RESUMEN
SHOX2 is a homeobox transcription factor associated with atrial fibrillation (AF) and sinus node dysfunction. Here, we generated two homozygous SHOX2 knock-out hiPSC lines from a healthy control line and a corrected AF patient line (disease-specific SHOX2 mutation corrected to WT) using CRISPR/Cas9. These cell lines maintained pluripotency, an ability to differentiate into all three germlayers and a normal karyotype, presenting a valuable tool to investigate the impact of a full SHOX2 knock-out with respect to arrhythmogenic diseases on a cellular level.
Asunto(s)
Fibrilación Atrial , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular , Mutación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Fibrilación Atrial/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Acoplamiento Excitación-Contracción , Señalización del Calcio , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismoRESUMEN
Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
Asunto(s)
Elementos de Facilitación Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Diferenciación Celular , Cromatina/metabolismo , Embrión de Mamíferos/citología , Regulación de la Expresión Génica , Células Germinativas/citología , Estratos Germinativos/citología , Masculino , Metilación , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones/citología , Mutación/genética , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , RNA-Seq , Análisis de la Célula Individual , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.
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Autofagia/fisiología , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Autofagia/genética , Cromatina , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/metabolismo , Epigenómica , Edición Génica , Expresión Génica , Síndrome de Hallermann/genética , Humanos , Mutación , FenotipoRESUMEN
PURPOSE: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1ß subunit of the cyclic AMP-dependent protein kinase A (PKA). METHODS: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development. RESULTS: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs. CONCLUSION: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.
Asunto(s)
Apraxias , Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Trastorno del Espectro Autista/genética , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Femenino , Humanos , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Dolor , EmbarazoRESUMEN
We undertook a functional dissection of chromatin remodeler BAZ1B in neural crest (NC) stem cells (NCSCs) from a uniquely informative cohort of typical and atypical patients harboring 7q11.23 copy number variants. Our results reveal a key contribution of BAZ1B to NCSC in vitro induction and migration, coupled with a crucial involvement in NC-specific transcriptional circuits and distal regulation. By intersecting our experimental data with new paleogenetic analyses comparing modern and archaic humans, we found a modern-specific enrichment for regulatory changes both in BAZ1B and its experimentally defined downstream targets, thereby providing the first empirical validation of the human self-domestication hypothesis and positioning BAZ1B as a master regulator of the modern human face. In so doing, we provide experimental evidence that the craniofacial and cognitive/behavioral phenotypes caused by alterations of the Williams-Beuren syndrome critical region can serve as a powerful entry point into the evolution of the modern human face and prosociality.
Asunto(s)
Cromosomas Humanos Par 7/genética , Domesticación , Dosificación de Gen , Factores de Transcripción/genética , Síndrome de Williams/genética , Línea Celular , Movimiento Celular , Bases de Datos Genéticas , Epigenoma , Evolución Molecular , Cara , Redes Reguladoras de Genes , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismoRESUMEN
MAGEL2 is a maternally imprinted, paternally expressed gene, located in the Prader-Willi region of human chromosome 15. Pathogenic variants in the paternal copy of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG), a neurodevelopmental disorder related to Prader-Willi syndrome (PWS). Patients with SHFYNG, like PWS, manifest neonatal hypotonia, feeding difficulties, hypogonadism, intellectual disability and sleep apnea. However, individuals with SHFYNG have joint contractures, greater cognitive impairment, and higher prevalence of autism than seen in PWS. Additionally, SHFYNG is associated with a lower prevalence of hyperphagia and obesity than PWS. Previous studies have shown that truncating variants in MAGEL2 lead to SHFYNG. However, the molecular pathways involved in manifestation of the SHFYNG disease phenotype are still unknown. Here we show that a Magel2 null mouse model and fibroblast cell lines from individuals with SHFYNG exhibit increased expression of mammalian target of rapamycin (mTOR) and decreased autophagy. Additionally, we show that SHFYNG induced pluripotent stem cell (iPSC)-derived neurons exhibit impaired dendrite formation. Alterations in SHFYNG patient fibroblast lines and iPSC-derived neurons are rescued by treatment with the mTOR inhibitor rapamycin. Collectively, our findings identify mTOR as a potential target for the development of pharmacological treatments for SHFYNG.
Asunto(s)
Autofagia , Síndrome de Prader-Willi/patología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/efectos de los fármacos , Dendritas/fisiología , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Síndrome de Prader-Willi/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Largo no Codificante/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.
Asunto(s)
Síndrome Branquio Oto Renal/genética , Variación Estructural del Genoma/genética , Mutación/genética , Cresta Neural/fisiología , Factor de Transcripción AP-2/metabolismo , Adolescente , Alelos , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Haploinsuficiencia , Humanos , Masculino , Ratones , Análisis de la Célula Individual , Factor de Transcripción AP-2/genéticaRESUMEN
Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Técnicas de Transferencia de Gen , Enfermedad Granulomatosa Crónica/patología , Células Madre Pluripotentes Inducidas/enzimología , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Diferenciación Celular , Células Cultivadas , Marcación de Gen , Terapia Genética , Vectores Genéticos , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/terapia , Humanos , Queratinocitos/citología , Glicoproteínas de Membrana/metabolismo , Mutación , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismoRESUMEN
SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Complejos Multiproteicos/metabolismo , Fenotipo , Animales , Western Blotting , Bovinos , Proteínas de Ciclo Celular/genética , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas Cromosómicas no Histona/genética , Cartilla de ADN/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones , Microscopía Fluorescente , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , CohesinasRESUMEN
Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1ß, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos de Superficie/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , Reactividad Cruzada , Citocinas/biosíntesis , Expresión Génica , Vectores Genéticos/genética , Inmunofenotipificación , Lentivirus/genética , Pulmón/inmunología , Activación de Linfocitos/inmunología , Ratones , Fenotipo , TransgenesRESUMEN
Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.
Asunto(s)
Eritropoyetina/farmacología , Neoplasias/metabolismo , Receptores de Eritropoyetina/biosíntesis , Transducción de Señal/efectos de los fármacos , Anticuerpos/inmunología , Western Blotting , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Proliferación Celular , Reacciones Cruzadas , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Fosforilación , ARN Mensajero/análisis , Receptores de Eritropoyetina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO-R) signaling operates as a potential tissue-protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO-R-mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO-R expression, and the effects of EPO on organ-cultured HFs were assessed in the presence/absence of a classical apoptosis-inducing chemotherapeutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up-regulate EPO transcription under hypoxic conditions, and express transcripts for EPO-R and the EPO-stimulatory transcriptional cofactor hypoxia-inducible factor-1alpha. Although EPO does not significantly alter human hair growth in vitro, it significantly down-regulates chemotherapy-induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahematopoietic site of EPO-R expression. They may recruit EPO/EPO-R signaling e.g., for modulating HF apoptosis under conditions of hypoxia and chemotherapy-induced stress.
Asunto(s)
Eritropoyetina/metabolismo , Folículo Piloso/metabolismo , Receptores de Eritropoyetina/metabolismo , Transducción de Señal/fisiología , Animales , Antineoplásicos Alquilantes/farmacología , Apoptosis , Ciclofosfamida/farmacología , Eritropoyetina/genética , Perfilación de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/efectos de los fármacos , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Riñón/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Órganos , Receptores de Eritropoyetina/genética , Cuero Cabelludo/anatomía & histologíaAsunto(s)
Biomarcadores de Tumor/análisis , Eritropoyetina/farmacología , Neoplasias de Cabeza y Cuello/patología , Receptores de Eritropoyetina/metabolismo , Biopsia con Aguja , Progresión de la Enfermedad , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Inmunohistoquímica , Neovascularización Patológica , Pronóstico , Receptores de Eritropoyetina/genética , Factores de Riesgo , Sensibilidad y Especificidad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Comparative analyses revealed considerable differences in embryonic pattern formation and cell-specification between Caenorhabditis elegans and Acrobeloides nanus, members of two neighboring nematode clades. While C.elegans develops very rapidly, A. nanus needs 4-5 times as long. To investigate whether differences during early embryogenesis could be related to developmental tempo, we studied three more slowly developing representatives of the genus Rhabditis, thus close relatives of C.elegans. Besides differences in body size and mode of reproduction, they differ from C.elegans in the order of cleavages, germline behavior and requirement for early zygotic transcription, showing evident similarities to A. nanus. The distinct variations in cell-cycle rhythms and arrest after inhibition of transcription appear to reflect a species-specific interplay in the timing between exhausting maternal supplies and making available newly transcribed gene products. Looking for the reversal of cleavage polarity in the germline present in C.elegans but not in A. nanus, two of the studied species express this distinct feature only in a later cell generation. We found that a C.elegans mutant in the mes-1 gene shows a similar deviation. Concerning specification of the gut cell lineage and the potential to compensate for lost cells, the three tested Rhabditis species behave less regulatively, like C.elegans; in contrast to A. nanus, the gut precursor EMS requires an inductive signal from the germline cell P2 and an experimentally eliminated EMS cell is not replaced by a neighboring blastomere. In conclusion, embryogenesis of the examined Rhabditis species includes features of both the fast-developing C. elegans and the slow-developing A. nanus.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis/genética , Nematodos/genética , Rabdítidos/genética , Animales , Blastocisto/metabolismo , Ciclo Celular , Linaje de la Célula , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Desarrollo Embrionario , Femenino , Rayos Láser , Mutación , Especificidad de la Especie , Factores de Tiempo , Transcripción GenéticaRESUMEN
The micro-opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. Using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that MOR1 and NK1 can form heterodimers in HEK 293 cells coexpressing the two receptors. Although NK1-MOR1 heterodimerization did not substantially change the ligand binding and signaling properties of these receptors, it dramatically altered their internalization and resensitization profile. Exposure of the NK1-MOR1 heterodimer to the MOR1-selective ligand [D-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAMGO) promoted cross-phosphorylation and cointernalization of the NK1 receptor. Conversely, exposure of the NK1-MOR1 heterodimer to the NK1-selective ligand substance P (SP) promoted cross-phosphorylation and cointernalization of the MOR1 receptor. In cells expressing MOR1 alone, beta-arrestin directs the receptors to clathrin-coated pits, but does not internalize with the receptor. In cells expressing NK1 alone, beta-arrestin internalizes with the receptor into endosomes. Interestingly, in cells coexpressing MOR1 and NK1 both DAMGO and SP induced the recruitment of beta-arrestin to the plasma membrane and cointernalization of NK1-MOR1 heterodimers with beta-arrestin into the same endosomal compartment. Consequently, resensitization of MOR1-dependent receptor functions was severely delayed in coexpressing cells as compared with cells expressing MOR1 alone. Together, our findings indicate that MOR1 by virtue of its physical interaction with NK1 is sequestered via an endocytotic pathway with delayed recycling and resensitization kinetics.
Asunto(s)
Receptores Opioides mu/metabolismo , Sustancia P/metabolismo , Arrestinas/metabolismo , Arrestinas/fisiología , Línea Celular , Dimerización , Endocitosis , Humanos , Cinética , Mediciones Luminiscentes , Pruebas de Precipitina , Transporte de Proteínas , Receptores Opioides mu/genética , Sustancia P/genética , Transfección , beta-ArrestinasRESUMEN
Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schröder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Höllt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.