M2 muscarinic receptor-receptor interaction enhanced by circulating antibodies from Chagas’ disease patients

BELTRAME, SABRINA P.; SÁNCHEZ, MANUEL A.; BILDER, CLAUDIO R.; AUGER, SERGIO R.; GOIN, JUAN C.

Buenos Aites

Congreso; III Iberoamerican Congress on Neuroimmunomodulation; 2009

NIM Argentina

Circulating IgG antibodies (Abs) against M2 muscarinic receptors (M2R) have been implicated in Chagas´disease (ChD) physiopathology. These Abs bind to and activate M2R, displaying agonist-like activity through an unclear mechanism. The ability of anti-M2R Abs to modulate M2 receptor-receptor interaction has been recently reported. In this work we characterized the specificity of this modulatory effect at the level of both IgG and M2R molecules using bioluminescence resonance energy transfer (BRET). HEK-293 cells expressing fusion proteins M2R-RLuc and M2R-YFP were treated with atropine, pilocarpine, carbachol or ChD-IgG and the BRET between RLuc and YFP was assessed by luminometry. Unlike muscarinic ligands, CH-IgG promoted a significant increase in the BRET signal suggesting that the ability to enhance M2R-M2R interaction is not related to its muscarinic activity and occurs through a different mechanism. Affinity purified Abs against the second extracellular loop (2nd ECL) of the human M2R could mimic the effect of the total ChD-IgG, fraction, indicating that the enhancement of M2R-M2R interaction is mediated by the recognition of the 2nd ECL of M2R. The monovalent Fab fragment from ChD IgG was unable to promote an increase in M2R-M2R interaction, suggesting that the effect of ChD-IgG results from receptor crosslinking by bivalent Abs.