**3.5 Receptor-receptor interactions**

Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs). GPCRs do not exist in isolation and interact with components of the bilayer, such as lipids and sterols, as well as with other GPCRs to form dimers and higher order oligomers, which are of functional significance as this affects the ligand binding and signaling properties of GPCRs [62, 63]. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs [64]. A large number of direct binding assays indicating negative or positive cooperativity suggest clustering of GPCRs [65]. Mansoor et al*.* [66] reported that GPCRs can come together in the presence of lipids. Oligomerization and the two monomers comprising a GPCR dimer could be nonequivalent, thereby allowing more refined regulation of GPCR activity [66–68]. Thus, GPCR dimerization can be the result of the receptors forming heterodimers as well as homodimers [69], with many dimers displaying modified pharmacology [70], altered responsiveness to viral entry through GPCRs [71], or attenuated signaling [72]. Therefore, it is apparent that the oligomeric potential of GPCRs allows further diversification of their repertoire as a result of more complex ligand-receptor relationships than envisioned for monomeric receptors due to a more complex ligand-receptor relationship [64]. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may constitute the functional unit, and oligomeric entities can be viewed as multiples of dimers [73].

**87**

interface [82].

the receptor.

*Immune Cell Activation: Stimulation, Costimulation, and Regulation of Cellular Activation*

Fluorescence correlation spectroscopy (FCS) studies suggest that μ-opioid receptors exist primarily as dimers that oligomerize with δ-opioid receptors into tetramers [74]. High-resolution crystallographic structures of the μ-opioid by Manglik et al*.* [75] showed that they exist as parallel dimers and/or tetramers. Some TM domains have been observed more often than others. TM5 and TM6 residues constituted the main interfaces for the μ-opioid receptor crystallized dimers, with extensive contacts throughout the length of these TM helices in μ-opioid receptor dimers. The μ-opioid dimers also showed a second, less prominent symmetric interface, involving TM1, TM2, and helix 8 (H8; the helix adjacent to TM7 running along the internal membrane surface [75]). For GPCRs, the majority of crystal structures that are currently available refer to antagonist-bound (inactive) structures. The inferred dimeric interfaces may, therefore, depend on specific conformational states. Furthermore, the TM5-TM6 interface inferred by the crystal structure of μ-opioid dimers could preclude either monomer from properly coupling to G protein, because the agonist-induced receptor-G protein interaction depends on rearrangements of TM5 and TM6 within the seven-helical domain bundle, suggesting that different receptor conformations stabilized with different ligands may also promote different dimeric interfaces [75]. Huang et al*.* [76] suggested that the comparison of the differences in the interfaces observed from the crystallized structures of the antagonist-bound μ-opioid and chemokine CXCR4 receptors and the ligand-free β1-adrenoceptor suggest that the TM5 interface can partner in the interaction with TM4 or TM6, depending on the conformation of

In δ-μ-OR heteromers, it was shown that binding and signaling by morphine or μ receptor agonists were potentiated by δ-OR antagonists, and reciprocally, binding and signaling by δ-OR agonists were potentiated by μ receptor selective antagonists [77, 78]. Studies carried out with the δ-opioid-cannabinoid CB1 receptor heteromer have also revealed allosteric modulations of cannabinoid CB1 receptor ligands on δ-OR ligand binding properties [79, 80]. They also showed that in recombinant systems expressing both receptors, as well as endogenous tissues, binding and consequently signaling by δ-OR could be potentiated by a low, nonsignaling dose of cannabinoid CB1 receptor agonist or a selective antagonist. In the δ-μ-OR heteromers, Gupta et al*.* [81] showed that morphine-induction increases heteromer abundance. Similarly, the δ-opioid-CB1 receptor heteromer increases in the brain after peripherally elicited neuropathic pain [79]. Studies by Zheng et al*.* [82] have revealed a complex interplay among cholesterol, palmitate, receptor dimerization, and G protein activation. They showed that reducing cholesterol levels or preventing palmitoylation of the μOR reduced receptor dimerization and Gα association. Additionally, preventing palmitoylation reduced the association of μOR with cholesterol, suggesting a functional complex of receptor, palmitate, and cholesterol. The authors also demonstrate that mutagenesis of the palmitoylated cysteine residue in μOR has no effect on ligand binding but decreased signaling efficiency, probably by impairing GPCR-G protein association. The same mutant had significantly reduced dimerization, and it was proposed that this was responsible for the reduced G protein coupling. Zhang et al*.* [83] demonstrated that the palmitate-free mutant associated more weakly with cholesterol. A model of the μOR dimer in which cholesterol and palmitate pack together to facilitate receptor dimerization reveals that cholesterol interactions contribute approximately 25% of the total interaction energy at the homodimer

*DOI: http://dx.doi.org/10.5772/intechopen.81568*

**3.6 Interactions of opioid receptors**

*Immune Cell Activation: Stimulation, Costimulation, and Regulation of Cellular Activation DOI: http://dx.doi.org/10.5772/intechopen.81568*

#### **3.6 Interactions of opioid receptors**

*Immune Response Activation and Immunomodulation*

half-life of the mature form of the variant receptor (~12 h) was shorter than that of the wild-type receptor (~28 h) showing its effect on protein stability. Thus, several lines of evidence suggest that the 118G variant may affect *OPRM1* gene expression in addition to mRNA translation, posttranslational processing, or turnover of the

Human genome has about 45,000-C-phosphate-G-(CpG) islands, many in the promoter regions of genes. The CpG islands are located upstream of the transcription start site to within the first exon [57]. Nielsen et al*.* [58] and Chorbov et al*.* [59] reported that in DNA obtained from peripheral lymphocytes, two of 16 CpG sites in a region of *OPRM1* gene promoter had significantly higher methylation in former heroin addicts than in controls. These two CpG sites are located in binding sites for the potential Sp1 transcription factor. Oertel et al*.* [60] showed that substitution of an A with a G at gene position +118 introduces a new CpG-methylation site at position +117, which leads to enhanced methylation of *OPRM1* gene resulting in decreased expression. Using m-fold software, Johnson et al*.* [61] showed that 118G variant demonstrated an altered folding that could affect mRNA stability. The epigenetic mechanism reported by Oertel et al*.* [60] impedes μ-OR upregulation in brain tissue, and they concluded that while in wild-type subjects, a reduced signaling efficiency associated with chronic heroin exposure was compensated for by an increased receptor density; this upregulation was absent in carriers of the 118G receptor variant due to diminished *OPRM1* mRNA transcription. The *OPRM1* 118A > G SNP variant not only reduces μ-OR signaling efficiency, but by a genetic-epigenetic interaction, also reduces OR expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure, providing evidence that a change in the genotype can

μ-opioid receptor protein, which can all effect signaling pathway/s.

**3.4 Epigenetics of OPRM1 gene and its impact on cell function**

cause a change in the epigenotype with major functional consequences.

Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs). GPCRs do not exist in isolation and interact with components of the bilayer, such as lipids and sterols, as well as with other GPCRs to form dimers and higher order oligomers, which are of functional significance as this affects the ligand binding and signaling properties of GPCRs [62, 63]. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs [64]. A large number of direct binding assays indicating negative or positive cooperativity suggest clustering of GPCRs [65]. Mansoor et al*.* [66] reported that GPCRs can come together in the presence of lipids. Oligomerization and the two monomers comprising a GPCR dimer could be nonequivalent, thereby allowing more refined regulation of GPCR activity [66–68]. Thus, GPCR dimerization can be the result of the receptors forming heterodimers as well as homodimers [69], with many dimers displaying modified pharmacology [70], altered responsiveness to viral entry through GPCRs [71], or attenuated signaling [72]. Therefore, it is apparent that the oligomeric potential of GPCRs allows further diversification of their repertoire as a result of more complex ligand-receptor relationships than envisioned for monomeric receptors due to a more complex ligand-receptor relationship [64]. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may constitute the functional unit, and oligomeric entities can be viewed as multiples of dimers [73].

**3.5 Receptor-receptor interactions**

**86**

Fluorescence correlation spectroscopy (FCS) studies suggest that μ-opioid receptors exist primarily as dimers that oligomerize with δ-opioid receptors into tetramers [74]. High-resolution crystallographic structures of the μ-opioid by Manglik et al*.* [75] showed that they exist as parallel dimers and/or tetramers. Some TM domains have been observed more often than others. TM5 and TM6 residues constituted the main interfaces for the μ-opioid receptor crystallized dimers, with extensive contacts throughout the length of these TM helices in μ-opioid receptor dimers. The μ-opioid dimers also showed a second, less prominent symmetric interface, involving TM1, TM2, and helix 8 (H8; the helix adjacent to TM7 running along the internal membrane surface [75]). For GPCRs, the majority of crystal structures that are currently available refer to antagonist-bound (inactive) structures. The inferred dimeric interfaces may, therefore, depend on specific conformational states. Furthermore, the TM5-TM6 interface inferred by the crystal structure of μ-opioid dimers could preclude either monomer from properly coupling to G protein, because the agonist-induced receptor-G protein interaction depends on rearrangements of TM5 and TM6 within the seven-helical domain bundle, suggesting that different receptor conformations stabilized with different ligands may also promote different dimeric interfaces [75]. Huang et al*.* [76] suggested that the comparison of the differences in the interfaces observed from the crystallized structures of the antagonist-bound μ-opioid and chemokine CXCR4 receptors and the ligand-free β1-adrenoceptor suggest that the TM5 interface can partner in the interaction with TM4 or TM6, depending on the conformation of the receptor.

In δ-μ-OR heteromers, it was shown that binding and signaling by morphine or μ receptor agonists were potentiated by δ-OR antagonists, and reciprocally, binding and signaling by δ-OR agonists were potentiated by μ receptor selective antagonists [77, 78]. Studies carried out with the δ-opioid-cannabinoid CB1 receptor heteromer have also revealed allosteric modulations of cannabinoid CB1 receptor ligands on δ-OR ligand binding properties [79, 80]. They also showed that in recombinant systems expressing both receptors, as well as endogenous tissues, binding and consequently signaling by δ-OR could be potentiated by a low, nonsignaling dose of cannabinoid CB1 receptor agonist or a selective antagonist. In the δ-μ-OR heteromers, Gupta et al*.* [81] showed that morphine-induction increases heteromer abundance. Similarly, the δ-opioid-CB1 receptor heteromer increases in the brain after peripherally elicited neuropathic pain [79]. Studies by Zheng et al*.* [82] have revealed a complex interplay among cholesterol, palmitate, receptor dimerization, and G protein activation. They showed that reducing cholesterol levels or preventing palmitoylation of the μOR reduced receptor dimerization and Gα association. Additionally, preventing palmitoylation reduced the association of μOR with cholesterol, suggesting a functional complex of receptor, palmitate, and cholesterol. The authors also demonstrate that mutagenesis of the palmitoylated cysteine residue in μOR has no effect on ligand binding but decreased signaling efficiency, probably by impairing GPCR-G protein association. The same mutant had significantly reduced dimerization, and it was proposed that this was responsible for the reduced G protein coupling. Zhang et al*.* [83] demonstrated that the palmitate-free mutant associated more weakly with cholesterol. A model of the μOR dimer in which cholesterol and palmitate pack together to facilitate receptor dimerization reveals that cholesterol interactions contribute approximately 25% of the total interaction energy at the homodimer interface [82].
