Quaternary Structure Determination of the M3 Muscarinic Acetylcholine Receptors Based on Spectral-FRET

G-protein-coupled receptors (GPCRs) such as human muscarinic acetylcholine receptor 3 (hM3) assume dimeric/oligomeric forms in living cells while maintaining their ability to bind and activate G-proteins. The precise stoichiometry, quaternary organization, and stability of these receptor complexes in living cells remain a subject of significant controversy. We have investigated the organization of hM3 receptors in living cells using spectrally resolved two photon microscopy (SR-TPM). Wild type hM3 and a synthetic-ligand-regulated form of this receptor (RASSL), were expressed either constitutively or in an antibiotic dependent manner in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. A so-called donor of energy (Cerulean Fluorescent Protein) fused to RASSL can get excited by laser light and transfers its energy to nearby (<10nm) acceptors (Yellow Fluorescent Protein) fused to wild type hM3 receptors through a non-radiative process called Forster Resonance Energy Transfer (FRET). Using our SR-TPM microscope, we were able to collect images showing distributions of single complexes of hM3-RASSL and hM3-WT formed at various levels of RASSL receptor expression and determined their apparent efficiency of energy transfer (Eapp). The calculated Eapp for individual complexes were binned according to their values in order to obtain a cumulative histogram of Eapp for all complexes. The experimental data thus obtained were fitted to theoretical models [Raicu et al, Nature Photonics, 2009] in order to determine the quaternary structure of the M3 receptor, which turned out to be a rhombus-shaped tetramer.