Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization

AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting

Arrestin-2 Interacts with the Ubiquitin-Protein Isopeptide Ligase Atrophin-interacting Protein 4 and Mediates Endosomal Sorting of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway

G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs

Efeito do trinexapac-etil associado a adubação nitrogenada elevada e parcelada na produtividade do trigo cultivar BRS-220

The use of high nitrogen doses, associated with the application of growth reducers, can be an alternative to wheat plants express high productive potential without the occurrence of lodging. For this reason, the aim of this study was to evaluate the effect of trinexapac-ethyl growth reducer associated with high and split nitrogen fertilization on yield and its components in wheat cultivar BRS 220. The experimental design was randomized blocks with two treatment and four replications, compose with the application of 110 kg N ha-1 with or without 0,4 g L-1 of trinexapac-ethyl. It was evaluated yield and its components, stem diameter, length of internode, plant height and stem length and the averages were submitted to Student t-test using Systat 13®. The application of the growth retardant contributed to a significant increase in yield and stem diameter of the plants, besides the reduction of height and length of stem and internode, when compared to the treatment without product application. El uso de altas dosis de fertilización nitrogenada, asociado con la aplicación de retardadores del crecimiento, puede ser una alternativa a las plantas de trigo capaces de expresar alto potencial productivo sin la aparición de otras plantas. Por esta razón, el objetivo de este estudio fue evaluar el efecto de reductor de crecimiento trinexapac-etil asociado con la fertilización nitrogenada elevada y parcelada sobre el rendimiento y sus componentes en el trigo cultivar BRS 220. El diseño experimental fue de bloques al azar con dos tratamientos y cuatro repeticiones, compuestos del aporte de 110 kg de N ha-1 asociado o no con 0,4 g L-1 trinexapac-etilo. Se evaluó el rendimiento y sus componentes, diámetro del tallo, longitud de entrenudos, altura de la planta y la longitud del tallo. Los datos fueron sometidos a la prueba t de Student en el programa Systat 13®. La aplicación del retardante de crecimiento contribuyó a un aumento significativo en el rendimiento y el diámetro de tallo de la planta, además de la reducción de la altura y de la longitud de los tallos en comparación con el tratamiento sin la aplicación del producto. O uso de altas doses de adubação nitrogenada, associado à aplicação de redutores de crescimento, pode ser uma alternativa para que plantas de trigo consigam expressar potencial produtivo elevado sem a ocorrência de acamamento. Por este motivo, o objetivo deste trabalho foi avaliar o efeito do redutor de crescimento trinexapac-etil associado à adubação nitrogenada elevada e parcelada no rendimento e seus componentes em plantas de trigo da cultivar BRS 220. O delineamento experimental foi de blocos ao acaso com dois tratamentos e quatro repetições, compostos da aplicação de 110 kg de N ha-1 associados ou não a 0,4 g L-1 de trinexapac-etil. Foram avaliados rendimento e seus componentes, diâmetro de colmo, comprimento de entrenó, altura de planta e comprimento do colmo. As médias foram submetidas ao teste-t de Student no programa Systat 13®. A aplicação do redutor de crescimento contribuiu com um aumento significativo no rendimento e no diâmetro do colmo das plantas, além da redução da altura, comprimento do colmo e do entrenó, quando comparado com o tratamento sem a aplicação do produto

Brief Communication Co-seismic displacement on October 26 and 30, 2016 (M_w 5.9 and 6.5) – earthquakes in central Italy from the analysis of discrete GNSS network

<p><strong>Abstract.</strong> On October 26<sup>th</sup> 2016, immediately north of the epicentral area affected by the M<sub>w</sub> 6.0, August 24<sup>th</sup> earthquake, a strong earthquake (<b><i>M</i></b><sub>w</sub> = 5.9), with a focal mechanism showing W-dipping normal faulting, occurred at the boundary between Marche and Umbria regions (central Apennines, Italy). Four days later (on October 30<sup>th</sup>), the main-shock (<b><i>M</i></b><sub>w</sub> = 6.5) of the whole seismic sequence occurred in the same area. The central Apennines are characterized by northeast-verging thrust-propagation folds, involving Mesozoic- Tertiary sedimentary successions. During the 2016 sequence, coseismic deformation has been recorded at the rear of the Sibillini Thrust which separates the main mountain chain from the Marche-Abruzzi foothills (Fig. 1). This contractional structure has been partly dissected and/or inverted by NNW-SSE trending Quaternary normal and oblique-slip faults. The major event (October 30) induced extensive geological effects at the surface and structural damages in the broader epicentral area up to a distance of 30 km. According to the report of the Istituto Nazionale di Geofisica e Vulcanologia (SUMMARY REPORT ON THE 30 OCTOBER, 2016 EARTHQUAKE IN CENTRAL ITALY Mw 6.5, Gruppo di Lavoro INGV sul Terremoto in centro Italia 10 November 2016), the hypocenter of major event was located at 42.8322° N, 13.1107° E at a depth of 9.2 km (Figs. 1 and 2). Following the August seismic events, we installed five new geodetic points located on both sides of the principal fracture zone and carried out two campaigns of GNSS measurements, the first one at the end of September (30-09/02-10, 2016), the second one early November (11/13-11, 2016) that covered the period of the October events. <br><br> In this brief communication, we provide the results of our geodetic campaigns that registered the co- seismic displacement occurred in the period between doy (day of year) 2016/274 and doy 2016/318, therefore documenting the two latter major shocks. We also compare our results with the available surface deformation field of the broader area obtained on the basis of the DInSAR technique and particularly the elaboration realized by CNR-IREA of Sentinel-1 radar imaging of Copernicus European Program of 26/10–1/11 (<a href=" http://www.irea.cnr.it/index.php?option=com_k2&view=item&id=761:nuovi-risultati-sul-terremoto- del-30-ottobre-2016-ottenuti-dai-radar-dei-satelliti-sentinel-1"target="_blank">http://www.irea.cnr.it/index.php?option=com_k2&view=item&id=761:nuovi-risultati-sul-terremoto- del-30-ottobre-2016-ottenuti-dai-radar-dei-satelliti-sentinel-1</a>). The comparison shows an overall good fit. It’s worth to note that these earthquakes occurred in a sector of the Central Apennines characterized by high geodetic strain-rates (e.g., D’Agostino 2014), where several continuous GNSS stations are operating.</p&gt

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe