Deconstructing G Protein-Coupled Receptor Dimer Pharmacology: Case Studies in Dopamine D1 and D2 Receptors.

摘要

Dopamine receptors mediate various important neurophysiological functions. At a molecular level, G protein coupling is considered the main activation mechanism for most of the receptor-mediated cellular processes. A number of studies using native tissue have supported the idea that receptors can interact to form dimers or higher order oligomers. Particularly in medium spiny neurons of the striatum, dopamine receptor subtypes are reported to form dimers with themselves or other receptors (e.g. adenosine receptor A2A). Although a functional relevance for these dimers has been proposed, current assay systems are not capable of teasing out dimer-specific signaling events from those from other receptor populations. We have developed an assay that allows investigation of receptor-effector coupling specifically with defined dimer pairs. Using this assay, we investigated putative dopamine D1-D2 and A2A-D2 receptor dimer functions and studied the issue of a purported G protein coupling switch in the D1-D2 receptor dimer in which the heteromer was proposed to activate Gq, unlike D1 or D2 receptor when expressed alone9. We were unable, however, to find evidence for Gq activation by the D1-D2 heteromer, as the protomers in the heteromer maintained fidelity of signaling to their cognate G proteins. We also developed and optimized a series of novel Gs biosensors to elucidate differences in heterotrimeric G protein conformational changes triggered by dopamine D1 and A2A receptors, two of the prominent pharmacological targets in the striatum.;In addition to G protein signaling, intracellular calcium is also involved in many important cellular functions in all cell types. In neurons, intracellular calcium is implicated in learning and memory (synaptic plasticity) as well as neurodegeneration (apoptosis). In medium spiny neurons, dopamine-mediated calcium release from internal stores has been reported to result from activation of phospholipase C (PLC). However, different subtypes of dopamine receptors and intermediary proteins have been proposed to play a role in this dopamine-mediated PLC activation, and the underlying mechanisms are unclear. We found that activation of D1 and D2 receptors expressed individually can mobilize calcium in a PLC-dependent manner. In parallel, we also examined D1 and D2 receptor colocalization in striatal brain slices as well as in cultured medium spiny neurons. Although we found evidence using bacterial artificial chromosome-D1 and D2 reporter mice that D1 and D2 receptors are co-expressed in a small number of brain regions, we failed to observe D1-D2 receptor colocalization, suggesting the possibility that in neurons the receptors are somehow segregated.