Abstract
The 78-kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bind gastrin, but the affinity of the N-terminal half for gastrin is 7.2-fold higher than the affinity of the C-terminal half. In order to define the gastrin-binding sites of the GBP in greater detail, we have constructed a truncation mutant lacking residues 221-318 of the N-terminal domain and a series of point mutants in which the lysine residues in the first 220 residues of the N-terminal domain were mutated to arginine residues. The effect of these mutations on both the extent of covalent cross-linking of iodinated gastrin2,17 and on the affinity for gastrin17 was investigated. Deletion of residues 221-318 of the GBP decreased the affinity 5.5-fold and reduced, but did not abolish, the extent of covalent cross-linking. Mutation of the 17 lysines in residues 1-220 of the GBP decreased the affinity for gastrin between 1.7- and 3.5-fold and in some cases reduced, but did not abolish, the extent of covalent cross-linking. We conclude that one or more lysine residues are involved in binding of gastrin to the GBP, but that no single lysine residue is the preferred target for covalent cross-linking of iodinated gastrin2,17 to the GBP.
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REFERENCES
Baldwin, G. S. (1993). Comp.Biochem.Physiol. 104B, 55–61.
Baldwin, G. S. (1994). Proc.Natl.Acad.Sci.USA 91, 7593–7597.
Baldwin, G. S. (1995). FEBS Lett. 359, 97–100.
Baldwin, G. S., Chandler, R., Scanlon, D. B., and Weinstock, J. (1986). J.Biol.Chem. 261, 12252–12257.
Baldwin, G. S., Chandler, R., Grego, B., Rubira, M. R., Seet, K. L., and Weinstock, J. (1994). Int.J.Biochem. 26, 529–538.
Baldwin, G. S., Murphy, V. J., Yang, Z., and Hashimoto, T. (1998). J.Pharm.Exp.Ther. 286, 1110–1114.
Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996). EMBO J. 15, 5135–5145.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989). Gene 77, 51–59.
Hoosein, N. M., Kiener, P. A., Curry, R. C., Rovati, L. C., McGilbra, D. K., and Brattain, M. G. (1988). Cancer Res. 48, 7179–7183.
King, J. and Laemmli, U. K. (1971). J.Mol.Biol. 62, 465–473.
Mantamadiotis, T., Sobieszczuk, P., Weinstock, J., and Baldwin, G. S. (1993). Biochim.Biophys.Acta 1170, 211–215.
Murphy, V. J., Mantamadiotis, T., and Baldwin, G. S. (1996). Int.J. Biochem.Cell Biol. 28, 1233–1240.
Seet, L., Fabri, L., Nice, E. C., and Baldwin, G. S. (1988). Biomed. Chromatogr. 2, 159–163.
Thumwood, C. M., Hong, J., and Baldwin, G. S. (1991). Exp.Cell Res. 192, 189–192.
Uchida, Y., Izai, K., Orii, T., and Hashimoto, T. (1992). J.Biol.Chem. 267, 1034–1041.
Weinstock, J. and Baldwin, G. S. (1988). Cancer Res. 48, 932–937.
Yang, S. Y., He, X.-Y., and Schulz, H. (1995). Biochemistry 34, 6441–6447.
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Rorison, K.A., Lee, D.J. & Baldwin, G.S. Mutation of Lysine Residues of the 78-kDa Gastrin-Binding Protein Reduces Gastrin Binding. J Protein Chem 20, 345–351 (2001). https://doi.org/10.1023/A:1012220501939
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DOI: https://doi.org/10.1023/A:1012220501939