RESUMO
INTRODUCTION: At night, the pineal gland produces the indoleamines, melatonin, N-acetylserotonin (NAS), and N-acetyltryptamine (NAT). Melatonin is accepted as a hormone of night. Could NAS and NAT serve that role too? METHODS: Concentration-response measurements with overexpressed human melatonin receptors MT1 and MT2 ; mass spectrometry analysis of norepinephrine-stimulated secretions from isolated rat pineal glands; analysis of 24-hour periodic samples of rat blood. RESULTS: We show that NAT and NAS do activate melatonin receptors MT1 and MT2 , although with lower potency than melatonin, and that in vitro, melatonin and NAS are secreted from stimulated, isolated pineal glands in roughly equimolar amounts, but secretion of NAT was much less. All three were found at roughly equal concentrations in blood during the night. However, during the day, serum melatonin fell to very low values creating a high-amplitude circadian rhythm that was absent after pinealectomy, whereas NAS and NAT showed only small or no circadian variation. CONCLUSION: Blood levels of NAS and NAT were insufficient to activate peripheral melatonin receptors, and they were invariant, so they could not serve as circulating hormones of night. However, they could instead act in paracrine circadian fashion near the pineal gland or via other higher-affinity receptors.
Assuntos
Ritmo Circadiano , Glândula Pineal/metabolismo , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Serotonina/análogos & derivados , Triptaminas/metabolismo , Animais , Células HEK293 , Humanos , Masculino , Melatonina/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismoRESUMO
The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.
Assuntos
Proteínas de Neoplasias/metabolismo , Proteólise , Receptores Adrenérgicos alfa 1/metabolismo , Células Hep G2 , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Domínios PDZ , Receptores Adrenérgicos alfa 1/genéticaRESUMO
Small molecules that target the adrenergic family of G protein-coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional α1B-adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous α1B-ARs are not detectable via either [3H]-prazosin-binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of α1B-ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating ß-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both α1B- and ß-ARs may represent valuable small-molecule therapeutics for the treatment of colon cancer.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Carcinoma , Neoplasias do Colo , Receptores Adrenérgicos alfa 1 , Biofarmácia/métodos , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Descoberta de Drogas , Humanos , Receptores Adrenérgicos alfa 1/análise , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estimulação QuímicaRESUMO
G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/metabolismo , Descoberta de Drogas/métodos , Células HEK293 , Humanos , Ligantes , Domínios PDZ , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/análise , Transdução de SinaisRESUMO
α(1D)-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α(1D)-ARs form a "signalosome" with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α(1D)-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α(1D)-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α(1D)-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α(1D)-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.
Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais/fisiologia , alfa Catenina/metabolismo , Proteínas Associadas à Distrofina/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , RNA Interferente Pequeno/metabolismo , Receptores Adrenérgicos alfa 1/genética , alfa Catenina/genéticaRESUMO
α(1D)-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α(1D)-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, ß(1) and ß(2)) interact with α(1D)-ARs and our previous studies suggest multiple isoforms are required for proper α(1D)-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α(1D)-AR function. Radioligand binding experiments reveal α-syntrophin enhances α(1D)-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate ß(2)-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α(1D)-AR/DAPC signalosome significantly alter the pharmacological properties of α(1)-AR ligands in vitro.
Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Proteínas Associadas à Distrofina/fisiologia , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Proteínas Associadas à Distrofina/genética , Células HEK293 , Humanos , Ligantes , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de SinaisRESUMO
We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives (t1/2) using LICOR NIR imaging-polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous ß-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous ß2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.
Assuntos
Ensaios de Triagem em Larga Escala , Receptor 5-HT2A de Serotonina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes de Fusão/química , Bortezomib/farmacologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Células HEK293 , Meia-Vida , Humanos , Imagem Molecular/métodos , Norepinefrina/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/genética , Receptores Adrenérgicos alfa 1/genética , Receptores de Interleucina-8A/genética , Receptores de Somatostatina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodosRESUMO
Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.
Assuntos
Hipófise/metabolismo , Receptores Acoplados a Proteínas G/genética , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Sequência de Bases , Linhagem Celular , Estrogênios/metabolismo , Estrogênios/farmacologia , Genes Reporter , Genoma , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Hipófise/citologia , Hipófise/efeitos dos fármacos , Receptores de Kisspeptina-1RESUMO
G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by post-translational modifications, including N-glycosylation of asparagine residues. α1D-adrenergic receptors (α1D-ARs) - key regulators of central and autonomic nervous system function - contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α1D-AR between transmembrane domain 2 and 3. Label-free dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α1D-AR N-terminal glycosylation is required for complete translation of nascent, functional receptor.
Assuntos
Retículo Endoplasmático/metabolismo , Mutação de Sentido Incorreto , Receptores Adrenérgicos alfa 1/metabolismo , Substituição de Aminoácidos , Retículo Endoplasmático/genética , Glicosilação , Células HEK293 , Humanos , Domínios Proteicos , Transporte Proteico/genética , Receptores Adrenérgicos alfa 1/genéticaRESUMO
Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.
Assuntos
Proteínas de Membrana/metabolismo , Domínios PDZ , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Imunoprecipitação , Interferometria , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)-a key regulator of cardiovascular, urinary and CNS function-binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.
RESUMO
Alpha(1)-adrenergic receptors are one of the three subfamilies of G protein coupled receptors activated by epinephrine and norepinephrine to control important functions in many target organs. Three human subtypes (alpha(1A), alpha(1B), alpha(1D)) are derived from separate genes and are highly homologous in their transmembrane domains but not in their amino or carboxyl termini. Recent advances in our understanding of these "non-identical triplets" include development of knockout mice lacking single or multiple subtypes, new insights into subcellular localization and trafficking, identification of allosteric modulators, and increasing evidence for an important role in brain function. Although all three subtypes activate the same G(q/11) signaling pathway, they also appear to interact with different protein binding partners. Recent evidence suggests they may also form dimers, and may initiate independent signals through pathways yet to be clearly elucidated. Thus, this subfamily represents a common phenomenon of a group of similar but non-identical receptor subtypes activated by the same neurotransmitter, whose individual functional roles remain to be clearly established.
Assuntos
Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Sistema Nervoso Central/fisiologia , Humanos , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/fisiologia , Transdução de Sinais/fisiologia , Frações Subcelulares/fisiologiaRESUMO
BACKGROUND: Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder precipitated by exposure to extreme traumatic stress. Yet, most individuals exposed to traumatic stress do not develop PTSD and may be considered psychologically resilient. The neural circuits involved in susceptibility or resiliency to PTSD remain unclear, but clinical evidence implicates changes in the noradrenergic system. METHODS: An animal model of PTSD called Traumatic Experience with Reminders of Stress (TERS) was developed by exposing C57BL/6 mice to a single shock (2 mA, 10 sec) followed by exposure to six contextual 1-minute reminders of the shock over a 25-day period. Acoustic startle response (ASR) testing before the shock and after the last reminder allowed experimenters to separate the shocked mice into two cohorts: mice that developed a greatly increased ASR (TERS-susceptible mice) and mice that did not (TERS-resilient mice). RESULTS: Aggressive and social behavioral correlates of PTSD increased in TERS-susceptible mice but not in TERS-resilient mice or control mice. Characterization of c-Fos expression in stress-related brain regions revealed that TERS-susceptible and TERS-resilient mice displayed divergent brain activation following swim stress compared with control mice. Pharmacological activation of noradrenergic inhibitory autoreceptors or blockade of postsynaptic α(1)-adrenoreceptors normalized ASR, aggression, and social interaction in TERS-susceptible mice. The TERS-resilient, but not TERS-susceptible, mice showed a trend toward decreased behavioral responsiveness to noradrenergic autoreceptor blockade compared with control mice. CONCLUSIONS: These data implicate the noradrenergic system as a possible site of pathological and perhaps also adaptive plasticity in response to traumatic stress.
Assuntos
Agressão/fisiologia , Modelos Animais de Doenças , Norepinefrina/fisiologia , Comportamento Social , Transtornos de Estresse Pós-Traumáticos/fisiopatologia , Estresse Psicológico/fisiopatologia , Estimulação Acústica/métodos , Agressão/efeitos dos fármacos , Animais , Mapeamento Encefálico/métodos , Clonidina/farmacologia , Estimulação Elétrica/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prazosina/farmacologia , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Estresse Psicológico/psicologiaRESUMO
G protein-coupled receptors initiate signaling cascades. M(1) muscarinic receptor (M(1)R) activation couples through Galpha(q) to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of PIP(2) closes PIP(2)-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M(1)R activation, M(1)R/Gbeta interaction, Galpha(q)/Gbeta separation, Galpha(q)/PLC interaction, and PIP(2) hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M(1)R activation (<100 ms) and M(1)R/Gbeta interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Galpha(q)/Gbeta separation and Galpha(q)/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP(2) hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Galpha(q)/PLC interaction. Evidently, channel release of PIP(2) and closure are rapid, and the availability of active PLC limits the rate of M current suppression.
Assuntos
Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/fisiologia , Animais , Bovinos , Células Cultivadas , Humanos , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Camundongos , Microscopia de Fluorescência , Fosfatidilinositóis/farmacocinética , Fotometria , Ligação Proteica/genética , RatosRESUMO
Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for alpha(1D)-ARs. We found that dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Distrofina/metabolismo , Hipertensão/metabolismo , Complexos Multiproteicos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1 , Animais , Aorta/metabolismo , Aorta/patologia , Linhagem Celular , Distrofina/genética , Proteínas Associadas à Distrofina/genética , Epinefrina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Hipertensão/genética , Hipertensão/patologia , Camundongos , Complexos Multiproteicos/genética , Tono Muscular/genética , Músculo Liso Vascular/patologia , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologia , Resistência Vascular/genéticaRESUMO
The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.
Assuntos
Substituição de Aminoácidos , Doenças Genéticas Inatas/metabolismo , Hipogonadismo/metabolismo , Mutação Puntual , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos/genética , Linhagem Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Doenças Genéticas Inatas/genética , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipogonadismo/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1RESUMO
To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-AR C terminus as bait was performed on a human brain cDNA library. Alpha-syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.
Assuntos
Proteínas Associadas à Distrofina/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/química , Linhagem Celular , Citoplasma/metabolismo , Proteínas Associadas à Distrofina/química , Humanos , Proteínas de Membrana/química , Camundongos , Proteínas Musculares/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
Heterologous expression of alpha(1D)-adrenergic receptors (alpha(1D)-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with alpha(1B)-ARs promotes surface localization of alpha(1D)-ARs. Here, we report that the alpha(1B)-/alpha(1D)-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of alpha(1B)-ARs and epitope-tagged alpha(1D)-ARs in both human embryonic kidney 293 and DDT(1)MF-2 cells, alpha(1D)-AR binding sites were not detectable with the alpha(1D)-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect alpha(1D)-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the alpha(1B)-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in alpha(1B)/alpha(1D)-AR-expressing cells, revealing that alpha(1D)-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in alpha(1B)/alpha(1D)-AR-coexpressing cells. Stable coexpression of an uncoupled mutant alpha(1B)-AR (Delta12) with alpha(1D)-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that alpha(1B)/alpha(1D)-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why alpha(1D)-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with alpha(1B)-ARs.
Assuntos
Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacologia , Sítios de Ligação , Linhagem Celular , Dimerização , Humanos , Piperazinas/farmacologia , Ensaio Radioligante , Receptores Adrenérgicos alfa 1/químicaRESUMO
G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.
Assuntos
Receptores Acoplados a Proteínas G/fisiologia , Animais , Dimerização , Desenho de Fármacos , Humanos , Ligantes , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.