Functional Expression of Single-Chain Heterodimeric G-Protein-Coupled Receptors for Adenosine and Dopamine

Abstract

ABSTRACT. The direct homo-and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A2AR) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A2AR/A2AR vs. A2AR/D2R). In order to stimulate the heteromerization of A2AR and D2R, we have designed a single-polypeptide-chain heterodimeric A2AR/D2R complex by fusing the C-terminus of the A2AR via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A2AR/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.

Design: An assay based on single-polypeptide-chain heterodimeric A2AR/D2R and non-oligomerized fusions forin vivoanalysis of their allosteric receptor-receptor interactions

“…In our previous report [9,10], among GPCR protein-protein interactions, such as the disulfide bond formation of the N-ter, coiled-coil interaction of the cytoplasmic C-ter, and TM interaction [90], a type II TM protein with a cytoplasmic N-ter segment, single TM, and extracellular C-ter tail, i.e., the Caenorhabditis elegans accessory protein of odorant receptor (odr4) [91] was first selected for connection between the N-ter receptor half (A2AR) and the C-ter receptor half (D2LR, the long form of D2R) of the [102,103].…”

“…Interestingly, in the phase of rational design and screening, fusing the two receptors was found to stimulate the receptor dimer formation [9,10]: In the study, by fusing the cytoplasmic carboxy-terminus (C-ter) of the human brain-type A2AR (that is derived from Dr. Shine's cDNA [30])( Fig.…”

“…Shine's cDNA [30])( Fig. S1) via the transmembrane domain (TM) of a type II TM protein (Section 3.3) with the extracellular amino-terminus (N-ter) of D2R in tandem, we created successful designs for a fusion receptor, single-polypeptide-chain (sc) heterodimeric GPCR complex A2AR/D2R, as a solution [9,10]. However, the resulting prototype scA2AR/D2LR has compact folding, i.e., fixed stoichiometry (the apparent ratios of A2AR to D2R binding sites), A2AR : D2R = 10:3 = 3~4:1 ([10]:…”

“…1A (pp. 140) in our previous report [9], with a written permission by the copyright owner, the Japan Society for Cell Biology.…”

“…In the striatum, how the G-protein-coupled receptor (GPCR) for adenosine, subtype A2A (A2AR) [4,5] contributes to the DA neurotransmission in the "volume transmission"/dual-transmission model [6] has been studied extensively [7]. In addition to a fusion receptor, the prototype single-polypeptide-chain (sc) heterodimeric A2AR/D2R complex (the GPCR for DA, subtype D2; [8]) [9], several types were created and tested experimentally [10], while referring to a relationship between nanoscale surface curvature and surface-bound protein oligomerization [11][12]. In order to further explore in vivo, we have thus designed a new molecular tool, supermolecule scA2AR/D2R.…”