The differential effects of beta-2-adrenergic and fibroblast growth factor receptor activation neuronal process outgrowth

Abstract

Clonal cell lines were established from the embryonic day (E) 15 rat basal forebrain using retroviral transduction of a temperature sensitive mutant of the SV40 large tumor antigen to study signals that direct the earliest events of neuronal process outgrowth. One neuronal progenitor cell line, AS583-8.E4.22, extends neurites in response to catecholamines and basic fibroblast growth factor (bFGF). The neurite outgrowth in response to catecholamines is mediated by ss2-adrenergic receptors and characterized by the rapid formation of multiple branched processes with large growth cone-like structures. Developing neurons from primary cultures of E15 basal forebrain were also found to respond to ss-adrenergic receptor agonists by enhancing growth cone formation. bFGF, in contrast, induces fewer, unbranched processes and smaller growth cone-like structures. Examination of cytoskeletal protein profiles using two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis (2D IEF-SDS PAGE) studies revealed different patterns of protein expression and/or post-translational modifications following isoproterenol (ISO), a non-specific ss-adrenergic receptor agonist, or bFGF. Immunoblots of 2D IEF-SDS PAGE demonstrated that the expression levels of acetylated and tyrosinated tubulin immunoreactive spots differed in response to ISO and bFGF. In addition, confocal microscopic analysis revealed that following ISO the distribution of tyrosinated tubulin extended to the distal ends of processes whereas the distribution of acetylated tubulin was diminished within distal ends. This pattern is associated with increased microtubule "dynamic instability", the state in which process outgrowth is facilitated. In contrast, following bFGF treatment the relative distributions of tyrosinated and acetylated tubulins were identical, a state associated with a diminution of dynamic instability and slow process outgrowth. Confocal analysis of F-actin, protein-tyrosine phosphorylation and paxillin, a focal adhesion protein, demonstrate that ISO and bFGF treatments result in different intracellular distributions. The results suggest that neurotransmitter and growth factor receptor activation provide cues that influence the differential expression and post-translational modification of cytoskeletal elements resulting in different varieties of neuronal process outgrowth, based on cytoskeletal composition. The neuronal progenitor cell line provides a useful model to study the signaling mechanisms coupling neurotransmitter and growth factor receptor activation to the cytoskeletal rearrangements and post-translational modifications that mediate process outgrowth.