Functional Homomers and Heteromers of Dopamine D2L and D3 Receptors Co-exist at the Cell Surface

Human dopamine D-2long and D-3 receptors were modified by N-terminal addition of SNAP or CLIP forms of O-6-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D-2long and D-3 receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D-2long-D-3 heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D-2long-D-3 heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D-2long and D-3 receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D-2long and D-3 receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D-2long and D-3 receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites.