Decoding the Signaling through Homomeric and Heteromeric Dopamine D2 and Cannabinioid CB1 Receptors

Abstract

G protein-coupled receptors (GPCRs) play critical roles in signal transduction and represent around half of drug targets. Accumulating evidence supports that GPCRs form both homomers and heteromers. Utilizing electron microscopic immunocytochemistry, cannabinoid 1 (CB1) receptors were found to co-localize with dopamine 2 (D2) receptors in the somata and dendrites of striato-pallidal GABA neurons. Fluorescence resonance energy transfer and co-immunoprecipitation studies have provided evidence for heteromerization of D2 and CB1 receptors. In addition, the D2-CB1 receptor complex shows an interesting switching behavior of its G protein coupling specificity from Gi/o to Gs signaling. Both homomeric D2 and CB1 receptors are preferentially Gi/o coupled and decrease cAMP levels induced by forskolin stimulation. However, application of CB1 receptor agonist in a cell line co-expressing D2 and CB1 receptors increased cAMP levels, consistent with Gs signaling.Electrophysiological assays using GIRK channels as reporters of Gs activity corroborated the switching behavior of the CB1 receptor, although not reproducibly. We sought additional assays to study the switch in signaling specificity of CB1 receptors in CB1-D2 complexes. G alpha 16 belongs to the Gq superfamily and its activation leads to phospholipase C activation and Ca2+ release. We constructed G alpha 16 chimeras in which the last 11 or 30 amino acids were replaced with the corresponding sequence of Go and Gs. HEK 293 cells were transfected transiently with D2 or CB1 receptor and chimeric G alpha 16 cDNA and calcium responses were monitored by Fluo4-AM in a FlexStation. Stimulation of the D2 receptor showed increases in intracellular calcium levels in cells co-transfected with the D2 receptor and G16/o chimeras but not with G16/s or G16 wt. Signaling through the CB1 receptor and D2-CB1 receptor complex is currently being tested.