Abstract
Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues usingin situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptorrelated proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.
Similar content being viewed by others
References
Miyata, A., Arimura, A., Dahl, R. R., Minamino, N., Uehara, A., Jiang, L., Culler, M. D., and Coy, D. H. (1989).Bioche. Biophys. Res. Commun. 164, 567–574.
Miyata, A., Jiang, L., Dahl, R. D., Kitada, C., Kubo, K., Fujino, M., Minamino, N., and Arimura, A. (1990).Biochem. Biophys. Res. Commun. 170, 643–648.
Gottschall, P. E., Tatsuno, I., Miyata, A., and Arimura, A. (1990).Endocrinology 127, 272–277.
Lam, H. C., Takahashi, K., Ghatei, M. A., Kanse, S. M., Polak, J. M., and Bloom, S. R. (1990).Eur. J. Biochem. 193, 725–729.
Cauvin, A., Robberecht, P., DeNeef, P., Gourlet, P., Vandermeers, A., Vandermeers-Piret, M. C., and Christophe, J. (1991).Regul. Pept. 35, 161–173.
Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993).Nature 365, 170–175.
Tatsuno, I., Somogyvari-Vigh, A., and Arimura, A. (1994).Peptides 15, 55–60.
Ohtaki, T., Watanabe, T., Ishibashi, Y., Kitada, C., Tsuda, M., Gottschall, P. E., Arimura, A., and Fujino, M. (1990).Biochem. Biophys. Res. Commun. 171, 838–844.
Gottschall, P. E., Tatsuno, I., and Arimura, A. (1991).FASEB J5, 194–199.
Tatsuno, I., Gottschall, P. E., and Arimura, A. (1991).Peptides 12, 617–621.
Hashimoto, H., Ishihara, T., Shigemoto, R., Mori, K., and Nagata, S. (1993).Neuron 11, 333–342.
Hosoya, M., Onda, H., Ogi, K., Masuda, Y., Miyamoto, Y., Ohtaki, T., Okazaki, H., Arimura, A., and Fujino, M. (1993).Biochem. Biophys. Res. Commun. 194, 133–143.
Morrow, J. A., Lutz, E. M., West, K. M., Fink, G., and Hamar, A. J. (1993).FEBS Lett. 329, 99–105.
Ogi, K., Miyamoto, Y., Masuda, Y., Habata, Y., Hosoya, M., Ohtaki, T., Masuo, Y., Onda, H., and Fujino, M. (1993).Biochem. Biophys. Res. Commun. 196, 1511–1521.
Pisegna, J. R. and Wank, S. A. (1993).Proc. Natl. Acad. Sci. USA 90, 6345–6349.
Svoboda, N., Tastenoy, M., Ciccarelli, R., Stievenart, M., and Christophe, J. (1993).Biochem. Biophys. Res. Commun. 195, 881–888.
Svoboda, N., Tastenoy, M., Van Rampelbergh, J., Goossens, J. F., De Neef, P., Waelbroeck, M., and Robberecht, P. (1994).Biochem. Biophys. Res. Commun. 205, 1617–1624.
Miyamoto, Y., Habata, Y., Ohtaki, T., Masuda, Y., Ogi, K., Onda, H., and Fujino, M. (1994).Biochem. Biophys. Acta 1218, 297–307.
Hashimoto, H., Yamamoto, K., Hagiwara, N., Ogawa, N., Nishino, A., Aino, H., Nogi, H., Imanishi, K., Matsuda, T., and Baba, A. (1996).J. Biochem. Biophys. Acta 1281, 129–133.
Pisegna, J. R. and Wank, S. A. (1996).J. Biol. Chem. 271, 17,267–17,274.
Pantaloni, C., Brabet, P., Bilanges, B., Dumuis, A., Houssami, S., Spengler, D., Bockaert, J., and Journot, L. (1996).J. Biol. Chem. 271, 22,146–22,151.
Chatterjee, T. K., Sharma, R. V., and Fisher, R. A. (1996).J. Biol. Chem. 271, 32,226–32,232.
Koves, K., Arimura, A., Somogyvari-Vigh, A., Vigh, S., and Miller, J. (1990).Endocrinology 127, 264–271.
Koves, K., Arimura, A., Gorcs, T. G., and Somogyvari-Vigh, A. (1991).Neuroendocrinology 54, 159–169.
Vigh, S., Arimura A., Koves, K., Somogyvari-Vigh, A., Sitton, J., and Fermin, C. D. (1991).Peptides 12, 313–318.
Yon, L., Freuilloley, M., Chartrel, N., Arimura, A., Conlon, J. M., Fournier, A., and Vaudry, H. (1992).J. Comp. Neurol. 324, 485–499.
Uddman, R., Luts, A., Arimura, A., and Sundler F. (1991).Cell Tissue Res. 265, 197–201.
Fridolf, T., Sundler, F., and Ahren, B. (1992).Cell Tissue Res. 269, 275–279.
Sundler, F., Ekblad, E., Absood, A., Hakanson, R., Koves, K., and Arimura, A. (1992).Neuroscience 46, 439–454.
Tabain, A., Chen, D., Hakanson, R., and Sunder, F. (1994).Neuroendocrinology 59, 113–119.
Arimura, A., Somogyvari-Vigh, A., Miyata, A., Mizuno, K., Coy, D. H., and Kitada, C. (1991).Endocrinology 129, 2787–2789.
Masuo, Y., Suzuki, N., Matsumoto, H., Tokito, F., Matsumoto, Y., Tsuda, M., and Fujino, M. (1993).Brain Res. 602, 57–63.
Shivers, B. D., Gorcs, T. J., Gottschall, P. E., and Arimura, A. (1991).Endocrinology 128, 3055–3065.
Masuo, Y., Ohtaki, T., Masuda, Y., Nagai, Y., Suno, M., Tsuda, M., and Fujino, M. (1991).Neurosci. Lett. 126, 103–106.
Masuo, Y., Tokito, F., Matsunoto, Y., Shimanoto, N., and Fujino, M. (1994).Neurosci. Lett. 170, 43–46.
Shioda, S., Shuto, Y., Somogyvari-Vigh, A., Legradi, G., Onda, H., and Arimura, A. (1994).Soc. Neurosci. Abstracts 20, 516, 221.14.
Hashimoto, H., Nogi, H., Mori, K., Ohishi, H., Shigemoto, R., Yamamoto, K., Matsuda, T., Mizuno, N., Nagata, S., and Baba, A. (1996).J. Comp. Neurol. 371, 567–577.
Suda, K., Smith, D. M., Ghatei, M. A., Murphy, J. K., and Bloom, S. R. (1991).J. Clin. Endocrinol. Metab. 72, 958–964.
Basile, M., Gonzalezk, B. J., Leroux, P., Jeandel, L., Gourner, A., and Vaudry, H. (1993).Neuroscience 57, 329–338.
Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., and Nagata, S. (1992).Neuron 8, 811–819.
Mayo, K. E. (1992).Mol. Endocrinol. 6, 1734–1744.
Engvall, E. and Perlmann, P. (1971).Immunohistochemistry 8, 871–874.
Kruseman, A. N. (1983).J. Clin. Pathol. 36, 406–410.
Ohtaki, T., Masuda, Y., Ishibashi, Y., Kitada, C., Arimura, A., and Fujino, M. (1993).J. Biol. Chem. 268, 26,650–26,657.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, M., Shioda, S., Somogyvári-Vigh, A. et al. Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors. Endocr 7, 183–190 (1997). https://doi.org/10.1007/BF02778140
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02778140