Simple SummaryDopamine receptor type 2 (DRD2) mediates antitumoral effects in different types of pituitary tumors with variable efficacy. DRD2′s functionally selective agonists are able to preferentially activate the canonical G-protein-mediated signaling arm or the β-arrestin 2 pathway, which have recently been implicated in antimitotic signaling of DRD2. In a PRL-secreting pituitary tumor cell line, the β-arrestin 2-biased DRD2 ligand UNC9994 induced a stronger reduction in cell proliferation compared to the unselective agonist cabergoline, the currently used drug for this type of tumor. Primary PRL-secreting human tumoral cells that were resistant to cabergoline showed good reduction in cell proliferation and hormone secretion after UNC9994 incubation. Overall, these data suggest that UNC9994 may represent an alternative pharmacological strategy for cabergoline-resistant or poorly responsive tumors. In contrast, this strategy is not beneficial for non-functioning pituitary tumors.AbstractThe molecular events underlying the variable effectiveness of dopamine receptor type 2 (DRD2) agonists in pituitary neuroendocrine tumors (PitNETs) are not known. Besides the canonical pathway induced by DRD2 coupling with Gi proteins, the β-arrestin 2 pathway contributes to DRD2′s antimitotic effects in PRL- and NF-PitNETs. A promising pharmacological strategy is the use of DRD2-biased agonists that selectively activate only one of these two pathways. The aim of the present study was to compare the effects of two biased DRD2 ligands, selectively activating the G protein (MLS1547) or β-arrestin 2 (UNC9994) pathway, with unbiased DRD2 agonist cabergoline in PRL- and NF-PitNET cells. In rat tumoral pituitary PRL-secreting MMQ cells, UNC9994 reduced cell proliferation with a greater efficacy compared to cabergoline (−40.2 ± 20.4% vs. −21 ± 10.9%,p< 0.05), whereas the G-protein-biased agonist induced only a slight reduction. β-arrestin 2 silencing, but not pertussis toxin treatment, reverted UNC9994 and cabergoline’s antiproliferative effects. In a cabergoline-resistant PRL-PitNET primary culture, UNC9994 inhibited cell proliferation and PRL release. In contrast, in NF-PitNET primary cultures (n= 23), biased agonists did not show better antiproliferative effects than cabergoline. In conclusion, the preferential activation of the β-arrestin 2 pathway by UNC9994 improves DRD2-mediated antiproliferative effects in PRL-PitNETs, suggesting a new pharmacological approach for resistant or poorly responsive tumors.