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Supplementary data
Supplementary data associated with this article can be found,
References and notes
1. Kotani, M.; Detheux, M.; Vandenbogaerde, A.; Communi, D.; Vanderwinden, J.-
M.; Le Poul, E.; Brézillon, S.; Tyldesley, R.; Suarez-Huerta, N.; Vandeput, F.;
Blanpain, C.; Schiffmann, S. N.; Vassart, G.; Parmentier, M. J. Biol. Chem. 2001,
276, 34631.
2. Muir, A. I.; Chamberlain, L.; Elshourbagy, N. A.; Michalovich, D.; Moore, D. J.;
Calamari, A.; Szekeres, P. G.; Sarau, H. M.; Chambers, J. K.; Murdock, P.;
Steplewski, K.; Shabon, U.; Miller, J. E.; Middleton, S. E.; Darker, J. G.; Larminie,
C. G. C.; Wilson, S.; Bergsma, D. J.; Emson, P.; Faull, R.; Philpott, K. L.; Harrison,
D. C. J. Biol. Chem. 2001, 276, 28969.
3. Lee, D. K.; Nguyen, T.; O’Neill, G. P.; Cheng, R.; Liu, Y.; Howard, A. D.; Coulombe,
N.; Tan, C. P.; Tang-Nguyen, A.-T.; George, S. R.; O’Dowd, B. F. FEBS Lett. 1999,
446, 103.
4. Ohtaki, T.; Shintani, Y.; Honda, S.; Matsumoto, H.; Hori, A.; Kanehashi, K.; Terao,
Y.; Kumano, S.; Takatsu, Y.; Masuda, Y.; Ishibashi, Y.; Watanabe, T.; Asada, M.;
Yamada, T.; Suenaga, M.; Kitada, C.; Usuki, S.; Kurokawa, T.; Onda, H.;
Nishimura, O.; Fujino, M. Nature 2001, 411, 613.
5. Clements, M. K.; McDonald, T. P.; Wang, R.; Xie, G.; O’Dowd, B. F.; George, S. R.;
Austin, C. P.; Liu, Q. Biochem. Biophys. Res. Commun. 2001, 284, 1189.
6. De Roux, N.; Genin, E.; Carel, J.-C.; Matsuda, F.; Chaussain, J.-L.; Milgrom, E.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10972.
Figure 3. Photocrosslinking experiments of GPR54 with Kp-14-based probes. (A)
GPR54-expressing HEK293 cells were labeled by Kp-14-based probes. Biotin on the
crosslinked GPR54 was detected by Western blot analysis. (B) GPR54-expressing
HEK293 cells were exposed to W7C (500 nM) in the presence (+) or absence (À) of
unlabeled Kp-10 (10 lM) for the competitive experiments.
7. Seminara, S. B.; Messager, S.; Chatzidaki, E. E.; Thresher, R. R.; Acierno, J. S., Jr.;
Shagoury, J. K.; Bo-Abbas, Y.; Kuohung, W.; Schwinof, K. M.; Hendrick, A. G.;
Zahn, D.; Dixon, J.; Kaiser, U. B.; Slaugenhaupt, S. A.; Gusella, J. F.; O’Rahilly, S.;
Carlton, M. B. L.; Crowley, W. F., Jr.; Aparicio, S. A. J. R.; Colledge, W. H. N. Eng. J.
Med. 2003, 349, 1614.
8. Funes, S.; Hedrick, J. A.; Vassileva, G.; Markowitz, L.; Abbondanzo, S.; Golovko,
A.; Yang, S.; Monsma, F. J.; Gustafson, E. L. Biochem. Biophys. Res. Commun. 2003,
312, 1357.
9. Gottsch, M. L.; Cunningham, M. J.; Smith, J. T.; Popa, S. M.; Acohido, B. V.;
Crowley, W. F.; Seminara, S.; Clifton, D. K.; Steiner, R. A. Endocrinology 2004,
145, 4073.
10. Matsui, H.; Takatsu, Y.; Kumano, S.; Matsumoto, H.; Ohtaki, T. Biochem. Biophys.
Res. Commun. 2004, 320, 383.
Figure 4. Deglycosylation experiments of labeled GPR54 by W47C (left) and W7C
(right). After photocrosslinking, the cell lysate was incubated in the presence (+) or
absence (À) of PNGase F. Biotin on the crosslinked GPR54 was detected by Western
blot analysis.
11. Irwig, M. S.; Fraley, G. S.; Smith, J. T.; Acohido, B. V.; Popa, S. M.; Cunningham,
M. J.; Gottsch, M. L.; Clifton, D. K.; Steiner, R. A. Neuroendocrinology 2004, 80,
264.
12. Shahab, M.; Mastronardi, C.; Seminara, S. B.; Crowley, W. F.; Ojeda, S. R.; Plant,
T. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2129.
13. Franceschini, I.; Lomet, D.; Cateau, M.; Delsol, G.; Tillet, Y.; Caraty, A. Neurosci.
Lett. 2006, 401, 225.
14. Oakley, A. E.; Clifton, D. K.; Steiner, R. A. Endocrinol. Rev. 2009, 30, 713.
15. Ohkura, S.; Uenoyama, Y.; Yamada, S.; Homma, T.; Takase, K.; Inoue, N.; Maeda,
K.-I.; Tsukamura, H. Peptides 2009, 30, 49.
16. Tomikawa, J.; Homma, T.; Tajima, S.; Shibata, T.; Inamoto, Y.; Takase, K.; Inoue,
N.; Ohkura, S.; Uenoyama, Y.; Maeda, K.; Tsukamura, H. Biol. Reprod. 2010, 82,
313.
17. Kinsey-Jones, J. S.; Li, X. F.; Luckman, S. M.; O’Byrne, K. T. Endocrinology 2008,
149, 1004.
18. Ohkura, S.; Takase, K.; Matsuyama, S.; Mogi, K.; Ichimaru, T.; Wakabayashi, Y.;
Uenoyama, Y.; Mori, Y.; Steiner, R. A.; Tsukamura, H.; Maeda, K.-I.; Okamura, H.
J. Neuroendocrinol. 2009, 21, 813.
19. Lee, J. H.; Miele, M. E.; Hicks, D. J.; Phillips, K. K.; Trent, J. M.; Weissman, B. E.;
Welch, D. R. J. Natl. Cancer Inst. 1996, 88, 1731.
20. Asami, T.; Nishizawa, N.; Ishibashi, Y.; Nishibori, K.; Horikoshi, Y.; Matsumoto,
H.; Ohtaki, T.; Kitada, C. Bioorg. Med. Chem. Lett. 2012, 22, 6328.
21. Asami, T.; Nishizawa, N.; Ishibashi, Y.; Nishibori, K.; Nakayama, M.; Horikoshi,
Y.; Matsumoto, S.; Yamaguchi, M.; Matsumoto, H.; Tarui, N.; Ohtaki, T.; Kitada,
C. Bioorg. Med. Chem. Lett. 2012, 22, 6391.
22. Kitada, C.; Asami, T.; Nishizawa, N. U.S. Patent 7,625,869, 2006.
23. Asami, T.; Nishizawa, N. U.S. Patent 7,960,348, 2007.
24. Niida, A.; Wang, Z.; Tomita, K.; Oishi, S.; Tamamura, H.; Otaka, A.; Navenot, J.;
Broach, J. R.; Peiper, S. C.; Fujii, N. Bioorg. Med. Chem. Lett. 2006, 16, 134.
25. Tomita, K.; Niida, A.; Oishi, S.; Ohno, H.; Cluzeau, J.; Navenot, J.; Wang, Z.;
Peiper, S. C.; Fujii, N. Bioorg. Med. Chem. 2006, 14, 7595.
To further rationalize the identification of GPR54 using Kp-54- and
Kp-14-based photoaffinity probes, deglycosylation experiments of
the crosslinked 65 kDa protein were carried out. After UV light
exposure in the presence of W47C or W7C (500 nM) for photo-
crosslinking, the cell lysates were subjected to deglycosylation
treatment using peptide N-glycosidase (PNGase F). SDS–PAGE
separation followed by Western blot analysis demonstrated that
the 65 kDa band was shifted to a mass indicative of a protein
that had undergone removal of N-linked oligosaccharide chains
(Fig. 4). This observation provides further evidence that the labeled
protein with the probes was GPR54.
In conclusion, we have designed and synthesized photoaffinity
probes based on two endogenous kisspeptin sequences (Kp-54
and Kp-14). Six Kp-54-based probes (P30C, L35C, K40C, Y45C,
N46C and W47C) and one Kp-14-based probe (W7C) clearly de-
tected GPR54 on living cells in a specific manner, suggesting these
residues constitute the secondary interactive sites of Kp-54 and
Kp-14 for receptor binding to GPR54. These probes should be
promising tools to identify the distributions of possible kisspeptin
receptors, including GPR54.
Acknowledgments
26. Tomita, K.; Oishi, S.; Ohno, H.; Peiper, S. C.; Fujii, N. J. Med. Chem. 2008, 51,
7645.
27. Fleming, S. A. Tetrahedron 1995, 51, 12479.
28. Brunner, J. Annu. Rev. Biochem. 1993, 62, 483.
29. Sadakane, Y.; Hatanaka, Y. Anal. Sci. 2006, 22, 209.
30. It cannot be ruled out that the N-terminal biotin tag in the probes prevented
the receptor binding itself.
31. Lyubimov, Y.; Engstrom, M.; Wurster, S.; Savola, J.-M.; Korpi, E. R.; Panula, P.
Neuroscience 2010, 170, 117.
32. Oishi, S.; Misu, R.; Tomita, K.; Setsuda, S.; Masuda, R.; Ohno, H.; Naniwa, Y.;
Ieda, N.; Inoue, N.; Ohkura, S.; Uenoyama, Y.; Tsukamura, H.; Maeda, K.-I.;
Hirasawa, A.; Tsujimoto, G.; Fujii, N. ACS Med. Chem. Lett. 2011, 2, 53.
This work was supported by Grants-in-Aid for Scientific Re-
search and Platform for Drug Discovery, Informatics, and Structural
Life Science from the Ministry of Education, Culture, Sports, Sci-
ence, and Technology of Japan; and Research Program on Innova-
tive Technologies for Animal Breeding, Reproduction, and Vaccine
Development (REP-2003) from the Ministry of Agriculture, For-
estry and Fisheries of Japan. R.M. is grateful for Research Fellow-
ships from the JSPS for Young Scientists.