Stimulation and conformational change of G(o)alpha induced by GAP-43.
GAP-43 and G(o) are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G(o)alpha was highly purified from Escherichia coli cotransformed with pQE60 G(o)alpha and pBB131 (NMT). GAP-43 stimulated GTPgammaS binding to G(o)alpha and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G(o)alpha GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G(o)alpha. In the presence of GAP-43, fluorescence spectrum of G(o)alpha GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 +/-0.22) x10(5) to (4.1+/-0.43) x 10(5) (M(-1)). However, no obvious changes of fluorescence spectra of G(o)alpha GTPgammaS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G(o)alpha GDP so as to accelerate GDP release and subsequent GTPgammaS 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.
Zhang
Ling
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China.
Huang
Youguo
Y