Abstract
A preliminary study on the interaction of G protein (guanine triphosphate binding protein) β1γ2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gβ1γ2. The cell membrane containing Gβ1γ2 was isolated through affinity chromatography column with Ni-NTAagarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gβ1γ2 could significantly stimulate AC2 activity. The interaction of β1γ2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gβ1γ2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gβ1γ2 was the same as AC2 activity domain which was stimulated by Gβ1γ2.
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References
Chen Julian, Weng Ge-zhi, Ni Hanxiang, The advancement of G protein and coupled signal transduction pathways, Chinese J. Biotechnology (in Chinese), 2001, 17(2): 113–117.
Ford, C. E., Skiba, N. P., Bae, H. et al., Molecular basis for interactions of G protein βγ subunits with effectors, Science, 1998, 280(5367): 1271–1274.
Alfredoulloa, A. P., Conn, M., G-protein coupled receptors and the G-protein family, in Handbook of Physiology, Section 7: The Endocrine system molecular cell endocrinology, Chapter 6 Volume I: Cellular Endocrinology (ed. Goodman, M. H.), New York: Oxford University Press, 1998.
Schuck, P., Kinetics of ligand binding to receptor immobilized in a polymer matrix as detected with an evanescent wave biosensor (I)—A computer simulation of the influence of mass transport, Biophys. J., 1996, 70: 1230–1249.
Schuck, P., Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules, Annu. Rev. Biophy. Biomol. Struct., 1997, 26: 541–566.
Weng, G., Chen, Y., Iyengar, R., Mammalian Gs-stimulated adenylyl cyclases, in Handbook of Physiology, Section 7: The endocrin. system, Vol. I: Cellular Endocrinology (ed. Conn, P. M.), New York: Oxford Univ. Press, 1998, 165–176.
Simonds, W. F., G protein regulation of adenylate cyclase, Trends in Pharmacological Sciences, 1999, 20(2): 66–72.
Yan, S., Hahn, D., Huang, Z. et al., Two cytoplasmic domains of mammalian adenylyl cyclase form a Gsα-and forskolin-activated enzyme in vitro, J. Biol. Chem., 1996, 271(18): 10941–10945.
Tang, W., Gilman, A. G., Construction of a soluble adenylyl cyclase activated by Gsα-and forskolin, Science, 1995, 268: 1769–1772.
Chen, J., De Vivo, M., Dingus, J. et al., A region of adenylyl cyclase 2 critical for regulation by G protein Gγ subunits, Science, 1995, 268: 1166–1169.
Weng, G., Li, J., Dingus, J., Gβ subunit interacts with a peptide encoding region 956–982 of adenylyl cyclase 2, Cross-linking of the peptide to free Gβγ but not the heterotrimer, J. Biol. Chem., 1996, 271(43): 26445–26448.
Kozasa, T., Gilman, A. G., Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits, J. Biol. Chem., 1995, 270(4): 1734–1741.
Tang, W., Hurley, J., Type specific regulation of mammalian adenylyl cyclases, Science, 1998, 54 (2): 231–240.
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Chen, J., Ni, H., Sun, J. et al. G protein β1γ2 subunits purification and their interaction with adenylyl cyclase. Sci. China Ser. C.-Life Sci. 46, 212–223 (2003). https://doi.org/10.1360/03yc9023
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DOI: https://doi.org/10.1360/03yc9023