Analysis of various types of single-polypeptide-chain (sc) heterodimeric A(2A)R/D2R complexes and their allosteric receptor receptor interactions

作者：Kamiya Toshio^*; Yoshioka Kazuaki; Nakata Hiroyasu

来源：Biochemical and Biophysical Research Communications, 2015, 456(2): 573-579.

DOI：10.1016/j.bbrc.2014.11.098

• Oligomerization
• Adenosine A(2A) receptor
• Dopamine D-2 receptor
• Fluorescence resonance energy transfer
• Fusion protein
• Striatum

Adenosine A(2A) receptor (A(2A)R) heteromerizes with dopamine D-2 receptor (D2R). However, these class A G protein-coupled receptor (GPCR) dimers are not fully formed, but depend on the equilibrium between monomer and dimer. In order to stimulate the heteromerization, we have previously shown a successful design for a fusion receptor, single-polypeptide-chain (Sc) heterodimeric A(2A)R/D2R complex. Here, using whole cell binding assay, six more different scA(2A)R/D2R constructs were examined. Not only in scA(2A)R/ D2R 'liberated' with longer spacers between the two receptors, which confer the same configuration as the prototype, the A(2A)R-odr4TM-D2LR, but differ in size (Forms 1-3), but also in scA(2A)R/D2LR (Form 6) fused with a transmembrane (TM) of another type II TM protein, instead of odr4TM, neither of their fixed stoichiometry (the apparent ratios of A(2A)R to D2R binding sites) was 1, suggesting their compact folding. This suggests that type II TM, either odr4 or another, facilitates the equilibrial process of the dimer formation between A(2A)R and D2LR, resulting in the higher-order oligomer formation from monomer of scA(2A)R/D2LR itself. Also, in the reverse type scA(2A)R/D2LR, i.e., the D2LR-odr4TM-A(2A)R, counter agonistindependent binding cooperativity (cooperative folding) was found to occur (Forms 4 and 5). In this way, the scA(2A)R/D2LR system has unveiled the cellular phenomenon as a snapshot of the molecular behavior in A(2A)R/D2LR dimer. Thus, these results indicate that the various designed types of functional A(2A)R/D2R exist even in living cells and that this fusion expression system would be useful to analyze as a model of the interaction between class A GPCRs.

• 出版日期2015-1-9