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Investigations of MOR and DOR trafficking and crosstalk
Jessica Anand
Opioid drugs interact with opioid receptors, which belong to the superfamily of GPCRs. There are three different types of opioid receptor, mu, delta and kappa. Opioid drugs bind with varying affinities to these receptors and the physiological effects of opioid drugs vary depending on which class of receptor they bind most strongly. However, the range of physiological effects is much greater than the effects determined for each type of receptor alone. This may indicate that ligands can interact with combinations of receptors and produce varying physiological effects. It has been suggested that a heterodimer of the mu and delta opioid receptors would allow for effective treatment of pain without addiction liability when treated with a mu agonist and a delta antagonist. Opioid receptor heterodimers may influence the development of tolerance to and dependence on opioid drugs such as morphine. Understanding how opioid receptors organize within the cell could allow us to use a rational approach toward designing opioid drugs which are analgesic, but do not produce tolerance, dependence and addiction. This idea is explore further in “development of mixed efficacy opioid ligands.”

Our goal is to design potent, specific, fluorescent opioid ligands with which to probe opioid receptor crosstalk and co-trafficking. By designing specific ligands which can be visualized fluorescently can have examined systems which force a monomeric state for receptors as well as live cell systems which resemble endogenous conditions.

We have already generated two selective fluorescent ligands, a mu agonist and a delta antagonist (developed by former group member, Dr. Mary Divin), and imaged live cells using confocal microscopy. We have demonstrated that our fluorescent delta antagonist binds to receptors on the surface and that our fluorescent mu agonist both binds on to surface receptors and causes internalization.


Figure 1: Imaging of mu opioid receptors in living cells using fluorescent agonist. 100nM [Lys 7, Cys(Cy3)8] dermorphin administered to C6MOR cells imaged 20 minutes after addition of drug at 37ºC. (A) Alexa Fluor 488 conjugated to wheat germ agglutinin binds to the plasma membrane in yellow. Hoescht nuclear stain is shown in cyan. (B) [Lys7, Cys(Cy3)8] dermorphin is shown internalized in red.



Figure 2: Imaging of delta opioid receptors in living cells using fluorescent antagonist. 100nM Dmt-Tic-Lys(Cy5)-OH administered to C6DOR cells imaged after 20 minutes at 37ºC. (A) Alexa Fluor 488 conjugated to wheat germ agglutinin binds to the plasma membrane in yellow. Hoescht nuclear stain is shown in cyan. (B) Dmt-Tic-Lys(Cy5)-OH is shown bound to the plasma membrane in green.


In the future we plan to explore live cell systems which express both the mu and delta opioid receptors to determine if different combinations change the trafficking patterns of the mu and delta receptors. The results from these experiments have the potential to shed light on how tolerance and addiction to opioid drugs develops and may help with the rational design of analgesic drugs without addiction liability.


Related Publications

Kuszak AJ, Pitchiaya S, Anand JP, Mosberg HI, Walter NG, Sunahara RK.
Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2.
J Biol Chem, 284(39): 26732-26741 (2009)

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Design and synthesis of biologically active opioid peptides and peptidomimetics
Investigations of MOR and DOR trafficking and crosstalk
Development of mixed efficacy opioid ligands
Peptides and proteins in membranes
Homology modeling of GPCRs, important drug targets
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