Receptor Conformations Involved in Dopamine D_2LReceptor Functional Selectivity Induced by Selected Transmembrane-5 Serine Mutations

Abstract

Although functional selectivity is now widely accepted, the molecular basis is poorly understood. We have studied how aspects of transmembrane region 5 (TM5) of the dopamine D 2L receptor interacts with three rationally selected rigid ligands (dihydrexidine, dinapsoline, and dinoxyline) and the reference compounds dopamine and quinpirole. As was expected from homology modeling, mutation of three TM5 serine residues to alanine (S5.42A, S5.43A, S5.46A) had little effect on antagonist affinity. All three mutations decreased the affinity of the agonist ligands to different degrees, S5.46A being somewhat less affected. Four functions [adenylate cyclase (AC), extracellular signal-regulated kinase 1/2 phosphorylation (MAPK), arachidonic acid release (AA), and guanosine 5Ј-O-(3-thio)triphosphate binding (GTP␥S)] were assessed. The intrinsic activity (IA) of quinpirole was unaffected by any of the mutations, whereas S5.42A and S5.46A mutations abolished the activity of dopamine and the three rigid ligands, although dihydrexidine retained IA at MAPK function only with S5.42A. Remarkably, S5.43A did not markedly affect IA for AC and MAPK for any of the ligands and eliminated AA activity for dinapsoline and dihydrexidine but not dinoxyline. These data suggest that this mutation did not disrupt the overall conformation or signaling ability of the mutant receptors but differentially affected ligand activation. Computational studies indicate that these D 2 agonists stabilize multiple receptor conformations. This has led to models showing the stabilized conformations and interhelical and receptor-ligand contacts corresponding to the different activation pathways stabilized by various agonists. These data provide a basis for understanding D 2L functional selectivity and rationally discovering functionally selective D 2 drugs.

Detection of New Biased Agonists for the Serotonin 5-HT2A Receptor: Modeling and Experimental Validation

“…This is in line with sitedirected mutagenesis studies at position 6.55 in other aminergic GPCRs. These studies revealed the influence of this position on biased signaling related to differential G protein coupling (Tschammer et al, 2011;Fowler et al, 2012). In parallel, the finding that serotonin establishes an interaction with residue S5.46 in helix 5, which is not seen in the dynamic binding profile of 2C-N, could justify the biased nature of the latter.…”

Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model

“…the presence of unique sets of scaffolding proteins (Abbas et al, 2009; Becamel et al, 2004). Thus, every ligand interacting with a GPCR may produce its own unique signaling signature in unique cellular contexts (Flordellis, 2012; Fowler et al, 2012). …”

Advances in receptor conformation research: the quest for functionally selective conformations focusing on the β2‐adrenoceptor

“…Using a mutagenic approach, Fowler et al . () have identified the three conserved serine residues on TM5, especially Ser194 5.43 , to be the other major determinants for biased signalling at the D 2L receptor. Further computation analysis has revealed multiple ligand‐specific receptor conformations.…”