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

Background The adenosine A 2A receptor (A 2A R) heteromerizes with the dopamine D 2 receptor (D 2 R). In order to explore their functional interaction, we engineered previously stable single-polypeptide-chain (sc) A 2A R/D 2L R: whether the molecular entity of the striatal A 2A R/D 2 R antagonism, i.e., scA 2A R/D 2 Rs are just A 2A R/D 2 R with the antagonism, remains unresolved. New Method To further clarify the heteromerization through the scA 2A R/D 2L R, we here designed supramolecularly ‘exclusive’ monomers and dimers, using the Cε2 domain of IgE-Fc or apoproteins of the bacterial light-harvesting antenna complex. Results A concept of the recptor protein assembly regulation, i.e., the selective monomer/non-obligate dimer formation was obtained. Although none of these new fusions were constructed or tested, we could aim at obtaining heterodimer-specific agents, using the scA 2A R/D 2 R. Whether the resulting designs were explained feasibly and rationally was addressed. The structure and function of the non-obligate dimer were here discussed through scA 2A R/D 2 R, focusing on the procedure of the membrane protein design and methods for transient protein-protein interactions. Summary and Outlook Given that upon being expressed and allosteric regulation occurs regardless of specific signal to non-specific noise (S/N) ratio, the supramolecular designs, allowing us to express selectively monomer/non-obligate dimer of class A GPCR, are experimentally testable and will be used to confirm in vivo that such low S/N ratio interaction between A 2A R and D 2L R functions in the dopamine neurotransmission in the striatum. Abbreviations: A 2A R adenosine A 2A receptor 3HA-A 2A R A 2A R tagged with a triple HA epitope Bchl bacteriochlorophyll BRET bioluminescence resonance energy transfer C carboxy-terminal CD cluster of differentiation D 2L R and D 2S R the long and short form of dopamine D 2 receptor, respectively Fab antigen binding fragment Fc Fc fragment FcεRI high affinity receptor for IgE FRET fluorescence resonance energy transfer G 4 S an amino acid sequence consisting of a four-glycine-repeat followed by a serine residue GABA γ-aminobutyric acid GABA B GABA type B receptor GPCR G protein-coupled receptor G t transducin HA hemagglutinin HIV human immunodeficiency virus IC intracellular loops Ig immunoglobulin LH light-harvesting antenna complex mAb monoclonal antibody M r molecular weight N amino-terminal PD Parkinson’s disease PS photosystem RC reaction center Rluc Renilla luciferase sc single-chain TM transmembrane 3D three-dimensional