Molecular biological analysis on regulatory mechanisms underlying the biosynthesis of GTP binding protein

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In this research, molecular mechanisms underlying the regulation of the biosynthesis of GTP-binding protein were investigated using primary cultured neurons prepared from the mouse cerebral cortex and the following results were obtained.1. A long-term exposure of neurons to atropine, a muscarinic receptor antagonist, produced the increase in muscarinic receptor as well as G protein. The latter increase was clarified by the measurement of [ ^3H] GppNHp binding and GTPase activity.2. Neurons used in this study possessed beta-adrenergic receptor and cyclic AMP (CAMP) generating system functionally coupled with beta-adrenergic receptor. In addition, the presence of MRNA for Gsalpha protein which is involved in the formation of CAMP, induced by the stimulation of beta-adrenergic receptor, was clarified in primary cultured neurons and its content was reached to a plateau in the early stage of neuronal development.3. A long-term exposure to propranolol, an antagonist specific for, beta-adrenergic receptor, induced an increase in the number of beta-adrenergic receptor. Under such conditions, cAMP formation in the presence of GppNHp, a non-hydrolyzed analogue of GTP, showed a significant increase in comparison with that in non-treated neurons. The increase in ADP-ribosylation of 45 KDa protein with [ ^3H] NAD in the presence of cholera toxin was also detected in propranolol-treated neurons. These results indicate that the increase of Gsalpha protein is associated with the up-regulation of beta-adrenergic receptor. On the other hand, the MRNA for Gsalpha protein had no significant changes. Based on these results, it is assumed to be necessary to examine the alteration of MRNA for Gsalpha protein in an early stage of the exposure to propranolol, because the turnover of MRNA is considered to be short. From these viewpoints, studies on the time course of the change of MRNA after the exposure to propranolol are underway in our laboratory.