**7. Effect of μOR coactivation on cell proliferation**

Receptor-receptor interactions and di/oligomerization are established pathways for altering response to any given ligand. As both homo- and heterodimers of μOR have been reported, it is speculated that dimerization of μOR may have a role in regulating receptor signaling. This intermolecular cross talk within receptor oligomers often results in allosterism between the different binding pockets of the individual monomers. Negative binding cooperativity has been observed for both GPCR homo- and heteromers using equilibrium binding and/or radio ligand dissociation [90].

Assays with TLR and μOR coactivation showed that agonist activation of μOR using DAMGO causes a significant lowering of LPS-induced cell division in cells coexpressing μOR and TLR. This has direct bearing on pathogen clearance as TLRs are present and expressed in sentinel cells such as macrophages and dendritic cells, which recognize structurally conserved molecules derived from microbes. These large phagocytes are found in essentially all tissues where they patrol for potential pathogens by amoeboid movement. These cells play a critical role in nonspecific

defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. Where a pathogen is involved, monocytes are commonly preceded by neutrophils, which release a range of toxic agents designed to kill extracellular pathogens. The macrophages then clear out both the dead pathogens and the dead neutrophils. The process of recruitment of neutrophils and macrophages involves the activation of resident macrophages. Activated resident macrophages respond to local stimuli by producing cytokines that make the endothelial cell surface more sticky (through induction of increased expression of cell adhesion molecules such as P-selectin) and chemokines, that promote and direct migration of inflammatory cells. Macrophages treated with LPS express high levels of cyclin D2 [91]. The CD14 and TLR signaling funnels into the activation of NF-κB, AP-1 and IRF3 pathways via the ERK1/2 and MEK1/2 channel, which is also activated by μOR signaling. Both signaling pathways funnel into the MAPK cascade and hence effect the activation of NFκΒ. Preliminary data from our study using both A549 and HepG2 cells show a significant decrease in LPS-induced cell division in the simultaneous presence of ligands for μOR. Cell cycle arrest was observed primarily in the S and the M phase of cell cycle with a significant decrease under conditions of costimulation with both the receptor agonists. Cell cycle arrest observed as a result of receptor coactivation needs to be further explored as it can have important therapeutic implications as on one hand a particular signaling pathway or end point is associated with a therapeutic response, such as analgesic effect of opioids, while on the other hand it is associated with unwanted side effects, namely cell cycle suppression on exposure to LPS and opioids together.

Receptor heteromers, with their allosteric properties, give rise to a new kind of pharmacological target. The ability of one of the protomers to act as an allosteric modulator of the second protomer in the receptor heteromer gives the possibility of finding selective ligands for the protomer acting as conduit of the allosteric modulation [73]. As opioid drugs are used in more than one clinical condition understanding opioid receptor interactions with other GPCRs on the surface of different cell types can prove to be very beneficial in managing adverse, unwanted effects of these important therapeutic molecules. The immunomodulatory effects of morphine have been characterized both in animal and human studies and was found to decrease several functions of both natural and acquired immunity, interfering with important intracellular pathways involved in immune regulation [92]. Opiates namely morphine, heroin, fentanyl and methadone all induce immune-suppression and affect both innate and adaptive immunity defining a role of μOR in these functions.
