Abstract
Οpioid receptors (OR) (subtypes μ, δ, κ and NOP) belong to the superfamily of the Heptahelical G protein-coupled receptors (7TM or GPCRs), the largest class of receptors in the human genome and common targets for therapeutics. ORs mediate their responses in the nervous system via coupling to members of the Gi/Go proteins to regulate the activity of various effector systems. Opioids are the most potent analgesics but prolonged administration leads to phenomena of tolerance and dependence thus there is a great interest towards understanding of OR signalling in an effort to develop new drugs devoid of adverse effects. Extended observations have demonstrated that the cytoplasmic face of the ORs is critical in mediating their signal through interactions not only with G proteins but also with multiple other proteins.These regulatory proteins play distinct roles in the regulation of the OR signalling,and in the fine tuning of these receptors. Regulators of G protein signalling (RGS) proteins ...
Οpioid receptors (OR) (subtypes μ, δ, κ and NOP) belong to the superfamily of the Heptahelical G protein-coupled receptors (7TM or GPCRs), the largest class of receptors in the human genome and common targets for therapeutics. ORs mediate their responses in the nervous system via coupling to members of the Gi/Go proteins to regulate the activity of various effector systems. Opioids are the most potent analgesics but prolonged administration leads to phenomena of tolerance and dependence thus there is a great interest towards understanding of OR signalling in an effort to develop new drugs devoid of adverse effects. Extended observations have demonstrated that the cytoplasmic face of the ORs is critical in mediating their signal through interactions not only with G proteins but also with multiple other proteins.These regulatory proteins play distinct roles in the regulation of the OR signalling,and in the fine tuning of these receptors. Regulators of G protein signalling (RGS) proteins is a class of proteins that modulate G protein signalling events by directly interacting with Gα subunits and accelerating the GTP hydrolysis, thus reducing GPCR signalling towards their effectors. RGS can also interact with many GPCRs, effectors and auxiliary proteinsthus playing a key role in the cell functions, making them highly attractive as pharmacological targets (Abramow-Newerly et al., 2006). Our previous in vitro studies have shown that a member of the B/R4 subfamily of RGS proteins, such as RGS4, interacts directly with μ-OR and δ-OR within a conserved region in their C-termini (μ-CT and δ-CT), forming a helix VIII, as well as within the δ-third intracellular loop (δ-i3L). RGS4 associates with μ-OR and δ-OR in living cells and forms selective complexes with Gαi/o proteins in a receptor dependent manner. Expression of RGS4 in HEK293 cells attenuated adenylyl cyclase inhibition mediated by μ-OR and agonist-mediated ERK1,2 phosphorylation for both receptors (Georgoussi et al., 2006- Leontiadis et al., 2009), suggesting for the first time that RGS4 is a negative modulator of μ-OR and δ-OR signalling. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of other members of the B/R4 subfamily of RGS proteins, such as RGS2, to interact with OR we generated fusion peptides encompassing the C-terminus of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 interacts with the κ-CT, the δ-CT and the δ-i3L but fails to interact with the μ-CT. RGS4-N-terminal domain is responsible for OR interaction. Mapping the sites ofRGS2 interaction indicated that RGS2 interacts with the non conserved portion of the C-termini of ORs exhibiting a different docking site as compared to that of RGS4. Co-precipitation studies in living cells indicated that RGS2 and RGS4 associate with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins in an RGS protein dependent manner. Expression of both RGS2 and/or RGS4, in 293F cells attenuated agonist mediated-adenylyl cyclase inhibition for κ-ΟR, but not δ-OR, with RGS2 exhibiting a more robust effect. RGS4 and RGS2 reduced κ-ΟR-mediated ERK1,2 phosphorylation whereas, RGS4 accelerated agonist-induced internalization of the δ-OR but not of the κ-OR. Collectively, our observations demonstrate that RGS2 and RGS4 are novel interacting partners and negative modulators of κ-ΟR and δ-OR signalling. These two RGS proteins display a differential modulatory effect in each signalling pathway tested and play a key functional role by conferring selectivity for both κ-OR and δ-OR coupling with a specific subset of G proteins. Therefore they can be considered as attractive new pharmacological targets to manipulate opioid receptors signalling.
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