BACKGROUND
a cholesterol-palmitoyl interaction at c <dig>  has been observed in the crystallographic dimeric interface of transmembrane helix   <dig> and helix  <dig> in the β2-adrenergic receptor  crystal structure  <cit> .
palmitoylation is a covalent attachment of palmitic acid to cysteine residues of membrane proteins.
palmitoylation of the rhodopsin sub-family of g protein-coupled receptors  has been universally reported, and similar cholesterol-palmitoyl interactions may exist within other gpcrs.
sequence alignment has identified cysteine residues in the carboxy termini as potential palmitoylation sites in about 78% of  <dig> gpcrs examined  <cit> .
however, these cysteines are not the only palmitoylation sites.
for example, although rat μ-opioid receptor  has two cysteines  in its c terminus, mutation of these cysteines did not decrease the palmitoylation of oprm <dig>  <cit> , suggesting that c <dig>   may be the palmitoylation site.
similarly, v1a vasopressin receptor also has palmitoylation sites outside its c terminal domain  <cit> .
normally, palmitoylation facilitates the membrane targeting and signaling of gpcrs  <cit> .
for instance, palmitoylation-dependent receptor-g protein interaction is observed with both the β2-adrenergic receptor and the m <dig> muscarinic acetylcholine receptor  <cit> .
although there is no definitive answer as to how receptor palmitoylation contributes to gpcr signaling, the cholesterol-palmitoyl interaction at the β2-ar crystallographic dimeric interface suggests that facilitation of homodimerization may be one possible scenario.
because of the enrichment of many gpcrs in lipid raft  microdomains in cell membranes  <cit> , cholesterol within such microdomains can be easily incorporated into the receptor dimer.
in addition, because the interaction surface appears to be too small for the gpcr monomer to interact with g proteins  <cit> , dimerization may facilitate g protein coupling.
in fact, dimerization of many gpcrs, including oprm <dig> and β2-ar, regulates receptor signaling  <cit> .
in the work described here, we tested the hypothesis that a specific cholesterol-palmitoyl interaction within the oprm <dig> signaling complex affects its signaling by facilitating homodimerization and g protein coupling.
cholesterol, an important component of lipid raft microdomains on the cell membrane, is critical for gpcr signaling  <cit> , and the localization of some gpcrs in lipid raft microdomains has been suggested to contribute to downstream signaling  <cit> .
for example, oprm <dig> locates in lipid raft microdomains on the cell membrane in the absence of agonist  <cit> .
extraction of cholesterol from cells disrupts the entirety of the lipid raft microdomains and inhibits oprm <dig> signal transduction in both morphine-induced adenylyl cyclase inhibition and erk phosphorylation  <cit> .
thus, if a cholesterol-palmitoyl interaction could be identified in the interface of the oprm <dig> homodimer, it would suggest that cholesterol and cholesterol-enriched lipid raft microdomains may be linked to receptor palmitoylation during regulation of receptor signaling.
further, if the involvement of receptor dimerization and g protein coupling could be determined, this finding would extend our understanding of the mechanisms that underlie gpcr signaling.
we identified the palmitoylation site on oprm <dig> and examined the ability of the cholesterol-palmitoyl interaction to contribute to receptor homodimerization, g protein coupling, and signaling.
in addition, we developed a computational model of oprm <dig> to calculate the contribution of the cholesterol-palmitoyl interaction to the total interaction energy at the homodimer interface.
RESULTS
cys <dig> is the palmitoylation site of oprm1
we used wild-type hek <dig> cells  and hekoprm <dig> cells  to validate the palmitoylation assay  <cit> .
ha-tagged receptors were precipitated with ha antibody and protein g agarose.
the following procedures were used to determine receptor palmitoylation: 1) free sulfhydryl groups in precipitated receptors were blocked with n-ethylmaleimide .
2) palmitoylated cysteines were hydrolyzed with hydroxylamine.
3) biotin was conjugated to the de-palmitoylated cysteines in the immunoprecipitated receptors with btn-bmcc  cyclohexane carboxamido] butane).
the amount of biotin linked to the receptors was determined by immunoblotting.
we observed intensive biotin labeling in the hekoprm <dig> cells but not in the hek cells, which suggested that the palmitoylation we detected was specific to oprm <dig> .
since nem was used to block free sulfhydryl groups, the immunoreactivity of biotin increased when the nem step was omitted .
in addition, the immunoreactivity of biotin decreased when the hydroxylamine step was omitted  and no biotin was detected when btn-bmcc was not used , which confirmed that the assay was suitable for detecting the palmitoylation of oprm <dig>  to further confirm that the assay could be used to detect palmitoylation specifically, we used 2-bromopalmitate , a palmitoylation inhibitor, to block all palmitoylation.
a low level of receptor palmitoylation was observed when hekoprm <dig> cells were pretreated with 2-bp for  <dig> h , which also indicated that the palmitoylation we detected was on oprm <dig>  since palmitoylation is important for cell function, prolonged treatment with or high concentrations of 2-bp will affect cell viability.
in the current study, the treatment time and concentration of 2-bp were determined empirically so as to inhibit receptor palmitoylation while having minimal effect on cell viability.
thus, the 2-bp treatment paradigm we used did not completely block receptor palmitoylation.
mutation of the two conserved cysteine residues  in the c terminus of oprm <dig> does not affect palmitoylation  <cit> .
thus, we predicted the only other intracellular cysteine, c <dig> , to be the putative palmitoylation site.
to confirm this hypothesis, each of these three cysteines was mutated to alanine individually, and the mutants were stably expressed in hek cells to obtain the following: hekc170a, hekc346a, and hekc351a.
although c170a is difficult to stably express in cho cells  <cit> , we were able to successfully express a high level of c170a in the cell membrane of hek cells, possibly because of differences between cell lines or our use of poly-l-lysine during cell culture.
as shown in figure 1b, lanes  <dig> and 5- <dig>  we detected similar amounts of palmitoylated receptor in hekoprm <dig>  hekc346a, and hekc351a cells.
furthermore, the amount of palmitoylated receptor in hekc170a cells was similar to that in 2-bp-pretreated hekoprm <dig> cells .
these results suggest that c <dig>  but not c <dig>  or c <dig>  is indeed the palmitoylation site of oprm <dig>
our subsequent -diprenorphine saturation binding assay using isolated cell membranes indicated that there was no difference in the amounts of receptors in the cell membranes of the hekoprm <dig>  hekc170a, hekc346a, and hekc351a cells .
our facs analysis using an antibody against the ha-tag further confirmed that there was no difference in the amounts of membrane receptor between the four cell lines .
since the ha-tag was located in the receptor n-terminus, and the cell membrane was not disrupted during the analysis, the results obtained from the facs assay should represent the actual amounts of membrane receptor.
lastly, immunoblotting also indicated that the overall receptor expression levels were similar between the four cell lines .
in light of these results, it is reasonable to conclude that c <dig>  is the major palmitoylation site of oprm <dig>  for the sake of consistency, we will now use "oprm1" to refer to the wild-type oprm <dig>  "c170a" to refer to the palmitoylation-deficient mutant, and "receptor" to indicate both the wild type and mutants.
of note, agonist treatment did not affect receptor palmitoylation when morphine and fentanyl were used to challenge the hekoprm <dig> cells .
since the studies described here focused on how receptor palmitoylation influences receptor signaling, the effects of agonists on receptor palmitoylation or other subsequent observations are not discussed in depth.
receptor palmitoylation stabilizes morphine-induced signaling and receptor-gαi <dig> coupling
to determine the influence of palmitoylation on receptor signaling, we monitored morphine-induced adenylyl cyclase inhibition and erk phosphorylation.
morphine-induced adenylyl cyclase inhibition is defined by the ability of morphine to inhibit the forskolin-induced increase in the intracellular camp level.
we analyzed morphine-induced erk phosphorylation by calculating the percentage increase of phosphorylated erk when compared to basal level.
as summarized in table  <dig>  no difference in the affinities for ligands  was detected between oprm <dig> and c170a.
for example, the ki of ctop was  <dig> ±  <dig>  nm in hekoprm <dig> cells, and was  <dig>  ±  <dig>  nm in hekc170a cells .
in addition, the expression levels of oprm <dig> and c170a in the cell membrane were similar .
however, morphine induced less signaling in hekc170a than in hekoprm <dig> cells.
the ability of morphine to induce adenylyl cyclase inhibition in hekc170a was approximately 75% of that in hekoprm <dig>  when maximum inhibition was analyzed .
the ability to induce erk phosphorylation in hekc170a was approximately 69% of that in hekoprm <dig> .
morphine also induced receptor signaling in 2-bp-treated hekoprm <dig> cells and in hekc170a cells .
these results suggest that palmitoylation blockage impairs receptor signaling induced by morphine.
the affinities of receptor for ligands were determined in hekoprm <dig> and hekc170a cells using an agonist binding assay.
one-site  or two-site  curve-fitting models were used in the analyses with graphpad prism  <dig> .
data were analyzed by one-way anova with post-hoc dunnett's test.
standard deviations are provided, and "*" represents significant changes .
the percentage inhibition of forskolin-induced camp increase by various concentrations of morphine was analyzed with graphpad prism  <dig> .
the results are presented as the maximum inhibition  and the ic <dig> values .
erk phosphorylation was indicated by the amount of phosphorylated erk after  <dig> min of treatment with  <dig> μm morphine .
the amount of total erk and the results under control conditions were used for normalization.
experiments were repeated at least four times.
data were analyzed by one-way anova with post-hoc dunnett's test.
standard deviations are provided, and "*" represents significant changes .
both of the signaling events that we monitored are mediated via gαi <dig>  <cit> .
in addition, up-regulation and down-regulation of gαi <dig> in hekoprm <dig> cells significantly affects adenylyl cyclase inhibition and erk phosphorylation induced by morphine.
thus, we thought it likely that the impaired signaling described above was indicative of a decrease in receptor-gαi <dig> coupling.
we next investigated the effect of receptor palmitoylation on gαi <dig> coupling.
when we analyzed the immunoreactivity of oprm <dig> and gαi <dig> on the cell membrane , the colocalization between receptor and gαi <dig> in hekoprm <dig> cells was more obvious than in hekc170a cells.
similar observations were noted in our co-immunoprecipitation experiments .
when we used a gαi <dig> antibody to perform co-immunoprecipitation, the amount of oprm <dig> co-immunoprecipitated with gαi <dig> was greater than that of c170a.
when ha antibody was used to immunoprecipitate the receptor, more gαi <dig> was co-immunoprecipitated with oprm <dig> than with c170a.
these results indicate that the interaction between gαi <dig> and c170a is weaker than that between gαi <dig> and oprm <dig>
we further investigated the interaction between receptor and gαi <dig> with fret analysis.
the normalized net fret between cfpoprm <dig> and yfpgαi <dig> was much higher than that between cfp and yfp, suggesting that oprm <dig> and gαi <dig> were in close proximity of each other .
we performed fret analysis on the cell membrane to exclude any possible influence from the intracellular expression of fluorescence constructs.
the normalized net fret between cfpoprm <dig> and yfpgαi <dig> was higher than that between cfpc170a and yfpgαi <dig> .
because 1) we kept the expression of the fluorescence constructs, like cfpoprma <dig> and yfpgαi <dig> to similar levels by titrating the amounts of plasmids used for transfection, 2) we used immunoblotting to monitor expression during our studies, and 3) we determined overall fluorescence intensities prior to our fret and colocalization studies, the fret difference supports the conclusion that blockage of receptor palmitoylation in the c170a mutant impairs gαi <dig> coupling.
the yfp/cfp tagged receptors had similar functions when either flag- or ha-tagged.
morphine-induced adenylyl cyclase inhibition in the cells caused transient expression of these receptor constructs with similar kis:  <dig>  ±  <dig>  nm ,  <dig>  ±  <dig>  nm ,  <dig>  ±  <dig>  nm , and  <dig>  ±  <dig>  nm .
thus, the fret experiments should be indicative of the functional characteristics of the receptors.
receptor palmitoylation facilitates homodimerization and subsequent gαi <dig> coupling
we investigated the possible contribution of oprm <dig> palmitoylation to homodimerization by performing fret analysis between cfpoprm1/cfpc170a and yfpoprm1/yfpc170a.
as indicated in figure 3a, the normalized net fret between cfpoprm <dig> and yfpoprm <dig> was  <dig>  ±  <dig> , whereas it was  <dig>  ±  <dig>  between cfpc170a and yfpc170a in the cell membrane.
in addition, when the hek cells were co-transfected with cfpoprm <dig> and yfpc170a or with cfpc170a and yfpoprm <dig>  the normalized net frets were  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
the colocalization and co-immunoprecipitation assays between haoprm1/hac170a and flagoprm1/flagc170a confirmed the results of the fret assay .
in summary, our colocalization, co-immunoprecipitation and fret studies suggest that the amount of the oprm1-oprm <dig> homodimer is greater than the amount of the oprm1-c170a dimer, and the amount of the oprm1-c170a dimer is greater than that of the c170a-c170a homodimer, when similar levels of receptors are expressed.
it is reasonable, therefore, to suggest that the ability of c170a to form a homodimer is lower than that of oprm <dig>
because the amounts of homodimer decreased sequentially from lane  <dig> to lane  <dig> in figure 3c, we used fret analysis to determine if the decrease affected receptor-gαi <dig> coupling .
we transiently transfected yfpgαi <dig> with either cfpoprm <dig> or cfpc170a into hekoprm <dig> and hekc170a cells.
we considered two caveats in these experiments and took the following steps to ensure the success of the studies: 1) we determined that hekoprm <dig> and hekc170a cells expressed similar amounts of membrane receptors; and 2) we tightly controlled transient transfection of cfpoprm <dig> and cfpc170a in order to reach similar expression levels.
according to our hypothesis, if receptor palmitoylation affects gαi <dig> coupling, a similar sequential decrease in receptor-gαi <dig> coupling should be observed between oprm <dig> homodimer, oprm1-c170a dimer and c170a homodimer.
as indicated in figure 4a, we observed that the normalized net fret between cfpoprm <dig> and yfpgαi <dig> was greater than that between cfpc170a and yfpgαi <dig> in both hekoprm <dig> and hekc170a cells.
the normalized net fret between cfpoprm <dig> and yfpgαi <dig>  as well as between cfpc170a and yfpgαi <dig>  was greater in hekoprm <dig> than in hekc170a.
these results suggest a positive correlation between the receptor palmitoylation and gαi <dig> coupling.
this correlation can be explained by two potential mechanisms.
one possibility is that the homodimer's affinity for gαi <dig> is much higher than the monomer's affinity for gαi2; this mechanism is supported by a previous report  <cit> .
a second possibility is that the c170a monomer's affinity for gαi <dig> is much lower than the oprm <dig> monomer's affinity for gαi <dig>  if the second mechanism was the dominant one, the fret between transiently transfected cfpc170a and yfpgαi <dig> would be smaller in hekoprm <dig> cells than in hekc170a cells, because oprm1's higher affinity for yfpgαi <dig> would result in a higher competition for gαi <dig> in hekoprm <dig> than in hekc170a.
however, our fret analysis produced the opposite result: the fret between cfpc170a and yfpgαi <dig> was higher in hekoprm <dig> cells than in hekc170a cells .
these observations suggest that the reduced receptor dimerization in the absence of palmitoylation leads to decreased gαi <dig> coupling.
our additional colocalization and co-immunoprecipitation studies further supported this hypothesis .
in total, these results indicate a correlation between receptor homodimerization and gαi <dig> coupling.
receptor palmitoylation facilitates cholesterol association in the receptor signaling complex
in order to determine the detailed mechanisms underlying these phenomena, we utilized the observed interaction between cholesterol and palmitoyl group in the crystal structure of β2-ar  <cit> .
before we could determine the existence of a similar cholesterol-palmitoyl interaction in the oprm <dig> complex, however, we first needed to quantify the existing cholesterol in the receptor complex.
because direct detection of cholesterol within the homodimer requires purification of the receptor to homogeneity, and there is no guarantee that the cholesterol-receptor association will stay intact during purification, we instead examined the amount of cholesterol incorporated into the receptor signaling complex using a new method, described below.
to determine cholesterol association with the receptor complex, we used ha-antibody to precipitate the ha-tagged receptor.
in this method, if cholesterol does associate with the receptor complex specifically, greater amounts of cholesterol should be precipitated by the ha antibody when compared to immunoprecipitation with no antibody.
to avoid possible influence from the usage of antibody, flag antibody was used as control antibody, as no protein was flag-tagged in current paradigm.
cholesterol association with the receptor signaling complex was indicated by the additional amount of cholesterol precipitated by the ha antibody compared with that precipitated by a control antibody.
extensive washing with lysis buffer containing triton x- <dig> and digitonin ensured the removal of cholesterol that was nonspecifically associated with the receptor signaling complex.
using this procedure, ha antibody precipitated more cholesterol in hekoprm <dig> cells than did flag antibody or pbs.
since the receptor was ha-tagged at the n-terminus, any detected cholesterol in the precipitated receptors could be identified as cholesterol associated with receptor signaling complex.
further, in control experiments using hek cells, the two antibodies and pbs precipitated similar amounts of cholesterol .
these results confirm that this assay detects cholesterol associated with the receptor signaling complex.
we also noted decreased cholesterol association in hekc170a cells .
the amount of cholesterol precipitated with the ha antibody was similar to that precipitated with the flag antibody, suggesting that the mutation on c <dig>  contributes to the cholesterol association.
although the assay could not distinguish between cholesterol that associates with the receptor directly and cholesterol that associates with other proteins within the signaling complex, receptor palmitoylation appears to regulate the amount of cholesterol that associates with the complex.
cholesterol association facilitates homodimerization and gαi <dig> coupling
because 1) a cholesterol-palmitoyl interaction has been suggested in the β2-ar crystal structure, and 2) we demonstrated that receptor palmitoylation facilitates receptor dimerization and g protein coupling of oprm <dig> , we sought to discover whether cholesterol has the same functions in the latter receptor.
to determine the contribution of cholesterol association to receptor signaling, we treated the cells with simvastatin, an hmg-coa reductase inhibitor.
we assayed receptor dimerization and g protein coupling with fret, colocalization and immunoprecipitation.
the cellular cholesterol content decreased on the cell membrane of hekoprm <dig> cells after treatment of the cells with  <dig>  μm simvastatin for  <dig> h. we were able to prevent the decreases in cholesterol content by including  <dig> ng/ml cholesterol during the simvastatin treatment .
as expected, simvastatin treatment also induced a decrease in cholesterol level on the membrane of hekc170a cells .
we also assessed how cholesterol depletion influences its association with the receptor signaling complex.
simvastatin treatment decreased the association of cholesterol with the receptor complex, but this could be prevented by including  <dig> ng/ml cholesterol in the culture medium .
further, simvastatin not only decreased the amount of cholesterol precipitated in the "pbs" group, it also impaired the ability of the ha antibody to precipitate more cholesterol than flag antibody.
since cholesterol association was not detected in the hekc170a cells, simvastatin treatment had no effect in these cells .
since simvastatin treatment decreased the cellular cholesterol content, we used the fret assay to determine whether cholesterol content affects receptor dimerization and g protein coupling.
the normalized net fret between cfpoprm <dig> and yfpoprm <dig> in simvastatin-treated hek cells was decreased compared to untreated cells and could be reversed by inclusion of cholesterol during the simvastatin treatment .
a similar simvastatin-mediated decrease was observed with cfpoprm <dig> and yfpgαi <dig> and could also be reversed by the inclusion of cholesterol during the simvastatin treatment .
however, the cholesterol depletion induced by simvastatin did not affect the homodimerization  or g protein coupling of c170a .
therefore, the presence of cholesterol within the receptor signaling complex is critical for receptor homodimerization and gαi <dig> coupling.
we further illustrated the relationship between receptor palmitoylation, cholesterol association, and receptor dimerization by incubating cells with the palmitoylation inhibitor 2-bp.
we observed a decrease in cholesterol associated with the oprm <dig> signaling complex after 2-bp treatment .
because of the inhibitory effect of palmitoylation blockage on gαi <dig> membrane targeting  <cit> , the influence of 2-bp on gαi <dig> coupling was not investigated.
we also saw a reduction in the normalized net fret between cfpoprm <dig> and yfpoprm <dig> after 2-bp treatment .
in addition, 2-bp treatment did not affect cholesterol association with c170a or the homodimerization of c170a, since palmitoylation blockage in c170a already impaired these two functions to basal levels .
computational modeling suggests that palmitoyl-cholesterol interaction stabilizes the oprm <dig> homodimer
we undertook modeling studies to confirm that a specific cholesterol interaction with palmitoylated c <dig>  may enhance the interactions at the homodimer interface of oprm <dig>  the oprm <dig> model we developed for the modeling studies reported here is a homology model that uses the β2-ar crystal structure as a template  <cit> .
as mentioned in methods, oprm <dig> has two tmhs that differ in the position of helix deforming residues from the template β2-ar .
our conformational memories  calculations revealed that the location of p <dig>  in oprm <dig> causes the pitch of tmh <dig> to change after the proline such that residue  <dig>  faces into the binding pocket.
this same residue position in the β2-ar resides in the tmh2/ <dig> interface.
these results are consistent with the conformation of tmh <dig> in the cxcr <dig> crystal structure   <cit> .
the tmh <dig> region from  <dig>  to  <dig>  is ssaiglp in oprm <dig>  our cm calculations showed that the presence of the g <dig>  so close to p <dig>  causes a wider turn in tmh <dig> than is seen in β2-ar.
the net result is that tmh <dig> leans more towards tmh <dig>  one result of this change is the lipid exposure of residue  <dig> , a key residue in the tmh <dig> dimer interface .
these two key helix changes, along with the resulting changes in helix packing, distinguish the oprm <dig> binding pocket  from that of β2-ar.
our detailed modeling procedures are described in methods.
figure 8a illustrates the position of cholesterol relative to the palmitoyl and the tmh bundle.
due to the extreme tilt of tmh <dig> in the tmh bundle, the intracellular end of tmh <dig>  is between the intracellular ends of tmh <dig>  and tmh <dig> .
this position of tmh <dig> allows the cholesterol to pack between the c <dig>  palmitoyl and tmh <dig>  figure 8b provides an extracellular view of the final energy-minimized oprm <dig> homodimer.
in the resultant dimer, cholesterol is packed against the tmh <dig> interface and tmh <dig>  the palmitoyl at c <dig>  is packed against the cholesterol with tmh <dig>  blocking cholesterol from leaving the interface.
table  <dig> provides a summary of the resultant interaction energies for the palmitoylated oprm <dig> homodimer/cholesterol complex.
it is clear here that the major energetic contributions to the interaction energies between the protomers are van der waals  energies.
the homodimer interface residues with vdw contributions are n <dig> , i <dig> , c <dig> , i <dig> , and i <dig> , with a total energy of - <dig>  kcal/mol.
the cholesterol associated with protomer a interacts with protomer b residues r <dig> , n <dig> , k <dig> , i <dig> , and v <dig> , contributing an additional - <dig>  kcal/mol, and the cholesterol associated with protomer b contributes an additional - <dig>  kcal/mol.
thus, the total cholesterol interactions  contribute  <dig> % to the total interaction energy at the homodimer interface , suggesting that the interaction between cholesterol and palmitoyl facilitates oprm <dig> homodimerization
residues in bold are residue positions shown to be involved in the tmh <dig> homodimer inactive state interface of the dopamine d <dig> receptor  <cit> .
discussion
in summary, our experimental studies suggest that a cholesterol-palmitoyl interaction facilitates homodimerization and g protein coupling.
this conclusion is further supported by computational models of the oprm <dig> homodimer, which show that palmitoyl linked to c <dig>  can trap cholesterol at the interface of oprm <dig> homodimer; this subsequently stabilizes the homodimerization.
c <dig>  is highly conserved in the class a rhodopsin gpcrs, especially the peptide, opsin, olfactory, thyrotropin-releasing hormone and melatonin receptor families  <cit> .
c <dig>  on the melatonin type  <dig> receptor contributes to g protein activation  <cit> .
the critical role of the homologous residues in g protein coupling/activation has also been confirmed in the m <dig> muscarinic receptor, the α1b-adrenergic receptor, the at <dig> angiotensin receptor, and the interleukin- <dig> receptor, as summarized in the g protein-coupled receptor data base   <cit> .
although palmitoylation of the other two opioid receptor, δ-opioid receptor and κ-opioid receptor, has not been reported yet, the palmitoylation site in oprm <dig> is conserved in the other two opioid receptors: c <dig>  in δ-opioid receptor and c <dig>  in κ-opioid receptor.
the location of δ-opioid receptors in cholesterol-rich lipid rafts and its formation of a heterodimer oprm <dig> suggests the possible involvement of the palmitoylation on c <dig>  in its signaling  <cit> .
the palmitoylation inhibitor 2-bp decreased the normalized net fret between cfpoprm <dig> and yfpoprm <dig> to the level of that between cfpc170a and yfpc170a .
2-bp also decreased the cholesterol association with the signaling complex .
we did not determine the influences of 2-bp on receptor signaling and g protein coupling, because of its inhibitory effect on the membrane targeting of the highly palmitoylated g proteins  <cit> .
since the effects of 2-bp were consistent with the effects of the c <dig>  mutation, any conformational changes other than depalmitoylation are not significant in our current observations.
the dimerization of gpcrs, including the higher order oligomeric state of rhodopsin, has been long reported  <cit> .
the interface of monomer gpcr has been suggested to be too small for g protein coupling  <cit> .
however, the interaction between gpcr monomer and g protein still cannot be excluded.
here, the fret between cfpc170a and yfpc170a was about 13% of that between cfpoprm <dig> and yfpoprm <dig> , whereas the fret between cfpc170a and yfpgαi <dig> was about 28% of that between cfpoprm <dig> and yfpgαi <dig> .
this difference suggests that the receptor monomer can still interact with gαi <dig>  though with a lower affinity than the homodimer.
in the β2-ar crystal structure, cholesterol is found situated at the intracellular side of the tmh1-tmh <dig> bundles  <cit> .
thus, it is probable that localization of oprm <dig> within cholesterol-enriched domains such as lipid rafts regulates the cholesterol content within the receptor complex and receptor signaling.
therefore, the cholesterol-enriched lipid raft microdomain may be essential for the ability of the cholesterol-palmitoyl interaction to stabilize receptor homodimerization and g protein coupling.
in our studies, c <dig>  palmitoylation affected the amount of cholesterol associated with the signaling complex.
as reported previously, gαi <dig> anchors oprm <dig> to the lipid raft microdomains.
thus, by facilitating gαi <dig> coupling, cholesterol associated with oprm <dig> increases the percentage of receptor in lipid raft microdomains.
the modeling studies reported here show that the c <dig>  palmitoylation site is located very near the class a gpcr inactive homodimer interface identified by guo and colleagues for dopamine d <dig> receptor  <cit> .
for an oprm <dig> homodimer formed at this interface, the cholesterol associated with c <dig>  is ideally placed to contribute to the total energy of interaction for the homodimer.
as lambert discusses in his recent review  <cit> , the interaction energies at the homodimer interface are likely weak but sufficient to promote dimer formation transiently.
we report here that the enthalpic component of the interaction between oprm <dig> homodimers is - <dig>  kcal/mol and that the presence of cholesterol at the oprm <dig> homodimer interface raises the total interaction enthalpy to - <dig>  kcal/mol.
this modest interaction energy is derived predominantly from vdw interactions, as would be expected for the hydrophobic residues as well as the hydrophobic cholesterols in the homodimer interface.
by identifying a consensus cholesterol binding motif in the tmh2-tmh <dig> region that predicts cholesterol binding for 44% of human class a receptors, hanson and co-workers suggest that specific sterol binding is important to the structure and stability of many gpcrs  <cit> .
however, this consensus motif is not present in oprm <dig> and does not involve c <dig> .
in conclusion, both our experimental data and computational models delineate a cascade from cholesterol-palmitoyl interaction to receptor homodimerization and then to g protein coupling/activation.
conceivably, by regulating the cholesterol-palmitoyl interaction, either by the control of cholesterol metabolism or receptor palmitoylation, the stability of gpcr homodimers is altered, leading to the uncoupling of g protein.
in this respect, the cellular cholesterol content, specifically the cholesterol associated with the receptor, represents an additional target through which the signaling of gpcrs can be regulated.
CONCLUSIONS
c <dig>  is the palmitoylation site of oprm1
oprm <dig> is highly palmitoylated.
the c <dig>  has been indirectly suggested as the palmitoylation site, as it has been established that the only other two cysteines  are not the palmitoylation site  <cit> .
our current studies provide the first direct evidence that c <dig>  is the palmitoylation site of oprm <dig>
a cholesterol-palmitoyl interaction can be identified in oprm <dig> complex
a cholesterol-palmitoyl interaction has been identified both in the β2-ar crystal structure  <cit>  and, now, in oprm <dig>  although this is not the first identification of a cholesterol-palmitoyl interaction in a gpcr, our studies suggest that such interactions may be observed in the signaling complexes of many gpcrs.
cholesterol-palmitoyl interactions contribute to oprm <dig> signaling by facilitating receptor homodimerization and g protein coupling
our studies also represent the first report on the contributions of cholesterol-palmitoyl interaction to receptor signaling.
in addition, by the using multiple assays, including fret, we illustrate the mechanism underlying these contributions.
this understanding provides additional information on receptor homodimerization and g protein coupling.
computational modeling of oprm <dig> homodimer supports the conclusions listed above
to support the conclusions mentioned above, we generated a computational model of oprm <dig> homodimer based on the structure of other relevant gpcrs.
our model suggests that the cholesterol-palmitoyl interaction provides additional energy to stabilize the homodimer, which is consistent with our other observations.

 a cholesterol-palmitoyl interaction at c has been observed in the crystallographic dimeric interface of transmembrane helix and helix in the β2-adrenergic receptor crystal structure. palmitoylation is a covalent attachment of palmitic acid to cysteine residues of membrane proteins. palmitoylation of the rhodopsin sub-family of g protein-coupled receptors has been universally reported, and similar cholesterol-palmitoyl interactions may exist within other gpcrs. sequence alignment has identified cysteine residues in the carboxy termini as potential palmitoylation sites in about 78% of gpcrs examined. however, these cysteines are not the only palmitoylation sites. for example, although rat μ-opioid receptor has two cysteines in its c terminus, mutation of these cysteines did not decrease the palmitoylation of oprm, suggesting that c may be the palmitoylation site. similarly, v1a vasopressin receptor also has palmitoylation sites outside its c terminal domain. normally, palmitoylation facilitates the membrane targeting and signaling of gpcrs. for instance, palmitoylation-dependent receptor-g protein interaction is observed with both the β2-adrenergic receptor and the m muscarinic acetylcholine receptor. although there is no definitive answer as to how receptor palmitoylation contributes to gpcr signaling, the cholesterol-palmitoyl interaction at the β2-ar crystallographic dimeric interface suggests that facilitation of homodimerization may be one possible scenario. because of the enrichment of many gpcrs in lipid raft microdomains in cell membranes, cholesterol within such microdomains can be easily incorporated into the receptor dimer. in addition, because the interaction surface appears to be too small for the gpcr monomer to interact with g proteins, dimerization may facilitate g protein coupling. in fact, dimerization of many gpcrs, including oprm and β2-ar, regulates receptor signaling. in the work described here, we tested the hypothesis that a specific cholesterol-palmitoyl interaction within the oprm signaling complex affects its signaling by facilitating homodimerization and g protein coupling. cholesterol, an important component of lipid raft microdomains on the cell membrane, is critical for gpcr signaling, and the localization of some gpcrs in lipid raft microdomains has been suggested to contribute to downstream signaling. for example, oprm locates in lipid raft microdomains on the cell membrane in the absence of agonist. extraction of cholesterol from cells disrupts the entirety of the lipid raft microdomains and inhibits oprm signal transduction in both morphine-induced adenylyl cyclase inhibition and erk phosphorylation. thus, if a cholesterol-palmitoyl interaction could be identified in the interface of the oprm homodimer, it would suggest that cholesterol and cholesterol-enriched lipid raft microdomains may be linked to receptor palmitoylation during regulation of receptor signaling. further, if the involvement of receptor dimerization and g protein coupling could be determined, this finding would extend our understanding of the mechanisms that underlie gpcr signaling. we identified the palmitoylation site on oprm and examined the ability of the cholesterol-palmitoyl interaction to contribute to receptor homodimerization, g protein coupling, and signaling. in addition, we developed a computational model of oprm to calculate the contribution of the cholesterol-palmitoyl interaction to the total interaction energy at the homodimer interface.  cys is the palmitoylation site of oprm1 we used wild-type hek cells and hekoprm cells to validate the palmitoylation assay. ha-tagged receptors were precipitated with ha antibody and protein g agarose. the following procedures were used to determine receptor palmitoylation: 1) free sulfhydryl groups in precipitated receptors were blocked with n-ethylmaleimide. 2) palmitoylated cysteines were hydrolyzed with hydroxylamine. 3) biotin was conjugated to the de-palmitoylated cysteines in the immunoprecipitated receptors with btn-bmcc cyclohexane carboxamido] butane). the amount of biotin linked to the receptors was determined by immunoblotting. we observed intensive biotin labeling in the hekoprm cells but not in the hek cells, which suggested that the palmitoylation we detected was specific to oprm. since nem was used to block free sulfhydryl groups, the immunoreactivity of biotin increased when the nem step was omitted. in addition, the immunoreactivity of biotin decreased when the hydroxylamine step was omitted and no biotin was detected when btn-bmcc was not used, which confirmed that the assay was suitable for detecting the palmitoylation of oprm to further confirm that the assay could be used to detect palmitoylation specifically, we used 2-bromopalmitate, a palmitoylation inhibitor, to block all palmitoylation. a low level of receptor palmitoylation was observed when hekoprm cells were pretreated with 2-bp for h, which also indicated that the palmitoylation we detected was on oprm since palmitoylation is important for cell function, prolonged treatment with or high concentrations of 2-bp will affect cell viability. in the current study, the treatment time and concentration of 2-bp were determined empirically so as to inhibit receptor palmitoylation while having minimal effect on cell viability. thus, the 2-bp treatment paradigm we used did not completely block receptor palmitoylation. mutation of the two conserved cysteine residues in the c terminus of oprm does not affect palmitoylation. thus, we predicted the only other intracellular cysteine, c, to be the putative palmitoylation site. to confirm this hypothesis, each of these three cysteines was mutated to alanine individually, and the mutants were stably expressed in hek cells to obtain the following: hekc170a, hekc346a, and hekc351a. although c170a is difficult to stably express in cho cells, we were able to successfully express a high level of c170a in the cell membrane of hek cells, possibly because of differences between cell lines or our use of poly-l-lysine during cell culture. as shown in figure 1b, lanes and 5- we detected similar amounts of palmitoylated receptor in hekoprm hekc346a, and hekc351a cells. furthermore, the amount of palmitoylated receptor in hekc170a cells was similar to that in 2-bp-pretreated hekoprm cells. these results suggest that c but not c or c is indeed the palmitoylation site of oprm our subsequent -diprenorphine saturation binding assay using isolated cell membranes indicated that there was no difference in the amounts of receptors in the cell membranes of the hekoprm hekc170a, hekc346a, and hekc351a cells. our facs analysis using an antibody against the ha-tag further confirmed that there was no difference in the amounts of membrane receptor between the four cell lines. since the ha-tag was located in the receptor n-terminus, and the cell membrane was not disrupted during the analysis, the results obtained from the facs assay should represent the actual amounts of membrane receptor. lastly, immunoblotting also indicated that the overall receptor expression levels were similar between the four cell lines. in light of these results, it is reasonable to conclude that c is the major palmitoylation site of oprm for the sake of consistency, we will now use "oprm1" to refer to the wild-type oprm "c170a" to refer to the palmitoylation-deficient mutant, and "receptor" to indicate both the wild type and mutants. of note, agonist treatment did not affect receptor palmitoylation when morphine and fentanyl were used to challenge the hekoprm cells. since the studies described here focused on how receptor palmitoylation influences receptor signaling, the effects of agonists on receptor palmitoylation or other subsequent observations are not discussed in depth. receptor palmitoylation stabilizes morphine-induced signaling and receptor-gαi coupling to determine the influence of palmitoylation on receptor signaling, we monitored morphine-induced adenylyl cyclase inhibition and erk phosphorylation. morphine-induced adenylyl cyclase inhibition is defined by the ability of morphine to inhibit the forskolin-induced increase in the intracellular camp level. we analyzed morphine-induced erk phosphorylation by calculating the percentage increase of phosphorylated erk when compared to basal level. as summarized in table no difference in the affinities for ligands was detected between oprm and c170a. for example, the ki of ctop was ± nm in hekoprm cells, and was ± nm in hekc170a cells. in addition, the expression levels of oprm and c170a in the cell membrane were similar. however, morphine induced less signaling in hekc170a than in hekoprm cells. the ability of morphine to induce adenylyl cyclase inhibition in hekc170a was approximately 75% of that in hekoprm when maximum inhibition was analyzed. the ability to induce erk phosphorylation in hekc170a was approximately 69% of that in hekoprm. morphine also induced receptor signaling in 2-bp-treated hekoprm cells and in hekc170a cells. these results suggest that palmitoylation blockage impairs receptor signaling induced by morphine. the affinities of receptor for ligands were determined in hekoprm and hekc170a cells using an agonist binding assay. one-site or two-site curve-fitting models were used in the analyses with graphpad prism. data were analyzed by one-way anova with post-hoc dunnett's test. standard deviations are provided, and "*" represents significant changes. the percentage inhibition of forskolin-induced camp increase by various concentrations of morphine was analyzed with graphpad prism. the results are presented as the maximum inhibition and the ic values. erk phosphorylation was indicated by the amount of phosphorylated erk after min of treatment with μm morphine. the amount of total erk and the results under control conditions were used for normalization. experiments were repeated at least four times. data were analyzed by one-way anova with post-hoc dunnett's test. standard deviations are provided, and "*" represents significant changes. both of the signaling events that we monitored are mediated via gαi. in addition, up-regulation and down-regulation of gαi in hekoprm cells significantly affects adenylyl cyclase inhibition and erk phosphorylation induced by morphine. thus, we thought it likely that the impaired signaling described above was indicative of a decrease in receptor-gαi coupling. we next investigated the effect of receptor palmitoylation on gαi coupling. when we analyzed the immunoreactivity of oprm and gαi on the cell membrane, the colocalization between receptor and gαi in hekoprm cells was more obvious than in hekc170a cells. similar observations were noted in our co-immunoprecipitation experiments. when we used a gαi antibody to perform co-immunoprecipitation, the amount of oprm co-immunoprecipitated with gαi was greater than that of c170a. when ha antibody was used to immunoprecipitate the receptor, more gαi was co-immunoprecipitated with oprm than with c170a. these results indicate that the interaction between gαi and c170a is weaker than that between gαi and oprm we further investigated the interaction between receptor and gαi with fret analysis. the normalized net fret between cfpoprm and yfpgαi was much higher than that between cfp and yfp, suggesting that oprm and gαi were in close proximity of each other. we performed fret analysis on the cell membrane to exclude any possible influence from the intracellular expression of fluorescence constructs. the normalized net fret between cfpoprm and yfpgαi was higher than that between cfpc170a and yfpgαi. because 1) we kept the expression of the fluorescence constructs, like cfpoprma and yfpgαi to similar levels by titrating the amounts of plasmids used for transfection, 2) we used immunoblotting to monitor expression during our studies, and 3) we determined overall fluorescence intensities prior to our fret and colocalization studies, the fret difference supports the conclusion that blockage of receptor palmitoylation in the c170a mutant impairs gαi coupling. the yfp/cfp tagged receptors had similar functions when either flag- or ha-tagged. morphine-induced adenylyl cyclase inhibition in the cells caused transient expression of these receptor constructs with similar kis: ± nm, ± nm, ± nm, and ± nm. thus, the fret experiments should be indicative of the functional characteristics of the receptors. receptor palmitoylation facilitates homodimerization and subsequent gαi coupling we investigated the possible contribution of oprm palmitoylation to homodimerization by performing fret analysis between cfpoprm1/cfpc170a and yfpoprm1/yfpc170a. as indicated in figure 3a, the normalized net fret between cfpoprm and yfpoprm was ±, whereas it was ± between cfpc170a and yfpc170a in the cell membrane. in addition, when the hek cells were co-transfected with cfpoprm and yfpc170a or with cfpc170a and yfpoprm the normalized net frets were ± and ±, respectively. the colocalization and co-immunoprecipitation assays between haoprm1/hac170a and flagoprm1/flagc170a confirmed the results of the fret assay. in summary, our colocalization, co-immunoprecipitation and fret studies suggest that the amount of the oprm1-oprm homodimer is greater than the amount of the oprm1-c170a dimer, and the amount of the oprm1-c170a dimer is greater than that of the c170a-c170a homodimer, when similar levels of receptors are expressed. it is reasonable, therefore, to suggest that the ability of c170a to form a homodimer is lower than that of oprm because the amounts of homodimer decreased sequentially from lane to lane in figure 3c, we used fret analysis to determine if the decrease affected receptor-gαi coupling. we transiently transfected yfpgαi with either cfpoprm or cfpc170a into hekoprm and hekc170a cells. we considered two caveats in these experiments and took the following steps to ensure the success of the studies: 1) we determined that hekoprm and hekc170a cells expressed similar amounts of membrane receptors; and 2) we tightly controlled transient transfection of cfpoprm and cfpc170a in order to reach similar expression levels. according to our hypothesis, if receptor palmitoylation affects gαi coupling, a similar sequential decrease in receptor-gαi coupling should be observed between oprm homodimer, oprm1-c170a dimer and c170a homodimer. as indicated in figure 4a, we observed that the normalized net fret between cfpoprm and yfpgαi was greater than that between cfpc170a and yfpgαi in both hekoprm and hekc170a cells. the normalized net fret between cfpoprm and yfpgαi as well as between cfpc170a and yfpgαi was greater in hekoprm than in hekc170a. these results suggest a positive correlation between the receptor palmitoylation and gαi coupling. this correlation can be explained by two potential mechanisms. one possibility is that the homodimer's affinity for gαi is much higher than the monomer's affinity for gαi2; this mechanism is supported by a previous report. a second possibility is that the c170a monomer's affinity for gαi is much lower than the oprm monomer's affinity for gαi if the second mechanism was the dominant one, the fret between transiently transfected cfpc170a and yfpgαi would be smaller in hekoprm cells than in hekc170a cells, because oprm1's higher affinity for yfpgαi would result in a higher competition for gαi in hekoprm than in hekc170a. however, our fret analysis produced the opposite result: the fret between cfpc170a and yfpgαi was higher in hekoprm cells than in hekc170a cells. these observations suggest that the reduced receptor dimerization in the absence of palmitoylation leads to decreased gαi coupling. our additional colocalization and co-immunoprecipitation studies further supported this hypothesis. in total, these results indicate a correlation between receptor homodimerization and gαi coupling. receptor palmitoylation facilitates cholesterol association in the receptor signaling complex in order to determine the detailed mechanisms underlying these phenomena, we utilized the observed interaction between cholesterol and palmitoyl group in the crystal structure of β2-ar. before we could determine the existence of a similar cholesterol-palmitoyl interaction in the oprm complex, however, we first needed to quantify the existing cholesterol in the receptor complex. because direct detection of cholesterol within the homodimer requires purification of the receptor to homogeneity, and there is no guarantee that the cholesterol-receptor association will stay intact during purification, we instead examined the amount of cholesterol incorporated into the receptor signaling complex using a new method, described below. to determine cholesterol association with the receptor complex, we used ha-antibody to precipitate the ha-tagged receptor. in this method, if cholesterol does associate with the receptor complex specifically, greater amounts of cholesterol should be precipitated by the ha antibody when compared to immunoprecipitation with no antibody. to avoid possible influence from the usage of antibody, flag antibody was used as control antibody, as no protein was flag-tagged in current paradigm. cholesterol association with the receptor signaling complex was indicated by the additional amount of cholesterol precipitated by the ha antibody compared with that precipitated by a control antibody. extensive washing with lysis buffer containing triton x- and digitonin ensured the removal of cholesterol that was nonspecifically associated with the receptor signaling complex. using this procedure, ha antibody precipitated more cholesterol in hekoprm cells than did flag antibody or pbs. since the receptor was ha-tagged at the n-terminus, any detected cholesterol in the precipitated receptors could be identified as cholesterol associated with receptor signaling complex. further, in control experiments using hek cells, the two antibodies and pbs precipitated similar amounts of cholesterol. these results confirm that this assay detects cholesterol associated with the receptor signaling complex. we also noted decreased cholesterol association in hekc170a cells. the amount of cholesterol precipitated with the ha antibody was similar to that precipitated with the flag antibody, suggesting that the mutation on c contributes to the cholesterol association. although the assay could not distinguish between cholesterol that associates with the receptor directly and cholesterol that associates with other proteins within the signaling complex, receptor palmitoylation appears to regulate the amount of cholesterol that associates with the complex. cholesterol association facilitates homodimerization and gαi coupling because 1) a cholesterol-palmitoyl interaction has been suggested in the β2-ar crystal structure, and 2) we demonstrated that receptor palmitoylation facilitates receptor dimerization and g protein coupling of oprm, we sought to discover whether cholesterol has the same functions in the latter receptor. to determine the contribution of cholesterol association to receptor signaling, we treated the cells with simvastatin, an hmg-coa reductase inhibitor. we assayed receptor dimerization and g protein coupling with fret, colocalization and immunoprecipitation. the cellular cholesterol content decreased on the cell membrane of hekoprm cells after treatment of the cells with μm simvastatin for h. we were able to prevent the decreases in cholesterol content by including ng/ml cholesterol during the simvastatin treatment. as expected, simvastatin treatment also induced a decrease in cholesterol level on the membrane of hekc170a cells. we also assessed how cholesterol depletion influences its association with the receptor signaling complex. simvastatin treatment decreased the association of cholesterol with the receptor complex, but this could be prevented by including ng/ml cholesterol in the culture medium. further, simvastatin not only decreased the amount of cholesterol precipitated in the "pbs" group, it also impaired the ability of the ha antibody to precipitate more cholesterol than flag antibody. since cholesterol association was not detected in the hekc170a cells, simvastatin treatment had no effect in these cells. since simvastatin treatment decreased the cellular cholesterol content, we used the fret assay to determine whether cholesterol content affects receptor dimerization and g protein coupling. the normalized net fret between cfpoprm and yfpoprm in simvastatin-treated hek cells was decreased compared to untreated cells and could be reversed by inclusion of cholesterol during the simvastatin treatment. a similar simvastatin-mediated decrease was observed with cfpoprm and yfpgαi and could also be reversed by the inclusion of cholesterol during the simvastatin treatment. however, the cholesterol depletion induced by simvastatin did not affect the homodimerization or g protein coupling of c170a. therefore, the presence of cholesterol within the receptor signaling complex is critical for receptor homodimerization and gαi coupling. we further illustrated the relationship between receptor palmitoylation, cholesterol association, and receptor dimerization by incubating cells with the palmitoylation inhibitor 2-bp. we observed a decrease in cholesterol associated with the oprm signaling complex after 2-bp treatment. because of the inhibitory effect of palmitoylation blockage on gαi membrane targeting, the influence of 2-bp on gαi coupling was not investigated. we also saw a reduction in the normalized net fret between cfpoprm and yfpoprm after 2-bp treatment. in addition, 2-bp treatment did not affect cholesterol association with c170a or the homodimerization of c170a, since palmitoylation blockage in c170a already impaired these two functions to basal levels. computational modeling suggests that palmitoyl-cholesterol interaction stabilizes the oprm homodimer we undertook modeling studies to confirm that a specific cholesterol interaction with palmitoylated c may enhance the interactions at the homodimer interface of oprm the oprm model we developed for the modeling studies reported here is a homology model that uses the β2-ar crystal structure as a template. as mentioned in methods, oprm has two tmhs that differ in the position of helix deforming residues from the template β2-ar. our conformational memories calculations revealed that the location of p in oprm causes the pitch of tmh to change after the proline such that residue faces into the binding pocket. this same residue position in the β2-ar resides in the tmh2/ interface. these results are consistent with the conformation of tmh in the cxcr crystal structure. the tmh region from to is ssaiglp in oprm our cm calculations showed that the presence of the g so close to p causes a wider turn in tmh than is seen in β2-ar. the net result is that tmh leans more towards tmh one result of this change is the lipid exposure of residue, a key residue in the tmh dimer interface. these two key helix changes, along with the resulting changes in helix packing, distinguish the oprm binding pocket from that of β2-ar. our detailed modeling procedures are described in methods. figure 8a illustrates the position of cholesterol relative to the palmitoyl and the tmh bundle. due to the extreme tilt of tmh in the tmh bundle, the intracellular end of tmh is between the intracellular ends of tmh and tmh. this position of tmh allows the cholesterol to pack between the c palmitoyl and tmh figure 8b provides an extracellular view of the final energy-minimized oprm homodimer. in the resultant dimer, cholesterol is packed against the tmh interface and tmh the palmitoyl at c is packed against the cholesterol with tmh blocking cholesterol from leaving the interface. table provides a summary of the resultant interaction energies for the palmitoylated oprm homodimer/cholesterol complex. it is clear here that the major energetic contributions to the interaction energies between the protomers are van der waals energies. the homodimer interface residues with vdw contributions are n, i, c, i, and i, with a total energy of - kcal/mol. the cholesterol associated with protomer a interacts with protomer b residues r, n, k, i, and v, contributing an additional - kcal/mol, and the cholesterol associated with protomer b contributes an additional - kcal/mol. thus, the total cholesterol interactions contribute % to the total interaction energy at the homodimer interface, suggesting that the interaction between cholesterol and palmitoyl facilitates oprm homodimerization residues in bold are residue positions shown to be involved in the tmh homodimer inactive state interface of the dopamine d receptor. discussion in summary, our experimental studies suggest that a cholesterol-palmitoyl interaction facilitates homodimerization and g protein coupling. this conclusion is further supported by computational models of the oprm homodimer, which show that palmitoyl linked to c can trap cholesterol at the interface of oprm homodimer; this subsequently stabilizes the homodimerization. c is highly conserved in the class a rhodopsin gpcrs, especially the peptide, opsin, olfactory, thyrotropin-releasing hormone and melatonin receptor families. c on the melatonin type receptor contributes to g protein activation. the critical role of the homologous residues in g protein coupling/activation has also been confirmed in the m muscarinic receptor, the α1b-adrenergic receptor, the at angiotensin receptor, and the interleukin- receptor, as summarized in the g protein-coupled receptor data base. although palmitoylation of the other two opioid receptor, δ-opioid receptor and κ-opioid receptor, has not been reported yet, the palmitoylation site in oprm is conserved in the other two opioid receptors: c in δ-opioid receptor and c in κ-opioid receptor. the location of δ-opioid receptors in cholesterol-rich lipid rafts and its formation of a heterodimer oprm suggests the possible involvement of the palmitoylation on c in its signaling. the palmitoylation inhibitor 2-bp decreased the normalized net fret between cfpoprm and yfpoprm to the level of that between cfpc170a and yfpc170a. 2-bp also decreased the cholesterol association with the signaling complex. we did not determine the influences of 2-bp on receptor signaling and g protein coupling, because of its inhibitory effect on the membrane targeting of the highly palmitoylated g proteins. since the effects of 2-bp were consistent with the effects of the c mutation, any conformational changes other than depalmitoylation are not significant in our current observations. the dimerization of gpcrs, including the higher order oligomeric state of rhodopsin, has been long reported. the interface of monomer gpcr has been suggested to be too small for g protein coupling. however, the interaction between gpcr monomer and g protein still cannot be excluded. here, the fret between cfpc170a and yfpc170a was about 13% of that between cfpoprm and yfpoprm, whereas the fret between cfpc170a and yfpgαi was about 28% of that between cfpoprm and yfpgαi. this difference suggests that the receptor monomer can still interact with gαi though with a lower affinity than the homodimer. in the β2-ar crystal structure, cholesterol is found situated at the intracellular side of the tmh1-tmh bundles. thus, it is probable that localization of oprm within cholesterol-enriched domains such as lipid rafts regulates the cholesterol content within the receptor complex and receptor signaling. therefore, the cholesterol-enriched lipid raft microdomain may be essential for the ability of the cholesterol-palmitoyl interaction to stabilize receptor homodimerization and g protein coupling. in our studies, c palmitoylation affected the amount of cholesterol associated with the signaling complex. as reported previously, gαi anchors oprm to the lipid raft microdomains. thus, by facilitating gαi coupling, cholesterol associated with oprm increases the percentage of receptor in lipid raft microdomains. the modeling studies reported here show that the c palmitoylation site is located very near the class a gpcr inactive homodimer interface identified by guo and colleagues for dopamine d receptor. for an oprm homodimer formed at this interface, the cholesterol associated with c is ideally placed to contribute to the total energy of interaction for the homodimer. as lambert discusses in his recent review, the interaction energies at the homodimer interface are likely weak but sufficient to promote dimer formation transiently. we report here that the enthalpic component of the interaction between oprm homodimers is - kcal/mol and that the presence of cholesterol at the oprm homodimer interface raises the total interaction enthalpy to - kcal/mol. this modest interaction energy is derived predominantly from vdw interactions, as would be expected for the hydrophobic residues as well as the hydrophobic cholesterols in the homodimer interface. by identifying a consensus cholesterol binding motif in the tmh2-tmh region that predicts cholesterol binding for 44% of human class a receptors, hanson and co-workers suggest that specific sterol binding is important to the structure and stability of many gpcrs. however, this consensus motif is not present in oprm and does not involve c. in conclusion, both our experimental data and computational models delineate a cascade from cholesterol-palmitoyl interaction to receptor homodimerization and then to g protein coupling/activation. conceivably, by regulating the cholesterol-palmitoyl interaction, either by the control of cholesterol metabolism or receptor palmitoylation, the stability of gpcr homodimers is altered, leading to the uncoupling of g protein. in this respect, the cellular cholesterol content, specifically the cholesterol associated with the receptor, represents an additional target through which the signaling of gpcrs can be regulated.  c is the palmitoylation site of oprm1 oprm is highly palmitoylated. the c has been indirectly suggested as the palmitoylation site, as it has been established that the only other two cysteines are not the palmitoylation site. our current studies provide the first direct evidence that c is the palmitoylation site of oprm a cholesterol-palmitoyl interaction can be identified in oprm complex a cholesterol-palmitoyl interaction has been identified both in the β2-ar crystal structure and, now, in oprm although this is not the first identification of a cholesterol-palmitoyl interaction in a gpcr, our studies suggest that such interactions may be observed in the signaling complexes of many gpcrs. cholesterol-palmitoyl interactions contribute to oprm signaling by facilitating receptor homodimerization and g protein coupling our studies also represent the first report on the contributions of cholesterol-palmitoyl interaction to receptor signaling. in addition, by the using multiple assays, including fret, we illustrate the mechanism underlying these contributions. this understanding provides additional information on receptor homodimerization and g protein coupling. computational modeling of oprm homodimer supports the conclusions listed above to support the conclusions mentioned above, we generated a computational model of oprm homodimer based on the structure of other relevant gpcrs. our model suggests that the cholesterol-palmitoyl interaction provides additional energy to stabilize the homodimer, which is consistent with our other observations. 
in addition, both attenuation of receptor palmitoylation and inhibition of cholesterol synthesis impaired receptor signaling, possibly by decreasing receptor homodimerization and gαi coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer analyses. our findings suggest that this interaction contributes to oprm signaling by facilitating receptor homodimerization and g protein coupling. we sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor, and if so, to assess the role of cholesterol in this class of g protein-coupled receptor signaling. a computational model of the oprm homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate oprm homodimerization at the tmh4-tmh interface. c was determined to be the palmitoylation site of oprm mutation of this cys to ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. we demonstrate that c is the palmitoylation site of oprm and identify a cholesterol-palmitoyl interaction in the oprm complex. this conclusion is supported by computational modeling of the oprm homodimer. a cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure.
 a cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. we sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor, and if so, to assess the role of cholesterol in this class of g protein-coupled receptor signaling.  c was determined to be the palmitoylation site of oprm mutation of this cys to ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. in addition, both attenuation of receptor palmitoylation and inhibition of cholesterol synthesis impaired receptor signaling, possibly by decreasing receptor homodimerization and gαi coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer analyses. a computational model of the oprm homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate oprm homodimerization at the tmh4-tmh interface.  we demonstrate that c is the palmitoylation site of oprm and identify a cholesterol-palmitoyl interaction in the oprm complex. our findings suggest that this interaction contributes to oprm signaling by facilitating receptor homodimerization and g protein coupling. this conclusion is supported by computational modeling of the oprm homodimer. palmitoylationcholesterolhomodimerizationg protein coupling 