μ-opioid receptor oligomerization and functional importance

Abstract

G protein-coupled receptors (GPCR)s are molecular sensors that transduce signals from the environment into cellular responses. GPCRs also participate in cellular homeostasis and very small changes in the activity of these receptors can contribute to a number of complex diseases such as cancer, depression, and schizophrenia. The oligomerization of GPCRs has received intense research interest because of its potential importance in cellular signaling and pharmacology. Although dimers of class C GPCR are established, there is intense debate regarding the quaternary structure of class A and B GPCRs. We focused on the μ-opioid receptor (MOR), a prototypic class A GPCR and the primary target of opioid analgesic drugs. Understanding the stoichiometry and dynamics of homo- and hetero-multimerization of MORs can provide the opportunity to address the unmet need for superior painkiller drugs that produce analgesia without dangerous adverse effects such as tolerance, dependence, and respiratory depression. The quaternary state of MOR in the cell membrane of live cells was studied using complementary methodologies. Fluorescence/luminescence-based target engagement assays confirmed homodimer stoichiometry of MOR and cannabinoid receptor 1, CB1R, as well as their cross interaction in a heterodimeric conformation. The mutation of critical residues in the interface of homodimer and heterodimer as well as interruption with the μ- and CB1R-TM-TAT peptides were employed to study the homo- /heteromeric interface interactions. These studies confirmed for the first time that MOR homodimer interactions are sensitive to the interruption of TM5/TM6 interface, while the TM4/TM5/TM6 interfaces are important for heterodimer interactions. II Upon activation with DAMGO, a MOR full agonist, the homodimer conformation transitioned to a short-lived monomer state, which reverted back to homodimer conformation, and its kinetics correlated with b-arrestin recruitment. We hypothesize that monomer formation upon activation results in G-protein binding and signaling activation. By recruitment of b-arrestin the MOR monomers either return to the dimer state or are internalized. Also, ligands with apparent biased activity for b-arrestin recruitment such as endomorphin-1 stabilize the homodimer conformation that result in attenuation of G protein binding. Functional studies revealed the effect of CB1R heterodimerization on agonist-induced activity of MOR; a biased positive allosteric signaling via the Gi protein pathway as well as a negative allosteric effect on b-arrestin-2 recruitment. A mechanism for the enhanced G protein signaling in the heterodimer might be the stabilization of MOR monomeric state in the heteromer complex.--Author's abstract