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1.
Science ; 315(5812): 663-6, 2007 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-17272726

RESUMO

Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide-binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that beta-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. beta-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, beta-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the beta-arrestin-DGK complex to activated 7TMRs. The dual function of beta-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3',5'-monophosphate phosphodiesterases to Gs-coupled beta2-adrenergic receptors. Thus, beta-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.


Assuntos
Arrestinas/metabolismo , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Receptor Muscarínico M1/metabolismo , Transdução de Sinais , Animais , Células COS , Carbacol/farmacologia , Linhagem Celular , Chlorocebus aethiops , Diacilglicerol Quinase/genética , Humanos , Mutação , Ácidos Fosfatídicos/metabolismo , Ligação Proteica , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro , Transfecção , beta-Arrestinas
2.
Mol Endocrinol ; 19(10): 2579-90, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15928315

RESUMO

G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.


Assuntos
Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Hormônios Hipofisários/metabolismo , Mutação Puntual , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Sequência de Aminoácidos , Transporte Biológico Ativo , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Expressão Gênica , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
3.
J Biol Chem ; 280(12): 11560-8, 2005 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-15653688

RESUMO

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.


Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/química , Animais , Células CHO , Hormônio Liberador da Corticotropina/metabolismo , Cricetinae , Endocitose , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismo
4.
Cell Signal ; 16(9): 1001-11, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15212761

RESUMO

GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a beta-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged beta-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/beta-PIX complexes that have equal amounts of beta-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfoproteínas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Haplorrinos , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Mutagênese , Fosfoproteínas/genética , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
J Biol Chem ; 279(22): 23214-22, 2004 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-15054093

RESUMO

Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.


Assuntos
Arrestinas/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas CC/metabolismo , Humanos , Ligantes , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Conformação Proteica , Receptores CCR7 , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/genética , beta-Arrestina 2 , beta-Arrestinas
6.
Exp Parasitol ; 103(3-4): 112-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12880587

RESUMO

Trypanosoma cruzi Tc13 antigens belong to the trans-sialidase superfamily. Their sequences have been described only partially and, up to now, their physiological activity has not been elucidated. Here we present two new members of this family from the Tulahuén strain (Tc13 Tul) and the CL Brener clone (Tc13 CL), being the latter the first Tc13 sequence fully described. Alignment of all Tc13 sequences allowed us to define two sub-families that differ in the number of repeats and the presence or absence of the GPI addition site. Chromoblots demonstrate that Tc13 antigens are mainly located in chromosome III and its homologous. Pull down assays suggest that recombinant MBP-Tc13 Tul interacts with the second extracellular loop of the beta(1)-adrenergic receptor. This is the first evidence that a Tc13 antigen acts as a ligand interacting with a neurotransmitter receptor. These observations might add some light to the development of chagasic pathology.


Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Trypanosoma cruzi/classificação , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Sequência de Bases , Glicoproteínas , Humanos , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia
7.
Proc Natl Acad Sci U S A ; 100(3): 940-5, 2003 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-12552097

RESUMO

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Arrestinas/metabolismo , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores Adrenérgicos beta/metabolismo , Sulfonamidas , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Proteínas de Fluorescência Verde , Humanos , Isoquinolinas/farmacologia , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Miocárdio/citologia , Toxina Pertussis/farmacologia , Fosforilação , Ratos , Rolipram/farmacologia , Transdução de Sinais , Fatores de Tempo , Transfecção , beta-Arrestinas
8.
Science ; 298(5594): 834-6, 2002 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-12399592

RESUMO

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.


Assuntos
Arrestinas/metabolismo , AMP Cíclico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Arrestinas/genética , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Citosol/metabolismo , Humanos , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Camundongos , Mutação , Testes de Precipitina , Ratos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , beta-Arrestinas
9.
Trends Cell Biol ; 12(3): 130-8, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11859025

RESUMO

It is well established that the function of most heptahelical receptors (seven-transmembrane-span receptors; 7TMRs) is tightly regulated by the desensitizing actions of arrestins. Desensitization is the waning of 7TMR-mediated signals after prolonged exposure to agonist and occurs when arrestins bind to agonist-occupied and phosphorylated receptors, uncoupling the receptors from G proteins and preventing further signaling. Recently, there has been a marked shift in the focus of research into arrestin function because it has become clear that they not only prevent signaling from 7TMRs but also initiate and direct new signals from the very 7TMRs that they desensitize.


Assuntos
Arrestinas/fisiologia , Transdução de Sinais/fisiologia , Animais , Arrestinas/metabolismo , Arrestinas/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Humanos , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos
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