RESUMO
The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior.
Assuntos
Receptores de Dopamina D3/química , Receptores de Dopamina D3/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Agonistas de Dopamina/química , Agonistas de Dopamina/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Atividade Motora/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores de Dopamina D3/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Tetra-Hidronaftalenos/farmacologiaRESUMO
Cytochrome P450s are enzymes capable of metabolizing a wide variety of drugs. Their significant impact in drug discovery has led to extensive research, computationally and experimentally, in order to explore how a chemical entity responds to metabolizing enzymes. We present an overview of ligand-based and structure-based methodologies, along with pertinent information on the structures, biology, and relevance of these enzymes.