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. 2005 Jun 21;102(25):9050-5.
doi: 10.1073/pnas.0501112102. Epub 2005 Jun 2.

A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers

Maria Waldhoer  1 , Jamie Fong, Robert M Jones, Mary M Lunzer, Shiv K Sharma, Evi Kostenis, Philip S Portoghese, Jennifer L Whistler
Affiliations

A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers

Maria Waldhoer et al. Proc Natl Acad Sci U S A. .

Abstract

There has been much speculation regarding the functional relevance of G protein-coupled receptor heterodimers, primarily because demonstrating their existence in vivo has proven to be a considerable challenge. Here we show that the opioid agonist ligand 6'-guanidinonaltrindole (6'-GNTI) has the unique property of selectively activating only opioid receptor heterodimers but not homomers. Importantly, 6'-GNTI is an analgesic, thereby demonstrating that opioid receptor heterodimers are indeed functionally relevant in vivo. However, 6'-GNTI induces analgesia only when it is administered in the spinal cord but not in the brain, suggesting that the organization of heterodimers is tissue-specific. This study demonstrates a proof of concept for tissue-selective drug _targeting based on G protein-coupled receptor heterodimerization. Importantly, _targeting opioid heterodimers could provide an approach toward the design of analgesic drugs with reduced side effects.

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Figures

Fig. 1.
Fig. 1.
6′-GNTI specifically _targets KOP/DOP heterodimers. (a) Coexpression of opioid receptor types in HEK-293 cells was visualized by immunofluorescent staining of cells stably coexpressing HA-DOP-R (DOP) and FLAG-KOP-R (KOP) with antibodies directed to the respective epitope tags. (Scale bar = 10 μm.) (b) Ratio of opioid receptor heterodimers versus homomers by serial immunoprecipitation (IP). Cells coexpressing both HA-DOP-R (HADOP) and FLAG-KOP-R (FKOP) or each individually were lysed, and the receptors were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted (IB) with anti-HA antibody to detect KOP/DOP heterodimers (lanes 5 and 7). After the FLAG immunoprecipitation (F), the lysates were immunoprecipitated with anti-HA antibody and immunoblotted for HA to detect the DOP homomers/monomers remaining after immunoprecipitation of the heterodimers. Compare lanes 5 (heterodimers) and 6 (homomers/monomers). (c)6′-GNTI induced Ca2+ release in HEK-293 cells expressing one or two opioid receptor types. Agonist-mediated intracellular Ca2+ release was measured in cells expressing the chimeric G protein Δ6-Gqi4-myr (200 ng for every 40,000 cells) and MOP-R (▵), DOP-R (□), or KOP-Rs (○) alone or MOP/DOP-Rs (▪), KOP/MOP-Rs (▴), or KOP/DOP-Rs (•). Intracellular Ca2+ release was measured in a Flex apparatus (Molecular Devices), where relative light units (RLU) = maximum Ca2+ peak/cell number × 1,000. Shown are representative curves carried out in duplicate (n = 4). (Inset) Structure of 6′-GNTI. (d) Effects of receptor type-selective antagonists on 6′-GNTI-induced Ca2+ release in cells expressing the KOP/DOP-R heterodimer. Cells expressing the KOP/DOP-R heterodimer were preincubated for 30 min with increasing doses of NTI (○) or NorBNI (•) and stimulated with 100 nM 6′-GNTI. Agonist-induced Ca2+ release was assessed as described in c. Data are mean ± SEM measured in duplicates. (e and f) Effect of 6′-GNTI (e) and KOP-R- and DOP-R-selective antagonists (f) on competition for [3H]diprenorphine binding to cells expressing KOP/DOP-R heterodimers. Whole-cell competition binding experiments were performed on cells stably expressing the KOP/DOP-R heterodimers. Cells were incubated with 1.5 nM [3H]diprenorphine and increasing amounts of 6′-GNTI (▪) (e), NorBNI (•)(f), or NTI (○)(f). Shown are representative curves carried out in duplicate (n = 4). Note that error bars in e are too small to be visualized.
Fig. 2.
Fig. 2.
Mechanism of 6′-GNTI agonism. (a)6′-GNTI agonist activity on heterodimers is not due to synergy/cooperativity between two opioid receptor types. HEK-293 cells expressing the KOP/DOP-R heterodimer were transiently transfected with the chimeric G protein Δ6-Gqi4-myr (200 ng for every 40,000 cells) and increasing amounts of DOP-R (Left), KOP-R (Center), or CCR5 (Right). Cells were stimulated with 100 nM 6′-GNTI, and intracellular Ca2+ release was measured as described for Fig. 1c. Maximum stimulation in the presence of 200 ng of control pcDNA3 (Con) was set at 100%. All data sets were subjected to a one-way ANOVA analysis with Bonferroni's multiple comparison posttest. *, A significant difference from the control with P < 0.05. (b) Effects of receptor type-selective antagonists on agonist-induced Ca2+ release in cells expressing the KOP/DOP-R heterodimer. Cells expressing the KOP/DOP-R heterodimer were preincubated for 30 min with increasing doses of NTI (○) or NorBNI (•) and stimulated with 1 nM U69,593 (Left) or 50 nM DPDPE (Right). Agonist-induced Ca2+ release was assessed as described for Fig. 1c. Shown are representative curves (mean ± SEM) of at least three experiments carried out in duplicate.
Fig. 3.
Fig. 3.
6′-GNTI mediated analgesia i.t. but not intracerebroventricularly in mice. (a) Analgesia was measured by a tail-flick assay after injection of 6′-GNTI either i.t. (▪) or intracerebroventricularly (□) at the doses indicated. (b) Antagonism of 6′-GNTI-induced i.t. analgesia. NorBNI (•), NTI (○), or KDN-21 (▴) at the doses indicated was administered with 1 nmol of 6′-GNTI per mouse (a dose that produces 80% analgesia).
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References

    1. Pin, J. P., Kniazeff, J., Binet, V., Liu, J., Maurel, D., Galvez, T., Duthey, B., Havlickova, M., Blahos, J., Prezeau, L. & Rondard, P. (2004) Biochem. Pharmacol. 68, 1565–1572. - PubMed
    1. Kniazeff, J., Bessis, A. S., Maurel, D., Ansanay, H., Prezeau, L. & Pin, J. P. (2004) Nat. Struct. Mol. Biol. 11, 706–713. - PubMed
    1. Nelson, G., Chandrashekar, J., Hoon, M. A., Feng, L., Zhao, G., Ryba, N. J. & Zuker, C. S. (2002) Nature 416, 199–202. - PubMed
    1. Filipek, S., Krzysko, K. A., Fotiadis, D., Liang, Y., Saperstein, D. A., Engel, A. & Palczewski, K. (2004) Photochem. Photobiol. Sci. 3, 628–638. - PubMed
    1. Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A. & Palczewski, K. (2004) FEBS Lett. 564, 281–288. - PMC - PubMed

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