Determining the effects of prolonged morphine exposure on receptor organization might serve as a basis for understanding the mechanism of desensitization. The underlying cause of morphine tolerance is not understood but appears to culminate from a number of cellular effects. Several mechanisms of morphine desensitization have been proposed (see (1
)) and one of these involves uncoupling between μOR and G protein signaling cascade (see (5
)). Here, we directly measured the ability of morphine to alter G protein – receptor interactions using spectroscopic methods. Although we could not uncover evidence for μOR-G protein uncoupling, we instead found that prolonged treatment with morphine alters the oligomerization behavior of opioid receptor heteromers. We carried out these studies by expressing fluorescent tagged proteins in neuronal cells, and studied their association and movement. We first monitored the cellular localization of μOR, and G protein subunits. In accord with other studies, we find that these receptors are plasma membrane localized and remain on the membrane with morphine treatment. This result correlates well with biochemical and pharmacologic studies suggesting that desensitization is not due to a loss in receptor binding sites (see (1
We tested the idea that morphine treatment decreases the association between μOR and G proteins by measuring the changes in FRET between eYFP-μOR and eCFP-Gβ1 in real time in living cells. We used Gβ1 for these studies since proteomic data from rat studies show that this protein is down-regulated with morphine addiction (Abul-Husn and Devi, unpublished
), and since Gβ subunits do not undergo the large structural changes seen in the activation of Gα which could affect the degree of FRET. We used eCFP-Gβ1γ7 since it has been established to have wild type cell properties (15
). We note that FRET values between eCFP-Gαi and eYFP- μOR were unchanged with continuous morphine treatment similar to the behavior seen for Gβγ and the receptor, and that over-expression of Gαi did not affect the degree of FRET between eYFP-μOR and eCFP-Gβ1. Thus, our experiments show that G proteins remain coupled to μOR with morphine binding in sharp contrast to the behavior seen for other receptor- G protein systems(36
for the CFP/YFP is 30Å (35
), and thus a value of FRET greater than our negative controls will indicate that the proteins are physically associated when we consider the size of the proteins and the similar placement of the fluorescent tags. We note that the large uncertainty in probe orientation, and well as the potential contributions from multiple donors and acceptors, preclude us from drawing accurate distances from our FRET values. FRET studies between μOR and Gβ1 in two different neuronal cell lines suggest very minor changes with morphine treatment. This result argues against the idea that desensitization involves decoupling between receptor and G proteins. Thus, morphine must induce other changes in the receptor that alter its ability to generate cell signals.
Since GPCRs may form oligomers, we focused on the ability of morphine to affect receptor-receptor interactions. Using single point FCS, we find that μOR displays a diffusion coefficient close to those reported for other GPCRs and small integral membrane proteins (see (18
)), suggesting that the receptor is in the form of small oligomers. Addition of morphine does not significantly change this value. It is worthwhile to note that while previous FRAP and single point FCS studies show similar mobilities for GPCRs, different values can be found using other methods. For example, scanning FCS studies of a GFP-labeled bradykinin type 2 receptor (B2R) over-expressed in HEK293 cells can detect two additional populations with a ten-fold and 100-fold slower diffusion even though PCH analysis show the receptor is diffusing as a homodimer (18
). Those results suggest maintenance of a dimeric form of this receptor even when localized in large complexes. In contrast, recent single molecule studies of muscarinic acid receptors in COS cells that followed the fluorescence from a bound ligand showed unrestricted diffusion of the monomeric and dimeric, or possibly dimeric to tetrameric forms of the receptor (37
). Similar to previous B2R studies (18
), our studies show that the brightness of μOR matches an eGFP dimer, suggesting a unit of two closely spaced μOR units. Addition of morphine results in a small increase in brightness without a significant change in mobility. This behavior is consistent with a shift from a predominantly dimeric population to a predominantly tetrameric population.
μOR is the primary mediator of morphine signals (1
). Since its oligomerization with δOR (8
) is upregulated with chronic morphine (11
), we studied the interaction between these receptors using fluorescence methods in living cells. μOR and δOR were transfected under identical conditions and visual inspection of transfected cells indicate that the two receptors are similarly expressed. It is important to note that in our experiments, we viewed the receptors under over-expressed conditions which may promote and stabilize oligomers. Although this behavior is not observed (see below), we predict that endogenous receptors might have a higher tendency to dissociate under natural conditions due to their lower concentrations.
Our FRET measurements show that in the basal state, μOR appears to have the same propensity to bind to δOR as it does to itself, suggesting that both homo- and heteromers form. Interestingly, morphine appears to stabilize μOR-μOR tetramers but destabilize μOR-δOR tetramers even though our measurements suggest that G proteins remain associated with the receptors in the associated or dissociated states. Rapid dissociation and reassociation of GPCR subunits in membranes has been reported (37
), and the reduced tendency of liganded - μOR tetramers to dissociate most likely reflects a stabilization of μOR contacts that increases its persistence time. To interpret the fluorescence data in a structural context, we first created cartoon permutations of μOR-μOR and μOR-δOR receptor tetramers containing activated μOR structures. Based on our fluorescence measurements, we were able to narrow down to four model structures the 29 different possibilities to assemble μOR and δOR involving the literature-predicted TM regions at symmetric heteromeric interfaces (e.g., TM1-TM1, TM4-TM4, and TM4,5-TM4,5). These four model structures differed in the presence of TM4,5-TM4,5 or TM1-TM1 at heteromeric interfaces. Although more experiments must be carried out using a series of mutant constructs to discriminate among these structures, these 4 models serve as a basis for a more detailed study of the functional properties of morphine receptors.
It is unclear whether disruption of opioid receptors oligomers is linked to morphine sensitization. Previous studies have shown that chronic morphine treatment leads to increased cell surface expression of μOR-δOR heteromers (39
) and that dimerization with δOR leads to changes in the spatio-temporal dynamics of μOR signaling (5
). This behavior was mediated primarily by constitutive recruitment of arrestins to the heteromer. The fact that in this study we find association of Gβ1 with the heteromer under chronic morphine treatment suggests that the heteromer represents a multipart signaling complex that allows for the activation of heteromer-specific, distinct signaling pathway. This model could, at least in part, contribute to μOR desensitization seen during the chronic exposure to morphine.
Our studies showing that morphine treatment does not perturb μOR subunit interactions or μOR - G protein interactions, but destabilizes μOR-δOR oligomers can be interpreted in light of a recent study showing that activation of dOR in the heteromer leads to the μOR degradation rather than recycling in turn diminishing its cellular response (12
). One can speculate that the smaller μOR-δOR oligomers can transfer into the lysozomal pathway more easily than larger μOR homomers through more accessible sites for proteolysis or modifications, such as ubitquitination. It is thus possible that the μOR-δOR system might be of the prime examples of how the oligomerization state of a GPCR can mediate very different cellular responses.