did not cause Internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)- and U50,488H-induced Internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin- or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of Internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to ∼200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote Internalization of the hkor.
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differential regulation of the human κ opioid receptor by agonists etorphine and levorphanol reduced dynorphin a and u50 488h induced Internalization and phosphorylation
Journal of Pharmacology and Experimental Therapeutics, 2003Co-Authors: Fengqin Zhang, Xilu Jin, Leeyuan LiuchenAbstract:We previously observed that (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted Internalization and phosphorylation of the FLAG-tagged human kappa opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted Internalization of the FLAG-hkor in a time- and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor Internalization, effectively blocked dynorphin A(1-17)- and U50,488H-induced Internalization. Interestingly, the full agonists etorphine and levorphanol did not cause Internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)- and U50,488H-induced Internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin- or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of Internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to approximately 200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote Internalization of the hkor.
Peter J Roberts - One of the best experts on this subject based on the ideXlab platform.
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analysis of mglur1a constitutive Internalization using a pulse chase enzyme linked immuno sorbant assay elisa
Journal of Biochemical and Biophysical Methods, 2005Co-Authors: Giordano Pula, Stuart J Mundell, Peter J Roberts, Eamonn KellyAbstract:The surface expression of G protein-coupled receptors is regulated by Internalization. For many receptors, a constitutive level of Internalization in the absence of agonist has been reported. The constitutive Internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse–chase ELISA method that allows the investigation of mGluR1a constitutive Internalization. When investigated by pulse–chase ELISA, the constitutive Internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive Internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse–chase labelling procedure. The application of the pulse–chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive Internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive Internalization. Interestingly, only DMII underwent significant constitutive Internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse–chase ELISA appears to be an efficient tool to analyze the constitutive Internalization of different mGluR1 splice variants.
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agonist induced Internalization of metabotropic glutamate receptor 1a structural determinants for protein kinase c and g protein coupled receptor kinase mediated Internalization
Journal of Neurochemistry, 2003Co-Authors: Stuart J Mundell, Giordano Pula, Peter J Roberts, Katy Carswell, Eamonn KellyAbstract:To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced Internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid Internalization whilst Internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a Internalization, had no effect on the Internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent Internalization of mGlu1a, produced negligible Internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced Internalization of mGlu1a and DM-III, whilst Internalization of DM-I and DM-II was not significantly affected. The glutamate-induced Internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319–418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and Internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a Internalization requires the distal C terminus of the receptor (Ser894–Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869–Val893) in the proximal C-terminal tail.
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metabotropic glutamate receptor 1 Internalization induced by muscarinic acetylcholine receptor activation differential dependency of Internalization of splice variants on nonvisual arrestins
Molecular Pharmacology, 2002Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, David Holman, Peter J Roberts, Eamonn KellyAbstract:In this study, we characterized the glutamate- or second-messenger kinase-dependent Internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant Internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid Internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced Internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b Internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M1 muscarinic receptors in human embryonic kidney 293 cells, induced the Internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca2+ calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced Internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor Internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the Internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.
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agonist induced Internalization of the metabotropic glutamate receptor 1a is arrestin and dynamin dependent
Journal of Neurochemistry, 2001Co-Authors: Stuart J Mundell, Annelise Matharu, Giordano Pula, Peter J Roberts, Eamonn KellyAbstract:At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent Internalization to endosomes. Further quantification of receptor Internalization was provided by ELISA experiments which showed rapid agonist-induced Internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a Internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a Internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced Internalization of mGluR1a is an arrestin- and dynamin-dependent process.
