Our study provides evidence for an important physiological role of spinophilin in the regulation of MOR signaling and behavioral responses to opiate drugs of abuse. Deletion of the spinophilin gene causes reduced analgesic effects of acute morphine but enhanced adaptations to repeated morphine exposure, including increased morphine dependence, place conditioning, locomotor sensitization, and analgesic tolerance. These diverse effects of spinophilin may result not only from its broad distribution in the brain, but also from its multiple actions at the cellular level. Thus, spinophilin modulates MOR responsiveness via complex mechanisms that promote MOR internalization and recycling as well as MOR inhibition of adenylyl cyclase but oppose MOR activation of ERK. Our findings offer new insight into the cellular events underlying chronic opiate action and point to spinophilin as a promising new target for the optimization of the analgesic actions of opiate drugs with reduced drug tolerance and dependence.
Several studies have implicated signal transduction proteins in particular aspects of opiate action. Behavioral characterization of GRK3 knockout mice reveals that GRK3 is involved in analgesic tolerance but does not affect other aspects of morphine action (Terman et al., 2004
). Studies of genetic mutant mice suggest that RGS9-2 and β-arrestin-2 each act as a negative modulator of morphine’s effects (Bohn et al., 2000
; Zachariou et al., 2003
). RGS9-2 potently suppresses morphine reward, analgesia, and physical dependence, whereas β-arrestin-2 mainly affects analgesic responsiveness and tolerance but not physical dependence. The fact that β-arrestin-2 mutants show increased sensitivity to the analgesic effects of morphine but normal responses to other opioid drugs (Bohn et al., 1999
), indicates that signal transduction molecules beyond β-arrestin-2 modulate MOR analgesic responses. Our data suggest that spinophilin is one such additional modulator of MOR function, one that regulates MOR desensitization and affects the actions of all opiate analgesic drugs. Unlike the other signal transduction proteins studied to date, genetic ablation of spinophilin causes opposite effects on morphine reward and dependence versus analgesic responsiveness, a phenotype that mimics several features of addiction (exacerbated withdrawal, locomotor sensitization, place conditioning, and tolerance). The only other protein known to mediate this complex phenotype is the transcription factor, ΔFosB, acting in the nucleus accumbens (Zachariou et al., 2006
). However, knockout of spinophilin does not alter ΔFosB levels in this brain region, nor does ΔFosB regulate spinophilin expression (Zachariou et al., 2006
), suggesting that spinophilin and ΔFosB act through distinct mechanisms. Future studies are needed to determine whether modulation of dopamine receptor responsiveness by spinophilin, or possible spinal cord-specific actions of this protein, also contribute to the opiate phenotype.
Our data demonstrate an essential role of spinophilin in regulating the rate of MOR internalization: reducing spinophilin levels with siRNA in cultured striatal neurons delays internalization. Similar effects on MOR internalization are observed in cultured MEF cells from spinophilin knockout embryos, whereas overexpression of spinophilin in cell lines promotes MOR internalization. The facilitation of MOR internalization by spinophilin is particularly dramatic for morphine, which is normally associated with much slower receptor internalization in cultured cells compared to that induced by opioid peptides (Keith et al., 1998
; Whistler and von Zastrow, 1998
; Whistler et al., 1999
). Indeed, the high levels of expression of spinophilin in striatum could explain the rapid internalization of MOR observed in response to morphine in striatal neurons in vivo compared to cultured cells in vitro (Haberstock-Debic et al., 2003
Earlier in vitro studies indicated that the rate of MOR endocytosis is a critical factor controlling the development of analgesic tolerance (Keith et al., 1998
; Whistler and von Zastrow, 1998
; Whistler et al., 1999
), with reduced endocytosis associated with exacerbated tolerance. Thus, our findings that knockout of spinophilin reduces MOR internalization and facilitates tolerance (and other adaptations to repeated morphine exposure) are consistent with this model. Several more recent studies have provided important information on the molecular determinants of MOR endocytosis, as well as information on its occurrence in vivo (Haberstock-Debic et al., 2003
; Arttamangkul et al., 2006
; Zhao et al., 2006
). It will therefore be important in future studies to focus on interactions between spinophilin and other regulators of MOR trafficking and on local manipulation of spinophilin in particular brain regions, to determine the mechanism via which this protein modulates MOR function.
Our behavioral studies reveal that spinophilin knockout mice show increased sensitivity to the rewarding and locomotor sensitizing actions of morphine. We speculate that this increased sensitivity could be interpreted as earlier development of motivational dependence and are therefore consistent with our data on physical dependence. The increased sensitivity observed in the place preference test may also be related to interactions between spinophilin and PP1; however, if this were the case, this phenotype might be expected to be observed in neurabin knockout mice as well, as neurabin is also a ubiquitous PP1 interacting protein. Another possible mechanism via which spinophilin could modulate sensitivity to the rewarding actions of morphine would involve interactions with G protein Gβ subunits. Earlier reports indicated that spinophilin interacts with the βγ complex (Brady et al., 2003
; Wang et al., 2004
). We show here that exposure to opiate drugs leads to the rapid association of spinophilin with Gβ5. Gβ5 is one of the most abundant G protein β subunits in brain and, in particular, is enriched in striatum. Gβ5 is also an essential binding partner for RGS9, as it contributes to the protein’s stability (Chen et al., 2003
). The striatal-enriched protein, RGS9-2, is considered a key regulator of MOR responsiveness (Zachariou et al., 2003
; Psifogeorgou et al., 2007
) and, as expected, it is found in the same complex with spinophilin after activation of MOR. This is particularly interesting since lack of either RGS9 or spinophilin increases sensitivity to morphine reward and leads to more severe dependence. The fact that spinophilin knockout mice show decreased sensitivity to morphine’s analgesic effects, whereas RGS9 knockout animals show increased sensitivity, indicates that modulation of morphine analgesia by spinophilin may involve distinct actions at the cellular level or actions beyond the nucleus accumbens and dorsal striatum.
Modified ERK phosphorylation and cAMP activity in the brain have been associated with regulation of morphine reward and dependence (Nestler, 2001
; Valjent et al., 2006
). Opioids can increase or decrease ERK phosphorylation depending on the brain region involved (Eitan et al., 2003
; Muller and Unterwald, 2004
), but little information is available concerning the influence of ERK signaling in the nucleus accumbens on behavioral responses to morphine. Previous work showed that spinophilin accelerates ERK phosphorylation in cultured MEF cells after activation of α2
receptors (Wang et al., 2005
). Interestingly, in striatum, knockout of spinophilin enhances morphine-induced increases in phosphoERK. Further work is needed to explore the behavioral consequences of spinophilin regulation of ERK function in this brain region. Our studies also demonstrate that knockout of spinophilin reduces the ability of morphine to inhibit adenylyl cyclase in striatum, an effect which would be expected to influence behavioral responses to opiate drugs (Barrot et al., 2002
). It is striking that loss of spinophilin impairs MOR signaling via adenylyl cyclase but enhances MOR signaling via ERK. Opiate inhibition of adenylyl cyclase is thought to represent a direct effect of Gi on the enzyme, whereas the molecular pathway by which Gi-linked receptors activate the ERK pathway has remained incompletely understood. The opposite effects of spinophilin on these signaling pathways could provide unique insight into understanding MOR regulation of cAMP and ERK signaling.
In summary, we show that spinophilin opposes the consequences of repeated opiate exposure and that these actions could be achieved by promoting MOR internalization and recycling and by modulating signal transduction events that follow opiate activation of MOR. Spinophilin regulation of MOR is very different from that observed previously for other receptors. These findings thereby contribute to our understanding of receptor-selective functions of spinophilin and provide information on new molecular elements that regulate MOR responsiveness. The molecular actions of spinophilin suggest that it could serve as a new pharmacological target to promote opiate analgesia, while preventing cellular events that lead to addiction after repeated exposure to these drugs.