Identification of G Protein-Biased Agonists That Fail To Recruit β-Arrestin or Promote Internalization of the D1 Dopamine Receptor

Abstract

The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders, and D1R-selective ligands have potential as therapeutic agents. Previous studies have identified substituted benzazepines as D1R-selective agonists, but the in vivo effects of these compounds have not correlated well with their in vitro pharmacological activities. A series of substituted benzazepines, and structurally dissimilar D1R-selective agonists, were tested for their functional effects on D1R-mediated cAMP accumulation, D1R-promoted β-arrestin recruitment, and D1R internalization using live cell functional assays. All compounds tested elicited an increase in the level of cAMP accumulation, albeit with a range of efficacies. However, when the compounds were evaluated for β-arrestin recruitment, a subset of substituted benzazepines, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670, failed to activate this pathway, whereas the others showed similar activation efficacies as seen with cAMP accumulation. When tested as antagonists, the five biased compounds all inhibited dopamine-stimulated β-arrestin recruitment. Further, D1R internalization assays revealed a corroborating pattern of activity in that the G protein-biased compounds failed to promote D1R internalization. Interestingly, the biased signaling was unique for the D1R, as the same compounds were agonists of the related D5 dopamine receptor (D5R), but revealed no signaling bias. We have identified a group of substituted benzazepine ligands that are agonists at D1R-mediated G protein signaling, but antagonists of D1R recruitment of β-arrestin, and also devoid of agonist-induced receptor endocytosis. These data may be useful for interpreting the contrasting effects of these compounds in vitro versus in vivo, and also for the understanding of pathway-selective signaling of the D1R.

Luciferase Complementation Based-Detection of G-Protein-Coupled Receptor Activity

“…The antagonist also exhibited a high potency (pIC 50 9.220.42). All of the characteristics of D1R ligands are in agreement with previous reports [23,24]. D2R pharmacology was also in agreement with a previous study [25], in that norepinephrine showed less potency (pEC 50 5.220.14) relative to DA (pEC 50 6.610.10) and a selective D2R antagonist eticlopride showed complete blockade of 10M DA pre-activation and high potency (pIC 50 9.470.46).…”

Activation of D1/5 Dopamine Receptors: A Common Mechanism for Enhancing Extinction of Fear and Reward-Seeking Behaviors

“…Recent in vitro evidence from Conroy et al (2015) showed that the biased signaling properties of SKF 83822 and 83959 may be derived from the effects of these agonists on D1-receptorcoupled cAMP and β-arrestin pathways. D1/5 receptor agonists that promoted extinction, such as SKF 81297 and SKF 83822, strongly induced cAMP accumulation, β-arrestin recruitment, and D1 receptor internalization in Conroy et al (2015). However, SKF 83959, a D1/5 receptor agonist that is biased toward cAMP accumulation (SKF 83959) with little effect on β-arrestin or receptor internalization, had no effect on fear extinction.…”

“…For instance, although most D1 receptor agonists stimulate cyclic adenosine monophosphate (cAMP) accumulation in vitro, SKF 38393 and SKF 83959 do not strongly induce recruitment of arrestin or D1 receptor internalization, signaling mechanisms that are efficaciously activated by the full agonists SKF 81297 and SKF 83822 (Conroy et al, 2015). Thus, SKF 38393 and SKF 83959 are biased towards cAMP signaling.…”

From biased signalling to polypharmacology: unlocking unique intracellular signalling using pepducins

“…Since the concept was put forth, a number of synthetic GPCR ligands have been classified as biased modulators of their cognate receptor including the μ-opioid receptor, D1 and D2 dopamine receptors, parathyroid hormone receptor and angiotensin II 1A receptor along with others [15][16][17][18][19]. Moreover, the ability to bias GPCR signalling is becoming an important new direction in GPCR drug discovery with the movement of Oliceridine (or TRV130) and TRV027 (or TRV120027) through clinical trials.…”