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The Pharmaceutical Society of Korea (대한약학회)
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Homo- or Hetero-Dimerization of Muscarinic Receptor Subtypes is Not Mediated by Direct Protein-Protein Interaction Through Intracellular and Extracellular Regions

  • Kang, Yun-Kyung (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Center for Cell Signaling Research, Ewha Womans University) ;
  • Yoon, Tae-Sook (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Center for Cell Signaling Research, Ewha Womans University) ;
  • Lee, Kyung-Lim (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Center for Cell Signaling Research, Ewha Womans University) ;
  • Kim, Hwa-Jung (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Center for Cell Signaling Research, Ewha Womans University)
  • Published : 2003.10.01
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Abstract

The oligomerization of G-proteincoupled receptors (GPCRs) has been shown to occur by various mechanisms, such as via disulfide covalent linkages, non covalent (ionic, hydrophobic) interactions of the N-terminal, and/or transmembrane and/or intracellular domains. Interactions between GPCRs could involve an association between identical proteins (homomers) or non-identical proteins (heteromers), or between two monomers (to form dimers) or multiple monomers (to form oligomers). It is believed that muscarinic receptors may also be arranged into dimeric or oigomeric complexes, but no systematic experimental evidence exists concerning the direct physical interaction between receptor proteins as its mechanism. We undertook this study to determine whether muscarinic receptors form homomers or a heteromers by direct protein-protein interaction within the same or within different subtypes using a yeast two-hybrid system. Intracellular loops (i1, i2 and i3) and the C-terminal cytoplasmic tails (C) of human muscarinic (Hm) receptor subtypes, Hm1, Hm2 and Hm3, were cloned into the vectors (pB42AD and pLexA) of a two-hybrid system and examined for heteromeric or homodimeric interactions between the cytoplasmic domains. No physical interaction was observed between the intracellular domains of any of the Hm/Hm receptor sets tested. The results of our study suggest that the Hm1, Hm2 and Hm3 receptors do not form dimers or oligomers by interacting directly through either the hydrophilic intracellular domains or the C-terminal tail domains. To further investigate extracellular domain interactions, the N-terminus (N) and extracellular loops (o1 and o2) were also cloned into the two-hybrid vectors. Interactions of Hm2N with Hm2N, Hm2o1, Hm2o2, Hm3N, Hm3o1 or Hm3o2 were examined. The N-terminal domain of Hm2 was found to have no direct interaction with any extracellular domain. From our results, we excluded the possibility of a direct interaction between the muscarinic receptor subtypes (Hm1, Hm2 and Hm3) as a mechanism for homo- or hetero-meric dimerization/oligomerization. On the other hand, it remains a possibility that interaction may occur indirectly or require proper conformation or subunit formation or hydrophobic region involvement.

Keywords

References

  1. Abe, J., Suzuki, H., Notoya, M., Yamamoto, T., and Hirose, S., Ig-hepta, a novel member of the G protein-coupled heptahelical receptor (GPCR) family that has immunoglobulin-like repeats in a long N-terminal extracellular domain and defines a new subfamily of GPCRs. J. Biol. Chem., 274, 19957-19964 (1999) https://doi.org/10.1074/jbc.274.28.19957
  2. Angers, S., Salshpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M., Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA, 97, 3684-3689 (2000) https://doi.org/10.1073/pnas.060590697
  3. Bai, M., Trivedi, S., and Brown, E. M., Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 Cells. J. Biol. Chem., 273, 23605-23610 (1998) https://doi.org/10.1074/jbc.273.36.23605
  4. Bouvier, M., Oligomerization of$\gamma$protein coupled transmitter receptors. Nat. Rev. Neurosci., 2, 274-286 (2001) https://doi.org/10.1038/35067575
  5. Chidiac, P., Green, M. A., Pawagi, A. B., and Wells, J. W., Cardiac muscarinic receptors cooperativity as the basis for multiple states of affinity. Biochemistry, 36, 7361-7379 (1997) https://doi.org/10.1021/bi961939t
  6. Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S., The two-hybrid system; A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA, 88, 9578-9582 (1991) https://doi.org/10.1073/pnas.88.21.9578
  7. Cornea, A., Janovick, J. A., Maya-Nunez, G., and Conn, P. M., Gonadotropin-rleasing hormone receptor microaggregation. J. Biol. Chem., 276, 2153-2158 (2001) https://doi.org/10.1074/jbc.M007850200
  8. Cvejic, S. and Devi, L. A., Dimerization of the d opioid receptor: Implication for a role in receptor internalization. J. Biol. Chem., 272, 26959-26964 (1997) https://doi.org/10.1074/jbc.272.43.26959
  9. Fukushima, Y., Asano, T., Saitoh, T., Anai, M., Funaki, M., Ogihara, T., Katagiri, H., Matsuhashi, N., Yazaki, Y., and Sugano, K., Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells. FEBS lett., 409, 283-286 (1997) https://doi.org/10.1016/S0014-5793(97)00531-0
  10. Gomes, I., Jordan, B. A., Gupta, A, Rios, C., Trapaidze, N., and Devi, L. A., G protein coupled receptor dimerization: implications in modulating receptor function. J. Mol. Med., 79, 226-242 (2001) https://doi.org/10.1007/s001090100219
  11. Gouldson, P. R., Snell, C. R., Bywater, R. P., Higgs, C., and Reynolds, C. A., Domains swapping in G protein-coupled receptor dimers. Protein. Eng., 11, 1181-1193 (1998) https://doi.org/10.1093/protein/11.12.1181
  12. Gouldson, P. R., Higgs, C., Smith, R. E. Dean, M. K. Gkoutos, G. V., and Reynolds, C. A., Dimerization and domain swapping in$\gamma$protein-coupled receptors: a computational study. Neuropsychopharmacology, 23, S60-S76 (2000) https://doi.org/10.1016/S0893-133X(00)00153-6
  13. Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M., A peptide derived from a ${\beta}_2$-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem., 271, 16384-16392 (1996) https://doi.org/10.1074/jbc.271.27.16384
  14. Hirshberg, B. T. and Schimerlik, M. I., A kinetic model for oxotremorine M binding to recombinant porcine m2 muscarinic receptors expressed in chinese hamster ovary cells. J. Biol. Chem., 269, 26127-26135 (1994)
  15. Jones, K. A., Borowsky, B., Tamm, J. A., Craig, D., Durkin, M. M., Dai, M., Yao, W.-J., Johnson, M., Gunwaldsen, C., Huang, L. Y., Tang, C., Shen, Q., Salon, J. A., Morse, K., Laz, T. M., Smith, K. E., Nagarathnam, N., Noble, S. A., Branchek, T., and Gerald, C., GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature, 396, 674-679 (1998) https://doi.org/10.1038/25348
  16. Jordan, B. A. and Devi, L. A.,$\gamma$protein-coupled receptor heterodimerization modulates receptor function. Nature, 399, 697-700 (1999) https://doi.org/10.1038/21441
  17. Kapupman, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R., Karschin, A., and Bettler, B., GABAB-receptor subtypes assemble into functional heteromeric complexes. Nature, 396, 683-687 (1998) https://doi.org/10.1038/25360
  18. Kuner, R., Kohr, G., Grunewald, S., Eisenhardt, G., Bach, A., and Kornau, H. C., Role of heteromer formation in GABAB receptor function. Science, 283, 74-77 (1999) https://doi.org/10.1126/science.283.5398.74
  19. Li, M., Modulation of dopamine D2 receptor signaling by actin-binding protein (ABP-280). Mol. Pharmacol., 57, 446-452 (2000) https://doi.org/10.1124/mol.57.3.446
  20. Maggio, R., Vogel, Z., and Wess, J., Co-expression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular 'cross-talk' between$\gamma$protein linked receptors. Proc. Natl. Acad. Sci. USA, 90, 3103-3107 (1993a) https://doi.org/10.1073/pnas.90.7.3103
  21. Maggio, R., Vogel, Z., and Wess, J., Reconstitution of functional muscarinic receptors by co-expression of amino-terminal and carboxy terminal receptor fragments. FEBS Lett., 319, 195-200 (1993b) https://doi.org/10.1016/0014-5793(93)80066-4
  22. Maggio, R., Barbier, P., Fornai, F., and Corsini, G. U., Functional role of the third cytoplamic loop in muscarinic receptor dimerization. J. Biol. Chem., 271, 31055-31060 (1996) https://doi.org/10.1074/jbc.271.49.31055
  23. McVey, M., Ramsay, D., Kellet, E., Rees, S., Wilson, S., Pope, A. J., and Milligan, G., Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer: The human delta opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy. J. Biol. Chem., 276, 14092-14099 (2001) https://doi.org/10.1074/jbc.M008902200
  24. Milligan, G. and White, J. H., Protein-protein interactions at Gprotein-coupled receptors. Trends. Pharmacol. Sci., 22, 513-518 (2001) https://doi.org/10.1016/S0165-6147(00)01801-0
  25. Overton, M. C. and Blumer, K. J.,$\gamma$protein-coupled receptors function as oligomers in vivo. Curr. Biol., 10, 341-344 (2000) https://doi.org/10.1016/S0960-9822(00)00386-9
  26. Pace, A. J., Gama, L., and Breitwizer, G. E., Dimerization of the calcium-sensing receptor occurs within the extracellular domain and is eliminated by Cys(R)Ser mutations at Cys101 and Cys236. J. Biol. Chem., 274, 11629-11634 (1999) https://doi.org/10.1074/jbc.274.17.11629
  27. Potter, L. T., Ballesteros, L. A., Bichajian, L. H., Ferrendelli, C. A., Fisher, A., Hanchett, H. E., and Zhang, R., Evidence of paired M2 muscarinic receptors. Mol. Pharmacol., 39, 211-221 (1991)
  28. Rocheville, M., Lange, D. C., Patel, S. C., and Patel, Y. C., Receptors for dopamine and somatostatin: formation of heterooligomers with enhanced functional activity. Science, 288, 154-157 (2000) https://doi.org/10.1126/science.288.5463.154
  29. Romano, C., Yang, W. L., and O'Malley, K. L., Metabotropic glutamate receptor 5 is a disulfide-linked dimmer. J. Biol. Chem., 271, 28612-28616 (1996) https://doi.org/10.1074/jbc.271.45.28612
  30. Ward, D. T., Brown, E. M., and Harris, H. W., Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. J. Biol. Chem., 273, 14476-14483 (1998) https://doi.org/10.1074/jbc.273.23.14476
  31. Wess, J., Mutational analysis of muscarinic acetylcholiner receptors: Structural basis of ligand/receptor/G protein interaction. Life Sci., 53, 1447-1463 (1993) https://doi.org/10.1016/0024-3205(93)90618-D
  32. White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H., Heterodimerization is required for the formation of a functional GABAB receptor. Nature, 396, 679¯ 682 (1998)
  33. Wreggett, K. A. and Wells, J. W., Cooperactivity manifest in the binding properties of purified cardiac muscarinic receptors. J. Biol. Chem., 270, 22488-22499 (1995) https://doi.org/10.1074/jbc.270.38.22488
  34. Yoon, T. S. and Lee, K. L., Isoform-specific interaction of the cytoplasmic domain of Na,K-ATPase. Mol. Cell, 8, 606-613 (1998)
  35. Zeng, F. Y. and Wess, J., Identification and molecular characterization of m3 muscarinic receptor dimmers. J. Biol. Chem., 274, 19487-19489 (1999) https://doi.org/10.1074/jbc.274.27.19487