Bi‐directional cAMP signaling crosstalk between dopamine D2 receptors and the constitutively active orphan receptor GPR52

G protein-coupled receptors (GPCRs) are valuable drug targets with ~35% of FDA approved medicines targeting these receptors. However, nearly 120 GPCRs remain orphan receptors, whose endogenous ligands, G protein signaling, and therapeutic potential are unknown. Dopamine D2 receptor (D2R) antagonists are effective antipsychotics while D2R agonists correct motor dysfunction in Parkinson's disease. The novel human orphan receptor GPR52 was recently identified and determined to activate cAMP signaling cascades. Notably, GPR52 is primarily co-expressed in the striatum of the brain with dopamine D2 receptors. Here we elucidate G protein signaling mechanisms responsible for both constitutive and agonist-induced GPR52 cAMP signaling and examine signaling crosstalk between GPR52 and the D2R. Expression of human GPR52 in wildtype HEK293 cells profoundly elevated basal cAMP levels by over 100 fold (Glosensor assay), indicating high constitutive receptor activity. CRISPR/Cas9 stable knockout of Gs/olf G proteins in HEK293 cells eliminated the GPR52 constitutive activity. Subsequent addback of Gs to Gs/olf knockout cells rescued GPR52 constitutive activity to near wildtype levels of basal cAMP, while addback of Golf did not significantly elevate the constitutive activity. However, when treated with novel GPR52 agonist PW0787, both Gs and Golf showed strong agonist-induced responses with unchanged potency. These findings indicate high constitutive activity of GPR52 is mediated via Gs signaling, while agonist-induced GPR52 signaling occurs via either Gs or Golf. We hypothesized the high GPR52 constitutive signaling may set the basal tone of cAMP in cells to afford more robust effects from adenylyl cyclase inhibition by Gi/o-coupled receptors, such as the D2R. Expression of D2R alone in HEK293 cells produced low basal cAMP levels (~4000 light counts/sec, Glosensor assay), and treatments with agonist quinpirole and antagonist haloperidol both yielded minimal changes to cAMP levels (~1000-2000 light counts/sec). Notably, co-expression of GPR52 and D2R substantially increased basal cAMP levels (~500,000 light counts/sec). This elevated basal cAMP also allowed for larger windows of agonism and inverse agonism by D2R ligands quinpirole (decrease of ~500,000 light counts/sec) and haloperidol (increase of ~150,000 light counts/sec). When the cells were treated concurrently with the GPR52 agonist PW0787 to further elevate basal cAMP, the efficacies of quinpirole (decrease of ~1,500,000 light counts/sec) and haloperidol (increase of ~500,000 light counts/sec) grew even more significantly. These studies indicate that striatal orphan receptor GPR52 may set the tone of basal cAMP levels through constitutive activity to allow more dynamic changes in cAMP signaling by the D2R. These findings also suggest potential for GPR52 as a drug target by modulating D2R cAMP signaling in the striatum, with therapeutic possibilities for psychosis, Parkinson's disease, and substance use disorders.