The atypical antipsychotics clozapine and olanzapine promote down‐regulation and display functional selectivity at human 5‐HT₇ receptors

Abstract

Background and PurposeClassically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs-coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors.Experimental ApproachHEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured.Key ResultsThe agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants.Conclusions and ImplicationsTaken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor.

Allosteric Inhibition of Serotonin 5-HT7 Receptors by Zinc Ions

“…This result was consistent with the findings of Alberts et al [ 37 ], who showed that the agonist [ 3 H]5-CT labeled 60% of the 5-HT 7 receptors in comparison to the antagonist [ 3 H]mesulergine and postulated the presence of two affinity states in the 5-HT 7 receptor expressed in HEK293 cells. On the other hand, other data indicated similar B max values obtained for the agonist [ 3 H]5-CT, and the selective antagonist [ 3 H]SB-269970 [ 38 ], as well as for [ 3 H]mesulergine and [ 3 H]5-CT binding [ 39 ].…”

Related GPCRs couple differently to G s : preassociation between G protein and 5‐HT 7 serotonin receptor reveals movement of Gα s upon receptor activation

“…To determine whether constitutive 5‐HT 7 receptor activity influenced FRET between 5‐HT 7 ‐YFP and CFP‐Gγ 2 , we applied the inverse agonists, SB269970 and clozapine (19), and observed no alteration in FRET (Supplemental Fig. 1), which further supported that the ligand‐independent constitutive activity of 5‐HT 7 receptors was not responsible for the agonist‐induced reduction in FRET.…”

“…[ 3 H]GR113808 was from GE Healthcare (Chalfont St Giles, United Kingdom). The following plasmids were used: 5‐HT 4(b) (15), 3XHA‐5‐HT 4(b) , and 3XHA‐5‐HT 7(b) [5‐HT 4 and 5‐HT 7 receptors N‐terminally tagged with HA were obtained from the cDNA Resource Center (http://www.cdna.org)]; 5‐HT 7(b) (16); 5‐HT 7(b) yellow fluorescent protein (YFP) (19); β 1 AR‐YFP (6); Gβ 1 ; Gγ 2 ; cyan fluorescent protein (CFP)‐Gγ 2 ; Gαs; Gαs‐CFP (kindly provided by Catherine H. Berlot; Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania) (20); Gαs‐YFP (7); and Gβγ‐Venus (Gβ 1 and Gγ 2 labeled with Venus‐bimolecular fluorescence complementation that is only fluorescent when Gβγ are dimers; kindly provided by Nevin A. Lambert; Augusta University, Augusta, GA, USA) (21). The 5‐HT 4(b) ‐YFP receptor was constructed by mutating the stop codon of the human 5‐HT 4(b) receptor (15) and inserting the 5‐HT 4(b) coding sequence that was contained in an Nhe I/ Kpn I fragment into the 5‐HT 7(b) ‐YFP vector after mutating one Kpn I site to an Nhe I site and removing the Nhe I/ Kpn I fragment that contained the 5‐HT 7(b) coding sequence.…”

Preassociation between the 5‐HT 7 serotonin receptor and G protein G s : molecular determinants and association with low potency activation of adenylyl cyclase

“…Gg 2 -Venus was kindly provided by Gunnar Schulte (14). Construction of WT 5-HT 7(b) and 5-HT 4(b) with C-terminal CFP or YFP with or without the N-terminal HA tag have been previously described (17,27). Point mutations in Ga s -YFP and HA-5-HT 7 -CFP were inserted by using the In-Fusion HD Cloning Plus (Takara Bio USA, Mountain View, CA, USA) according to the manufacturer's protocol.…”