Abstract
GAP-43 and Go are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated Goα was highly purified from Escherichia coli cotransformed with pQE60 Goα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to Goα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that Goα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of Goα. In the presence of GAP-43, fluorescence spectrum of Goα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×105 to (4.1±0.43) × 105 (M−1). However, no obvious changes of fluorescence spectra of Goα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of Goα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.
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Zhang, L., Huang, Y. Stimulation and conformational change of Goα induced by GAP-43. Sci. China Ser. C.-Life Sci. 46, 174–183 (2003). https://doi.org/10.1360/03yc9019
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DOI: https://doi.org/10.1360/03yc9019