RESUMO
Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/ß, phosphorylate α1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α1-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Receptores Adrenérgicos alfa 1/metabolismo , Células HEK293 , Humanos , Fosforilação/fisiologia , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores Adrenérgicos alfa 1/genéticaRESUMO
In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α1A-Adrenergic receptor interaction with ß-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α1A-adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α1A-AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Proteólise , Receptores Adrenérgicos alfa 1/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espectrometria de Massas , Norepinefrina/farmacologia , Oximetazolina/farmacologia , Fosforilação/genética , Proteína Quinase C/genética , Receptores Adrenérgicos alfa 1/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Upon agonist stimulation, α1B-adrenergic receptors couple to Gq proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as ß-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α1B-AR vesicular traffic were investigated by studying α1B-adrenergic receptor-Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged α1B-ARs and enhanced green fluorescent protein--tagged Rab proteins, pharmacological protein kinase C activation mimicked α1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient α1B-AR-Rab5 FRET signal followed by a sustained α1B-AR-Rab9 interaction), suggesting brief receptor localization in early endosomes and transfer to late endosomes. This latter interaction was abrogated by blocking protein kinase C activity, resulting in receptor retention at the plasma membrane. Similar effects were observed when a dominant-negative Rab9 mutant (Rab9-GDP) was employed. When α1B-adrenergic receptors that had been mutated at protein kinase C phosphorylation sites (S396A, S402A) were used, phorbol ester-induced desensitization of the calcium response was markedly decreased; however, interaction with Rab9 was only partially decreased and internalization was observed in response to phorbol esters and sphingosine 1-phosphate. Finally, Rab9-GDP expression did not affect adrenergic-mediated calcium response but abolished receptor traffic and altered desensitization. Data suggest that protein kinase C modulates α1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.
Assuntos
Endocitose , Endossomos/metabolismo , Proteína Quinase C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fluorescência , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Upon natural agonist or pharmacological stimulation, G protein-coupled receptors (GPCRs) are subjected to posttranslational modifications, such as phosphorylation and ubiquitination. These posttranslational modifications allow protein-protein interactions that turn off and/or switch receptor signaling as well as trigger receptor internalization, recycling or degradation, among other responses. Characterization of these processes is essential to unravel the function and regulation of GPCR. METHODS: In silico analysis and methods such as mass spectrometry have emerged as novel powerful tools. Both approaches have allowed proteomic studies to detect not only GPCR posttranslational modifications and receptor association with other signaling macromolecules but also to assess receptor conformational dynamics after ligand (agonist/antagonist) association. RESULTS: this review aims to provide insights into some of these methodologies and to highlight how their use is enhancing our comprehension of GPCR function. We present an overview using data from different laboratories (including our own), particularly focusing on free fatty acid receptor 4 (FFA4) (previously known as GPR120) and α1A- and α1D-adrenergic receptors. From our perspective, these studies contribute to the understanding of GPCR regulation and will help to design better therapeutic agents.
Assuntos
Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genéticaRESUMO
Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with ligands. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces a capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR individually with drugs prevents the recruitment of both EGFR and FGFR. Our data reveals novel crosstalk between multiple unrelated receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.
RESUMO
The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor ß5-integrin. We show that ligand-activated EGFR, Grb2, Src, and ß5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to EGF into large flat plaques and provide a signaling platform that link EGFR and ß5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/ß5-integrin axis prevents both clathrin plaque growth and dampens receptor signaling. Our study reveals a reciprocal regulation between clathrin lattices and two different receptor systems to coordinate and enhance signaling. These findings have broad implications for the regulation of growth factor signaling, adhesion, and endocytosis.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/química , Proteína Adaptadora GRB2/metabolismo , Cadeias beta de Integrinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Endocitose , Receptores ErbB/metabolismo , Humanos , Microscopia Eletrônica , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismoRESUMO
Clathrin-mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of long-standing debate. Here, we use platinum replica and cryoelectron microscopy and tomography to present a structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a universal mechanistic model of clathrin-mediated endocytosis.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clatrina/metabolismo , Adesividade , Animais , Linhagem Celular , Colesterol/metabolismo , Microscopia Crioeletrônica , Humanos , Masculino , Camundongos , Modelos Biológicos , RatosRESUMO
Phosphorylation of the human α1B-adrenergic receptor (fused with the green fluorescent protein) was studied employing the inducible Flp-ln HEK293 T-Rex system for expression. Serine/alanine substitutions were performed in five sites corresponding to those previously identified as phosphorylation targets in the hamster ortholog. Desensitization was decreased in these mutants but receptor phosphorylation was still clearly detected. The protein phosphorylation of the wild-type receptor (fused to the green fluorescent protein) was studied, using mass spectrometry, under baseline and stimulated conditions (noradrenaline or phorbol myristate acetate). Basal phosphorylation was detected at sites located at the intracellular loop 3 and carboxyl terminus, and the number of sites detected increased under agonist activation and stimulation of protein kinase C. The phosphorylation patterns differed under the distinct conditions. Three of the phosphorylation sites detected in this work corresponded to those observed in the hamster receptor. The phosphorylation sites detected included the following: a) at the intracellular loop 3: serines 246, 248, 257, 267, and 277; and threonines 252, 264, and 268, and b) at the carboxyl terminus: serines 396, 400, 402, 406, 423, 425, 427, 455, and 470, and threonines 387, 392, 420, and 475. Our data indicate that complex phosphorylation patterns exist and suggest the possibility that such differences could be relevant in receptor function and subcellular localization.
Assuntos
Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Substituição de Aminoácidos , Animais , Cricetinae , MAP Quinases Reguladas por Sinal Extracelular , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Proteína Quinase C/metabolismoRESUMO
The human α1D-adrenergic receptor is a seven transmembrane-domain protein that mediates many of the physiological actions of adrenaline and noradrenaline and participates in the development of hypertension and benign prostatic hyperplasia. We recently reported that different phosphorylation patterns control α1D-adrenergic receptor desensitization. However, to our knowledge, there is no data regarding the role(s) of this receptor's specific phosphorylation residues in its subcellular localization and signaling. In order to address this issue, we mutated the identified phosphorylated residues located on the third intracellular loop and carboxyl tail. In this way, we experimentally confirmed α1D-AR phosphorylation sites and identified, in the carboxyl tail, two groups of residues in close proximity to each other, as well as two individual residues in the proximal (T442) and distal (S543) regions. Our results indicate that phosphorylation of the distal cluster (T507, S515, S516 and S518) favors α1D-AR localization at the plasma membrane, i. e., substitution of these residues for non-phosphorylatable amino acids results in the intracellular localization of the receptors, whereas phospho-mimetic substitution allows plasma membrane localization. Moreover, we found that T442 phosphorylation is necessary for agonist- and phorbol ester-induced receptor colocalization with ß-arrestins. Additionally, we observed that substitution of intracellular loop 3 phosphorylation sites for non-phosphorylatable amino acids resulted in sustained ERK1/2 activation; additional mutations in the phosphorylated residues in the carboxyl tail did not alter this pattern. In contrast, mobilization of intracellular calcium and receptor internalization appear to be controlled by the phosphorylation of both third-intracellular-loop and carboxyl terminus-domain residues. In summary, our data indicate that a) both the phosphorylation sites present in the third intracellular loop and in the carboxyl terminus participate in triggering calcium signaling and in turning-off α1D-AR-induced ERK activation; b) phosphorylation of the distal cluster appears to play a role in receptor's plasma membrane localization; and c) T442 appears to play a critical role in receptor phosphorylation and receptor-ß-arrestin colocalization.
Assuntos
Receptores Adrenérgicos alfa 1/análise , Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Fosforilação , Conformação Proteica , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
Lysophosphatidic acid (LPA) modulates the function of many organs, including the lung. A549 is a lung carcinoma-derived cell line, frequently used as a model for type II pneumocytes. Here we show that these cells expressed messenger RNA coding for LPA1-3 receptors with the following order of abundance: LPA1 > LPA2 > LPA3 and that LPA was able to increase intracellular calcium, extracellular signal-regulated kinases 1/2 phosphorylation, and cell contraction. These effects were blocked by Ki16425, an antagonist selective for LPA1 and LPA3 receptors, and by the LPA1-selective antagonist, AM095. Activation of protein kinase C inhibited LPA-induced intracellular calcium increase. This action was blocked by protein kinase C inhibitors and enzyme down-regulation. Phorbol myristate acetate and AM095, but not Ki16425, decreased the baseline intracellular calcium concentration. Ki16425 blocked the effect of AM095 but not that of phorbol myristate acetate. The data indicate that LPA1 receptors exhibit constitutive activity and that AM095 behaves as an inverse agonist, whereas Ki16425 appears to be a classic antagonist. Furthermore, the LPA agonist, 1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT, induced a weak increase in intracellular calcium, but was able to induce full ERK 1/2 phosphorylation and cell contraction. These effects were blocked by AM095. These data suggest that OMPT is a biased LPA1 agonist. A549 cells express functional LPA1 receptors and seem to be a suitable model to study their signaling and regulation.
Assuntos
Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Células A549 , Cálcio/metabolismo , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/genéticaRESUMO
Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated α1D-adrenoceptors were transfected into Rat-1, HEK293, and B103 cells, and changes in the phosphorylation state of extracellular signal-regulated kinase was assessed using biochemical and biophysical approaches. The phosphorylation state of other protein kinases (p38, MEK1, and Raf-1) was also studied. Noradrenaline-induced ERK phosphorylation in Rat-1 fibroblasts expressing amino termini-truncated α1D-adrenoceptors. However, in cells expressing receptors with both amino and carboxyl termini truncations, noradrenaline-induced activation was abrogated. Interestingly, ERK phosphorylation that normally occurs through activation of endogenous G protein-coupled receptors, EGF receptors, and protein kinase C, was also decreased, suggesting that downstream steps in the mitogen-activated protein kinase pathway were affected. A similar effect was observed in B103 cells but not in HEK 293 cells. Phosphorylation of Raf-1 and MEK1 was also diminished in Rat-1 fibroblasts expressing amino- and carboxyl-truncated α1D-adrenoceptors. Our data indicate that expression of carboxyl terminus-truncated α1D-adrenoceptors alters ERK and p38 phosphorylation state.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Ativação Enzimática , Receptores ErbB/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Mutação , Fosforilação , Domínios Proteicos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).