Various in vitro studies have demonstrated that GPCRs heteromerize altering their pharmacological and functional properties (1
). However, the evaluation of the in situ distribution of heteromers, and their functional relevance and modulation under pathological conditions, has been hampered by the lack of appropriate tools. We generated heteromer-selective antibodies using a subtractive immunization strategy in which cyclophosphamide was used to kill cells that generated antibodies to undesired antigens (in this case, HEK293 membranes), thereby increasing the exposure of the desired antigen (μ–δ heteromers) to antibody-producing cells. Our heteromer-selective antibody allowed the detection and isolation of μ–δ heteromers and blocked heteromer-mediated signaling. Thus, a subtractive immunization strategy could be used to generate antibodies against other GPCR heteromers and enable studies examining the abundance and regulation of receptor multimers in normal and pathological states.
The heteromer-selective antibody generated in this study blocked heteromer-mediated ligand binding and signaling and hence could be used to determine the biological contribution of heteromers compared to homomers following receptor activation. These findings are relevant to opiate action because the relative ratio of μ–δ heteromers to μ homomers could play a role in modulating the response to an opiate, and previous studies have shown that the heteromer transduces signaling through a pathway that is distinct from that of receptor homomers (9
). μ-δ receptors display decreased G-protein coupling and signaling compared to μ homomers and exhibit a β-arrestin–mediated μ receptor response (9
). This, in turn, leads to changes in the spatio-temporal dynamics of signaling as seen with phosphorylation of mitogen-activated protein kinase. In this assay μ homomers exhibited peak phosphorylation at 3–5 min and the activated kinase localized primarily to the nucleus while μ heteromers exhibited peak phosphorylation at 15 min and the activated kinase was retained in the cytoplasm (9
). This led to the differential activation of transcription factors (9
) that could be responsible for the differences in gene expression that occur with the development of morphine tolerance. Thus, the switch to the “β-arrestin–dependent” signaling cascade could contribute to the changes in morphine response that underlie tolerance. In addition, the absence of morphine tolerance in animals lacking δ receptors (23
) or in animals with reduced surface abundance of δ receptors due to deletion of the preprotachykinin gene (24
) is consistent with a requirement of μ–δ heteromer mediated signaling in the development of morphine tolerance. Furthermore, mouse knock-outs lacking β-arrestin-2 or δ receptors do not develop morphine tolerance (23
). Thus, μ–δ heteromers through their association with β-arrestin2 could underlie the development of tolerance to morphine. Although our data showing increased μ–δ heteromer abundance following chronic morphine administration would support this notion further studies are needed to examine how interactions between β-arrestin2 and μ–δ heteromers contribute to the development of morphine tolerance.
We found that μ–δ heteromer abundance was increased in the RVM following chronic morphine treatment. The RVM, a brain region containing both μ and δ opioid receptor mRNAs, is involved in antinociception through facilitation of the descending inhibitory pain pathways. Malfunction in this circuitry is thought to play a role in neuropathic pain, a condition characterized by the presence of hyperalgesia (supersensitivity to painful stimuli) and tactile allodynia (painful sensation by normally non-painful stimuli) (26
). Therefore, if neuropathic pain increases the likelihood of μ–δ heteromer formation in the RVM, this could account for the lack of analgesic potency of morphine in the treatment of neuropathic pain (28
), because the μ–δ heteromer signals through a β-arrestin-2 (9
) and studies have shown that functional deletion of β-arrestin-2 gene in mice leads to a potentiation as well as prolongation of the analgesic effects of morphine (29
). Therefore further studies are needed to examine the effects of neuropathic pain on μ–δ heteromers in the RVM. Glycinergic neurons of the MNTB, an auditory relay nucleus, also showed increased μ–δ heteromer abundance following chronic morphine administration. However, not much is known about the role of these receptors in auditory processing. Because chronic morphine administration causes increased μ–δ heteromer abundance in the MNTB, studies are needed to examine the role of these receptors in both acute and chronic pain states.
We observe co-localization of μ and δ receptors in cultured DRG neurons that is increased upon prolonged treatment with morphine. A recent study with mice with a knock-in of δ opioid receptor tagged with EGFP (δEGFP) used antibodies against GFP and μ receptors and showed that δEGFP colocalizes with μ opioid receptors in less than 5% of DRG neurons (30
). This degree of colocalization could be an underestimate because these mice have increased abundance of δ opioid receptors (31
), and the GFP antibody exhibits higher avidity for GFP than the μ antibody does towards μ receptors. This may result in an overestimation of δ opioid receptor receptor abundanve selective to μ receptors. In addition, the GFP tag at the C-terminus increases cell surface localization of the δ opioid receptor (32
). When taken together with the evidence that increased abundance of δ opioid receptor attenuates the maturation of the μ opioid receptor (33
), these results suggest that the low degree of colocalization between δEGFP and the μ opioid receptor (30
) could be due to alterations in δ opioid receptor maturation. Thus, as supported by our immunostaining data μ and δ opioid receptors may colocalize in DRGs, as well as in other regions of the brain.
In summary, we report the generation of a μ–δ heteromer-selective antibody that enabled us to examine chronic morphine treatment-mediated up-regulation of μ–δ heteromers in endogenous tissue. The subtractive immunization strategy used in the generation of the μ–δ heteromer-selective antibodies could be used to generate antibodies selective for other GPCR heteromers. This would help studies examining the role of GPCR heteromers in physiological and pathophysiological conditions.