N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias

G-protein-coupled receptors (GPCRs) are the largest class of mammalian signaling receptors and mediate vast physiological responses. The capacity to modulate GPCR signaling therapeutically is important for treatment of various diseases, and discovering new aspects of receptor signaling is critical for drug development. Protease-activated receptor-1 (PAR1) is GPCR for thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell. How a single GPCR can couple to multiple G-protein subtypes concurrently has remained an enigma. We demonstrate that N-linked glycosylation of PAR1 regulates G-protein coupling specificity and differentially controls cellular responses. Thus, the status of GPCR glycosylation is a critical determinant for specifying coupling to distinct G-protein subtypes

Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /

G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequent downstream signaling. Protease-activated receptor-1 (PAR1) is a member of a family of GPCRs that are proteolytically activated. PAR1 is a receptor for the coagulant protease thrombin, and is capable of coupling to multiple G-protein subtypes to elicit various cellular responses; however, the mechanisms that regulate this selectivity are not well understood. Palmitoylation is a post-translational modification that many GPCRs possess, and is defined as the addition of palmitate, a 16 carbon fatty acid, to a cysteine residue via a thioester linkage. Many GPCRs are palmitoylated on their C-terminal tails, but the role of this modification differs based on the GPCR being examined. In this dissertation, I examined the role of palmitoylation in PAR1 signaling and trafficking. I defined the sites of PAR1 palmitoylation to occur on conserved C-tail cysteine residues C387 and C388. I discovered that palmitoylation is important for other PAR1 post-translational modifications, specifically phosphorylation and ubiquitination. I also show that palmitoylation of PAR1 regulates the accessibility of a nearby tyrosine-based sorting motif to the adaptor-protein complex-2 (AP-2) and -3 (AP-3), which controls receptor internalization and degradation. Additionally, palmitoylation appears to be important for the regulation of selective PAR1-induced signaling pathways such as G[alpha]₁₂/₁₃ -induced RhoA activation, G[alpha]i coupling , and thrombin-stimulated p38 MAPK signaling pathways. However, thrombin-induced G[alpha]q-mediated phosphoinositide hydrolysis and ERK1/2 activation are unperturbed in the absence of PAR1 palmitoylation. Taken together, the studies summarized in this dissertation highlight the relevance of palmitoylation for PAR1 function, and suggest that palmitoylation governs a C-tail conformation that is important for accessibility of other proteins such as ligases, kinases, adaptor proteins, and G -proteins, ultimately regulating PAR1 signaling and traffickin

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins.

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein–coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser15, Ser78, and Ser82. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser15 and Ser78. Inhibition of both MK2 and MK3 was necessary to attenuate Ser82 phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo

Externado de Ginecología y Obstetricia - ME215 - 202101

1 Curso de la carrera de medicina, de carácter teórico-práctico del ciclo 10, en el que los estudiantes presentan una historia clínica con su respectivo plan diagnóstico, terapéutico y procedimientos en un paciente con una condición ginecológica u obstétrica, a través de la realización de actividades de simulación y metodología activa en aula. El curso del externado de ginecología y obstetricia, busca desarrollar las competencias específicas de profesionalismo-sentido ético y legal y responsabilidad profesional(nivel 3). El externado de ginecología y obstetricia, le permitirá al estudiante plantear un adecuado tratamiento y plan de trabajo según el diagnóstico de los principales problemas ginecológicos y obstétricos que le servirán en su vida futura profesional

Externado de Ginecología y Obstetricia - ME215 - 202102

Curso de la carrera de medicina, de carácter teórico-práctico del ciclo 10, en el que los estudiantes presentan una historia clínica con su respectivo plan diagnóstico, terapéutico y procedimientos en un paciente con una condición ginecológica u obstétrica, a través de la realización de actividades de simulación y metodología activa en aula. El curso del externado de ginecología y obstetricia, busca desarrollar las competencias específicas de profesionalismo-sentido ético y legal y responsabilidad profesional(nivel 3). El externado de ginecología y obstetricia, le permitirá al estudiante plantear un adecuado tratamiento y plan de trabajo según el diagnóstico de los principales problemas ginecológicos y obstétricos que le servirán en su vida futura profesional