Interacción del receptor muscarínico M2 con arrestinas no visuales y su modulación por anticuerpos séricos de pacientes chagásicos crónicos
Previous studies have demonstrated a strong correlation between serum levels of IgG autoantibod-ies against M2 muscarinic receptors (M2R Ab) and the extent of parasympathetic dysfunction in chronic chagasic patients. These antibodies recognize the second extracellular loop (II-ECL) of the human M2R...
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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2021
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7958 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7958.dir/7958.PDF |
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| Sumario: | Previous studies have demonstrated a strong correlation between serum levels of IgG autoantibod-ies against M2 muscarinic receptors (M2R Ab) and the extent of parasympathetic dysfunction in chronic chagasic patients. These antibodies recognize the second extracellular loop (II-ECL) of the human M2R and activate this receptor, thus eliciting negative inotropic and chronotropic effects through Gi protein activation in rat cultured cardiomyocytes and isolated atria. In contrast, preincu-bation of heterologous cells expressing M2R or rat atria with such antibodies, followed by the addi-tion of a muscarinic agonist, results in the inhibition of agonist-mediated effects. These data suggest that the chronic exposure of cardiac M2R to serum M2R Ab could attenuate of acetylcholine-medi-ated cardiac effects, thereby contributing to the development of parasympathetic dysfunction in chronic chagasic patients. Two mechanisms underlying the antibody-mediated inhibition of musca-rinic agonist activity through M2R have been proposed: (I) M2R desensitization/internalization me-diated by GRK and ?-arrestins; (II) negative allosteric modulation. In this study, the feasibility of these mechanisms was examined by assessing the effects of M2R Ab on the interaction between M2R and ?-arrestins (Arr-2 or Arr-3), in the presence or absence of agonist, using bioluminescence resonance energy transfer (BRET).\nCarbachol treatment of HEK 293T cells expressing M2R fused to Renilla luciferase (M2R -RLuc) and Arr-2 (or Arr-3) fused to yellow fluorescent protein (Arr-2-YFP or Arr-3-YFP) (BRET pair) stimu-lated the interaction between the M2R and Arr-2 (or Arr-3) only when GRK2 was coexpressed. In contrast, treatment of cells with serum IgG fractions from dysautonomic chagasic patients seroposi-tive for M2R Ab (Ch IgG+) or IgG from healthy noninfected individuals (control IgG) did not promote the translocation either ?-arrestin to the M2R. Preincubation of cells coexpressing the BRET pair and GRK2 with Ch IgG+ followed by the addition of carbachol resulted in carbachol-induced inhibition of Arr-2 recruitment (but not of that of Arr-3) to the M2R, while control IgG was unable to modulate the translocation of Arr-2 o Arr-3. The inhibitory effect of Ch IgG+ on agonist-induced Arr-2 translo-cation to the M2R (a) was neutralized by a synthetic peptide with the amino acid sequence of the II-ECL of the human M2R, thus demonstrating that serum inhibitory antibodies specifically interact with the M2R; (b) was dependent on the exposure time of M2R to muscarinic autoantibodies; (c) was consistent with a noncompetitive inhibition model, showing a decrease in agonist efficacy with no changes in potency; (d) was not mimicked by the Fab fraction derived from Ch IgG+, except in the presence of an anti-human Fab antibody, suggesting that this inhibitory effect is mediated by recep-tor crosslinking.\nAs regards the pharmacodynamic mechanism underlying the inhibitory effect of M2R Ab on agonist-induced M2R/Arr-2 interaction, the present data suggest that this effect does not likely oc-cur as a consequence of M2R homologous regulation. First, these antibodies cannot stimulate ?-arrestin recruitment to the M2R. Since agonist-induced M2R desensitization is associated with M2R/?-arrestin interaction, it is unlikely that these antibodies can promote M2R desensitization. Second, M2R Ab cannot induce arrestin-dependent or -independent M2R internalization in HEK 293T cells, according to recently published data. Third, agonist-induced Arr-3 translocation is not affected by M2R Ab, suggesting that the antibody-bound receptor is not desensitized nor internalized. Con-versely, the fact that M2R Ab interact with the II-ECL at the common allosteric site of the M2R and promote a selective, noncompetitive inhibition of agonist-induced Arr-2-recruitment suggests that these autoantibodies function as biased, negative allosteric modulators of agonist efficacy. |
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