RESUMEN
The cannabinoid receptor 2 (CB2R) has high, unexploited therapeutic potential in several central nervous system disorders due to its involvement in neuroinflammatory processes and pathologies like neurodegeneration. Dualsteric/bitopic ligands are currently developed to achieve receptor subtype selectivity and biased signaling. To obtain a molecular tool compound with photoswitchable potential dualsteric properties, we applied two different approaches to link a positive allosteric modulator with an orthosteric agonist via a photochromic unit. We characterized the photophysical properties of all compounds and determined efficacy in internalization, calcium mobilization, and BRET studies. We report the first potentially dualsteric photoswitchable ligand for studying molecular mechanisms of CB2R-associated pathologies. Compound 17-para is a submicromolar "cis-on" agonist with >10-fold higher potency compared to its trans photoisomer and allows high spatiotemporal control of CB2R activation.
Asunto(s)
Cannabinoides , Transducción de Señal , Receptores de Cannabinoides , Ligandos , Sitios de Unión , Cannabinoides/farmacología , Receptor Cannabinoide CB2RESUMEN
The cannabinoid 2 receptor (CB2 R) has high therapeutic potential for multiple pathogenic processes, such as neuroinflammation. Pathway-selective ligands are needed to overcome the lack of clinical success and to elucidate correlations between pathways and their respective therapeutic effects. Herein, we report the design and synthesis of a photoswitchable scaffold based on the privileged structure of benzimidazole and its application as a functionally selective CB2 R "efficacy-switch". Benzimidazole azo-arenes offer huge potential for the broad extension of photopharmacology to a wide range of optically addressable biological targets. We used this scaffold to develop compound 10 d, a "trans-on" agonist, which serves as a molecular probe to study the ß-arrestin2 (ßarr2) pathway at CB2 R. ßΑrr2 bias was observed in CB2 R internalization and ßarr2 recruitment, while no activation occurred when looking at Gα16 or mini-Gαi . Overall, compound 10 d is the first light-dependent functionally selective agonist to investigate the complex mechanisms of CB2 R-ßarr2 dependent endocytosis.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Cannabinoides , Arrestina beta 2/metabolismo , Cannabinoides/farmacología , Bencimidazoles/químicaRESUMEN
Activation of the human cannabinoid receptor type 1 (hCB1R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB1R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB1R (Ki (cis-form) = 0.18 µM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB1R Ki trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT ßarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB1R in disorders associated with the endocannabinoid system.
Asunto(s)
Amidas , Hexaclorobenceno , Endocannabinoides , Humanos , Indoles/química , Receptor Cannabinoide CB1 , Receptores de CannabinoidesRESUMEN
Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , Receptor Muscarínico M2/metabolismo , Ubiquitina/metabolismo , Animales , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cloroquina/farmacología , Agonistas Colinérgicos/farmacología , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/ultraestructura , Ganglios Espinales/citología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Microscopía Confocal , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Proteolisis , Ratas , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/genética , Transfección , Ubiquitina/efectos de los fármacos , UbiquitinaciónRESUMEN
Fidelity of signal transduction relies on cells expressing the appropriate number of functional receptors. Fluctuation in the total number of muscarinic acetylcholine receptors has been implicated in a range of physiological and pathophysiological processes, and the mechanisms responsible for this regulation represent potential molecular targets for therapeutic intervention. This article will review the current literature on the endocytic trafficking of muscarinic receptors and how knowledge of the trafficking of related receptors might influence future studies. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.
Asunto(s)
Receptores Muscarínicos/metabolismo , Animales , Endocitosis , Regulación de la Expresión Génica , Humanos , Transporte de ProteínasRESUMEN
Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes ß-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation.
Asunto(s)
Apoptosis , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Secuencia de Aminoácidos , Arrestinas/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de Formil Péptido/química , Receptores de Lipoxina/química , beta-ArrestinasRESUMEN
Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.
Asunto(s)
Transporte Axonal , Vectores Genéticos , Lentivirus/genética , Neuronas Motoras/metabolismo , Virus de la Rabia/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Endocitosis , Células HEK293 , Humanos , Neuronas Motoras/virología , Ratas , Proteínas del Envoltorio Viral/genéticaRESUMEN
VIDEO ABSTRACT: Some endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that µ-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor's first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached.
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Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Ubiquitinación , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Células HEK293 , Humanos , Fosforilación , Receptores Opioides mu/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
µ-Type opioid receptors (MORs) are members of the large seven-transmembrane receptor family which transduce the effects of both endogenous neuropeptides and clinically important opioid drugs. Prolonged activation of MORs promotes their proteolytic degradation by endocytic trafficking to lysosomes. This down-regulation process is known to contribute to homeostatic regulation of cellular opioid responsiveness, but mechanisms that mediate and control MOR down-regulation have not been defined. We show here that lysosomal down-regulation of MORs is ESCRT (endosomal sorting complex required for transport)-dependent and involves ubiquitin-promoted transfer of internalized MORs from the limiting endosome membrane to lumen. We also show that MOR down-regulation measured by conventional radioligand binding assay is determined specifically by ubiquitination in the first cytoplasmic loop. Surprisingly, we were unable to find any role of ubiquitination in determining whether internalized receptors recycle or are delivered to lysosomes. Instead, this decision is dictated specifically by the MOR C-tail and occurs irrespectively of the presence or absence of receptor ubiquitination. Our results support a hierarchical organization of discrete ubiquitin-independent and -dependent sorting operations, which function non-redundantly in the conserved down-regulation pathway to mediate precise endocytic control. Furthermore, they show that this hierarchical mechanism discriminates the endocytic regulation of naturally occurring MOR isoforms. Moreover, they are the first to reveal, we believe, for any seven-transmembrane receptor, a functional role of ubiquitination in the first cytoplasmic loop.
Asunto(s)
Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Lisosomas/metabolismo , Receptores Opioides mu/metabolismo , Ubiquitinación/fisiología , Animales , Células HEK293 , Humanos , Lisosomas/genética , Ratones , Estructura Secundaria de Proteína , Receptores Opioides mu/genéticaRESUMEN
Localization and trafficking of G protein-coupled receptors (GPCRs) is increasingly recognized to play a fundamental role in receptor-mediated signaling and its regulation. Individual receptors, including closely homologous subtypes with otherwise similar functional properties, can differ considerably in their membrane trafficking properties. In this chapter, we describe several approaches for experimentally assessing the subcellular localization and trafficking of selected GPCRs. Firstly, we describe a flexible method for receptor localization using fluorescence microscopy. We then describe two complementary approaches, using fluorescence flow cytometry and surface biotinylation, for examining receptor internalization and trafficking in the endocytic pathway.
Asunto(s)
Espacio Intracelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Biotinilación , Citometría de Flujo , Células HEK293 , Humanos , Transporte de Proteínas , Coloración y Etiquetado , Fijación del TejidoRESUMEN
The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Lisosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Receptores Opioides delta/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Línea Celular Transformada , Células Cultivadas , Regulación hacia Abajo , Endocitosis/genética , Endocitosis/fisiología , Células HEK293 , Humanos , Lisosomas/genética , Cuerpos Multivesiculares/genética , Transporte de Proteínas , Receptores Opioides delta/genéticaRESUMEN
Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin-dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin-dependent and -independent regulation that may be specific to animal cells.
Asunto(s)
Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Animales , Membrana Celular/metabolismo , Humanos , Transporte de Proteínas , Saccharomycetales/metabolismoRESUMEN
Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine delta-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized delta-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor's hydrophobic core.
Asunto(s)
Lisosomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitinación , Unión Competitiva , Biotinilación , Línea Celular , Electroforesis en Gel de Poliacrilamida , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Microscopía Fluorescente , Modelos Biológicos , Mutación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ensayo de Unión Radioligante , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Mu-type opioid receptors are physiologically important G-protein-coupled receptors that are generally thought to recycle after agonist-induced endocytosis. Here we show that several alternatively spliced receptor variants fail to do so efficiently because of splice-mediated removal of an endocytic sorting sequence that is present specifically in the MOR1 variant. All of the recycling-impaired receptor variants were found to undergo proteolytic down-regulation more rapidly than MOR1, irrespective of moderate differences in endocytic rate, indicating that alternative splicing plays a specific role in distinguishing the trafficking itinerary of receptors after endocytosis. The recycling-impaired MOR1B variant was similar to MOR1 in its ability to mediate opioid-dependent inhibition of adenylyl cyclase, and to undergo opioid-induced desensitization in intact cells. Functional recovery (resensitization) of MOR1B-mediated cellular responsiveness after opioid removal, however, was significantly impaired (4-fold reduction in rate) compared with MOR1. To our knowledge the present results are the first to establish a role of alternative RNA processing in specifying the post-endocytic sorting of G-protein-coupled receptors between divergent and functionally distinct membrane pathways.
Asunto(s)
Empalme Alternativo/fisiología , Membrana Celular/metabolismo , Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Receptores Opioides mu/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Línea Celular , Membrana Celular/genética , Humanos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/fisiología , Receptores Opioides mu/agonistas , Receptores Opioides mu/genéticaRESUMEN
Activation of seven-transmembrane receptors is typically followed by desensitization and arrestin-dependent internalization via vesicles that are pinched off by a dynamin collar. Arrestins also scaffold Src, which mediates dynamin-dependent internalization of beta2-adrenergic receptors. Type I mammalian gonadotropin-releasing hormone receptors (GnRHRs) do not rapidly desensitize or internalize (characteristics attributed to their unique lack of C-terminal tails) whereas non-mammalian GnRHRs (that have C-terminal tails) are rapidly internalized and desensitized. Moreover, internalization of Xenopus (X) GnRHRs is dynamin-dependent whereas that of human (h) GnRHRs is not, raising the possibility that binding of arrestin to the C-terminal tails of GnRHRs targets them to the dynamin-dependent internalization pathway. To test this we have compared wild-type GnRHRs with chimeric receptors (XGnRHR C-terminal tail added to the hGnRHR alone (h.XtGnRHR) or with exchange of the third intracellular loops (h.Xl.XtGnRHR)). We show that adding the XGnRHR C-terminal tail facilitates arrestin- and dynamin-dependent internalization as well as arrestin/green fluorescent protein translocation, but Src (or mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibition does not slow internalization, and h.XtGnRHR internalization is slower than that of the hGnRHR. Moreover, arrestin expression increased XGnRHR internalization even when dynamin was inhibited and h.Xl.XtGnRHR underwent rapid arrestin-dependent internalization without signaling to G(q/11). Thus, although the C-terminal tail can direct GnRHRs for arrestin- and dynamin-dependent internalization, this effect is not dependent on Src activation and arrestin can also facilitate dynamin-independent internalization.
Asunto(s)
Arrestina/metabolismo , Dinaminas/metabolismo , Receptores LHRH/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , ADN Complementario/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Cinética , Unión Proteica , Ingeniería de Proteínas , Receptores LHRH/química , Receptores LHRH/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , XenopusRESUMEN
Desensitization and internalization of G protein-coupled receptors can be mediated by phosphorylation within the C-terminal tail, facilitating beta-arrestin binding and targeting the receptor for internalization. Type II GnRH receptors (GnRH-Rs) show such regulation, but type I GnRH-Rs lack C-tails and are not rapidly desensitized or internalized. Here we show contrasting susceptibility of type I (human and sheep) and II (Xenopus) GnRH-Rs to regulation by protein kinase C (PKC). When human (h) or Xenopus (X) GnRH-Rs were expressed using recombinant adenovirus, PKC activation increased radioligand binding to XGnRH-Rs but not to hGnRH-Rs. A dominant-negative dynamin mutant (K44A) inhibited internalization of XGnRH-Rs (but not hGnRH-Rs) without influencing PKC regulation of XGnRH-R binding. PKC activation increased the affinity of XGnRH-Rs for the type II GnRH ligand and increased effects of low concentrations of GnRH-II on the [Ca(2+)](i) but had no effect on type I ligand binding to hGnRH-Rs, sGnRH-Rs or XGnRH-Rs, or to chimeric receptors with the XGnRH-R C-tail added to a type I receptor. Binding of type II ligand to human or sheep receptors was also unaffected but was increased in the chimeras. Mutation of both PKC-phosphorylation consensus sites in the XGnRH-R tail did not prevent the PKC-mediated increases in binding or alter agonist-induced translocation of beta-arrestin2/green fluorescent protein or inhibition of inositol phosphate accumulation by beta-arrestin2/green fluorescent protein. Thus, it appears that there are two distinct active conformations of XGnRH-Rs (differing in affinity for type I and II ligands) and that these cells exhibit a novel form of inside-out signaling in which PKC feeds back to influence receptor affinity.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Proteína Quinasa C/fisiología , Receptores LHRH/metabolismo , Transducción de Señal , Sitios de Unión , Células HeLa , Humanos , Fosforilación , Conformación Proteica , Receptores LHRH/química , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
GnRH receptors (GnRH-Rs) mediate direct antiproliferative effects on hormone-dependent cancer cells. GnRH-Rs can be grouped according to ligand specificity (for GnRH-I and -II), and there is evidence that type II GnRH ligands and/or receptors can inhibit proliferation. Type I GnRH-Rs (e.g. human and sheep) lack the C-terminal tails found in other G protein-coupled receptors including type II GnRH-Rs (e.g. Xenopus; XGnRH-R). This underlies the remarkable resistance of type I GnRH-Rs to desensitization and may be important for chronic effects on proliferation. To test this, we have compared the antiproliferative effects of GnRH-Rs expressed in MCF7 breast cancer cells using recombinant adenovirus (Ad). Endogenous GnRH-Rs were not detected, but infection with Ad-expressing sheep GnRH-Rs (sGnRH-R) facilitated proliferation inhibition by Buserelin, and maximum inhibition required only 10,000-20,000 sGnRH-Rs. XGnRH-Rs were much less efficient at inhibiting proliferation and were internalized faster than sGnRH-Rs. Thus, the type II GnRH-R is less efficient at inhibiting proliferation, presumably because it is rapidly desensitized and/or internalized. Moreover, comparisons of human GnRH-R, sGnRH-R, and XGnRH-R, as well as chimeric receptors (type I GnRH-Rs with C-terminal tails from XGnRH-Rs), revealed that C-terminal tail addition increases receptor expression and thereby increases the efficiency with which the vector facilitates the antiproliferative effect.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptores LHRH/metabolismo , Transducción de Señal , Animales , Antineoplásicos Hormonales/farmacología , Buserelina/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quimera , Femenino , Técnicas de Transferencia de Gen , Humanos , Conformación Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Receptores LHRH/genética , Ovinos , Especificidad de la Especie , Xenopus laevisRESUMEN
Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this "ubiquitination-independent" GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.