Double fluorescent knock-in mice to investigate endogenous mu-delta opioid heteromer subscellular distribution.

The heteromerization of Mu (MOP) and delta (DOP) opioid receptors has been extensively studied in heterologous systems. These studies demonstrated significant functional interaction of MOP and DOP evidenced by new pharmacological properties and intracellular signalling in transfected cells co-expressing the receptors. Due to the lack of appropriate tools for receptor visualization, such as specific antibodies, the pharmacological and functional properties of MOP-DOP heteromers in cells naturally expressing these receptors remains poorly understood. To address endogenous MOP-DOP heteromer trafficking and signalling in vivo and in primary neuronal cultures, we generated a double knock-in mouse line expressing functional fluorescent versions of DOP and MOP receptors. This mouse model has successfully been used to map the neuroanatomic distribution of the receptors and to identify brain regions in which the MOP-DOP heteromers are expressed. Here, we describe a method to quantitatively and automatically analyze changes in the subcellular distribution of MOP-DOP heteromers in primary hippocampal culture from this mouse model. This approach provides a unique tool to address specificities of endogenous MOP-DOP heteromer trafficking

Heteromerization Modulates mu Opioid Receptor Functional Properties in vivo.

Mu opioid receptors modulate a large number of physiological functions. They are in particular involved in the control of pain perception and reward properties. They are also the primary molecular target of opioid drugs and mediate their beneficial analgesic effects, euphoric properties as well as negative side effects such as tolerance and physical dependence. Importantly, mu opioid receptors can physically associate with another receptor to form a novel entity called heteromer that exhibits specific ligand binding, signaling, and trafficking properties. As reviewed here, in vivo physical proximity has now been evidenced for several receptor pairs, subsequent impact of heteromerization on native mu opioid receptor signaling and trafficking identified and a link to behavioral changes established. Selective targeting of heteromers as a tool to modulate mu opioid receptor activity is therefore attracting growing interest and raises hopes for innovative therapeutic strategies.journal articlereview20182018 11 13importe

G protein-coupled receptor heteromers are key players in substance use disorder.

G protein-coupled receptors (GPCR) represent the largest family of membrane proteins in the human genome. Physical association between two different GPCRs is linked to functional interactions which generates a novel entity, called heteromer, with specific ligand binding and signaling properties. Heteromerization is increasingly recognized to take place in the mesocorticolimbic pathway and to contribute to various aspects related to substance use disorder. This review focuses on heteromers identified in brain areas relevant to drug addiction. We report changes at the molecular and cellular levels that establish specific functional impact and highlight behavioral outcome in preclinical models. Finally, we briefly discuss selective targeting of native heteromers as an innovative therapeutic option.journal articlereviewresearch support, non-u.s. gov't2019 112018 09 29importe

Heteromerization of Endogenous Mu and Delta Opioid Receptors Induces Ligand-Selective Co-Targeting to Lysosomes.

IIncreasing evidence indicates that native mu and delta opioid receptors can associate to form heteromers in discrete brain neuronal circuits. However, little is known about their signaling and trafficking. Using double-fluorescent knock-in mice, we investigated the impact of neuronal co-expression on the internalization profile of mu and delta opioid receptors in primary hippocampal cultures. We established ligand selective mu-delta co-internalization upon activation by 1-[[4-(acetylamino)phenyl]methyl]-4-(2-phenylethyl)-4-piperidinecarboxylic acid, ethyl ester (CYM51010), [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO), and deltorphin II, but not (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), morphine, or methadone. Co-internalization was driven by the delta opioid receptor, required an active conformation of both receptors, and led to sorting to the lysosomal compartment. Altogether, our data indicate that mu-delta co-expression, likely through heteromerization, alters the intracellular fate of the mu opioid receptor, which provides a way to fine-tune mu opioid receptor signaling. It also represents an interesting emerging concept for the development of novel therapeutic drugs and strategies.journal article2020 Sep 302020 09 30importe