RESUMO
BACKGROUND: Rasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored. RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation. CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.
RESUMO
Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.
