dGTP-CONTAINING MICROTUBULES IN NEURITES OF NGF-TREATED NEURONS

Tubulin is typically considered to be a guanine nucleotide-binding protein that contains both exchangeable and nonexchangeable sites for GTP and/or GDP. During the course of studies on the nucleotide composition of intact taxol-stabilized microtubules from neuronal cells, we discovered the nearly stoichiometric presence of 2'-deoxy-GTP in the non-exchangeable sites of neuritic microtubules. Indeed, after treatment of PC12 pheochromocytoma cells or embryonic chick DRG neurons with nerve growth factor, we observed that the induced extension of neurites paralleled the increased dGTP content of isolated intact microtubules. (By contrast, adult bovine brain MTs lack dGTP.)

We are currently investigating the events that lead to dGTP accumulation in neuritic microtubules using the following hypothesis: NGF-mediated changes in tubulin biosynthesis occur on the same time-scale as NGF-induced depletion of cellular GTP and GDP pools through (a) increased Ras GTPase (which converts GTP to GDP), (b) activation of ribonucleotide reductase (which irreversibly converts GDP to dGDP), and (c)coordinate control of de novo GMP synthesis (blocking GDP and GTP synthesis). Such a mechanism may serve to reset the G-protein poise in growth factor-activated cells such as NGF-sensitive cells of neural crest origin.

This work is supported by NIH grant P01CA497120001.

Rat PC12 Pheochromocytoma Cells Cultured in the Absence (top panel) and Presence (bottom panel) of Mouse Nerve Growth Factor

PC-12 Cells Cultured in the Absence of Mouse NGF
PC12 Cells Cultured in the Presence of Mouse NGF