Heteromerization of μ-opioid receptor and cholecystokinin B receptor through the third transmembrane domain of the μ-opioid receptor contributes to the anti-opioid effects of cholecystokinin octapeptide

Abstract

Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats. Co-immunoprecipitation (Co-IP) and fluorescence lifetime-imaging-microscopy-based fluorescence resonance energy transfer (FLIM-FRET) assays showed that MOR heteromerized with CCKBR directly in transfected HEK293 cells. Combined with MOR mutant construction, the third transmembrane domain of MOR (TM3MOR) was demonstrated to participate in heteromerization with CCKBR. Receptor ligand binding, ERK phosphorylation and cAMP assays showed that MOR heteromerization with CCKBR weakened the activity of MOR. A cell-penetrating interfering peptide consisting of TM3MOR and TAT (a transactivator of HIV-1) sequences from the N terminal to the C terminal disrupted the MOR–CCKBR interaction and restored the activity of MOR in transfected HEK293 cells. Furthermore, intrathecal application of the TM3MOR-TAT peptide alleviated CCK-8-injection-induced antagonism to morphine analgesia in rats. These results suggest a new molecular mechanism for CCK-8 antagonism to opioid analgesia in terms of G-protein-coupled receptor (GPCR) interaction through direct heteromerization. Our study may provide a potential strategy for pain management with opioid analgesics.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn

“…It has also been reported that CCK contributes to the development of tolerance to the analgesic effects of transcutaneous electrical nerve stimulation in a rat model of inflammatory arthritis, again involving an anti‐opioid mechanism (DeSantana, da Silva, & Sluka, ). A recent report suggests that the anti‐opioid action of CCK may involve hetero‐dimerization of μ‐opioid and CCK B receptors (Yang et al, ). As the highest density of μ‐opioid receptors is in the superficial dorsal horn (Arvidsson et al, ), it is surprising that CCK‐expressing cells are far more numerous in laminae III‐IV.…”

Mu Opioid Receptor Heterodimers Emerge as Novel Therapeutic Targets: Recent Progress and Future Perspective

“…Previous studies found the antagonism of cholecystokinin octapeptide (CCK8) to opioid analgesia (Pu et al, 1994), and studies using L-365,260 (a specific antagonist of CCKBR) showed that CCK-8 inhibited opioid analgesia through CCKBR (Dourish et al, 1990). Recently, Yang et al (2018) demonstrated that MOR and CCKBR could form heterodimer and MOR-CCKBR heterodimer may underlie the antagonism of CCK8 to opioid analgesia. They first validated the co-localization of MOR and CCKBR in neurons in spinal cord dorsal horn and the DRGs by using double-labeling immunofluorescence staining.…”

“…Previous study showed that there exists a conformational antagonistic crosstalk between a2AAR and MORs in their downstream signaling upon co-activation (Vilardaga et al, 2008). Yang et al (2018) demonstrated that these receptors, either singly or as a heterodimer, activate common signal transduction pathways mediated through the inhibitory Gai/o. Using FRET microscopy, they showed that within the MOR-a2AAR heterodimer, the MOR and a2AAR communicate with each other through a cross-conformational switch that permits direct inhibition of one receptor by the other (Vilardaga et al, 2008).…”

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor

“…For example, CCK2R has been confirmed as being expressed in dorsal root ganglion neurons to mediate the inhibition of a Kv current (I A ) to increase pain sensation [16]. The CCK2R antagonist is known to reduce the need for opioid administration in relieving pain in a mouse model of burn-induced pain [17], likely by antagonizing CCK2R-µ-opioid receptor (MOR) heterodimerization, which inhibits MOR signaling [18]. CCK infusion into amygdala in rats has been found to activate a descending CCK2R-mediated pathway and to inhibit spinal neuron discharge, leading to hindpaw analgesia [19].…”

“…On the contrary, agonist stimulation of coexpressed CCK1R and CCK2R has been found to form CCK1R-CCK2R heterodimers [29]. Further, the heterodimerization of CCK2R and the µ-opioid receptor (MOR) has been found to reduce or antagonize opioid agonist-induced MOR activation, downstream signaling, and MOR analgesia [18]. Although agonist activation and associated changes in CCK receptor oligomerization status are interesting, the detailed chemical basis for such activation is lacking.…”