Characterization of M4 muscarinic acetylcholine receptor homo- and hetero-oligomerization by bioluminescence resonance energy transfer in live cells

BAYONÉS, LUCAS; BELTRAME, SABRINA ; WALDNER, CLAUDIA; GOIN, JUAN CARLOS

Huerta Grande

Congreso; XXVII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias; 2012

Sociedad Argentina de Investigación en Neurociencias

Recent data suggest that M4 muscarinic acetylcholine receptors (M4R) form constitutive homo-oligomers. This study used bioluminescence resonance energy transfer (BRET) to assess the ability of M4R to form homo- and heterooligomers in live HEK 293 cells. Cotransfection of cells with a fixed amount of Renilla Luciferase-tagged M4R and increasing amounts of YFP-tagged M4R resulted in a gradual increase in BRET upon addition of coelenterazine h. The resulting saturation curve could be fitted into a hyperbolic function, indicating that M4R can interact with one another to form constitutive homo-oligomers (BRETmax: 93,9 ± 0.4 mB; BRET50: 1.4 ± 0.2). Quantitative BRET analysis revealed that the M4R population is predominantly composed of high affinity homodimers (84%). BRET saturation curves showed that M4R can also interact with other muscarinic receptor subtypes like M2 and M3 to form heterooligomers, but not with the distantly related seven transmembrane receptor Smoothened. Short-term agonist exposure (Carbachol, 10 micromolar, 5-30 min) did not significantly modify the oligomeric status of M4R-M4R interaction. These data reveal new pharmacological properties of M4R and encourage future research to elucidate the role of oligomerization in muscarinic receptor pharmacology