G Protein Biased Signaling by Non‐catechol Dopamine D1 Receptor Agonists
Authors: Nilson Ashley N. , Wang Pingyuan , Jain Manish K. , Zhou Jia , Allen John A. 
Published: April 2020

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and working memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, until very recently all previously developed D1R selective agonists have a catechol moiety and catechol agonists have poor pharmacokinetic properties. We recently discovered the first non‐catechol D1R selective agonists with drug‐like properties and unexpectedly these ligands showed G protein biased signaling. Here, we define a novel mechanism of action for the non‐catechol D1R agonists involving impaired receptor endocytosis. First, HEK293 cells that have beta‐arrestin1/2 knocked out using CRISPR/Cas9 demonstrated that beta‐arrestins are required for agonist‐induced D1R endocytosis. Beta‐arrestin1/2 knockout significantly reduced agonist‐induced D1R endocytosis and re‐expressing beta‐arrestin1 or 2 rescued endocytosis in imaging and cell surface ELISA assays. Next, catechol and non‐catechol D1R agonists were tested in cAMP Glosensor and beta‐arrestin Tango assays to investigate potential biased signaling. The unbiased catechol D1R full agonist SKF‐81297 was used as the reference compound in all studies. The non‐catechol D1R agonists dose‐dependently increased cAMP production in HEK293 cells similar to the full agonist SKF‐81297 (Emax 100%), but did not engage beta‐arrestin. Interestingly, one non‐catechol agonist (PW0441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited beta‐arrestin (Emax = 100%, EC50 = 100 nM). The catechol agonist A‐77636 dose‐dependently increased full cAMP production (Emax = 104%, EC50 = 3.1 nM) but was a super agonist for beta‐arrestin recruitment (Emax = 130%, EC50 = 35nM). To determine the effect of G protein biased agonists on D1R endocytosis, the catechol and non‐catechol D1R agonists were tested in imaging and cell surface ELISA assays. The non‐catechol G protein biased agonists all induced significantly less total D1R endocytosis than the catechol agonist SKF‐81297. The pure G protein biased agonists PF‐1119 and PW0464 maximally induced 5% and 11% loss of cell surface D1R, respectively. In contrast, the catechol A‐77636 maximally induced 47% loss of cell surface D1R and induced significantly more total endocytosis than SKF‐81297. Finally, to elucidate the effect of G protein bias downstream of cAMP/PKA, HEK293 cells were treated from 0–360 min to assess CREB phosphorylation (pCREB) by western blotting. Preliminary results indicate G protein biased agonists have increased and prolonged pCREB levels compared to unbiased agonists. Together, these results indicate that G protein biased D1R agonists do not induce endocytosis and prolong G protein‐mediated signaling. Furthermore, these results suggest that G protein biased D1R agonists may signal longer in the G protein pathway due to reduced agonist‐induced receptor endocytosis. These results report a novel unbiased non‐catechol D1R agonist (PW0441), a D1R beta‐arrestin super agonist (A‐77636) and also elucidate a mechanism of action for G protein biased agonists in which G protein signaling is potentiated while beta‐arrestin mediated endocytosis is impaired.Support or Funding InformationSupported by NIH DA047643 and institutional funding from UTMB to JAA