Fenner Conference FGF signal transduction in PC12 cells: Comparison of the responses induced by endogenous and chimeric receptors ERIK D FOEHR, 1 SIMONA RAFFIONI, 1 RITSUKO FUJI 1 and RALPH A BRADSHAW 1,2 Departments of 1 Physiology & Biophysics and 2 Anatomy & Neurobiology, College of Medicine, University of California, Irvine, California, USA Summary Rat phaeochromocytoma (PC12) cells respond to many growth factors and produce dierent phenotypes, including neurite outgrowth. Receptor tyrosine kinases (RTK), which activate multiple signalling pathways in response to ligand binding, initiate many of these. One such family of receptors, the ®broblast growth factor receptor (FGFR), has four dierent members and expresses at least three of these in PC12 cells. A chimeric tyrosine kinase receptor, consisting of the extracellular domain of human plasma-derived growth factor receptor-b (hPDGFR-b) and the transmembrane and intracellular region of FGFR1 (designated PFR1), was constructed and was stably transfected into cloned PC12 cell lines. This chimera, which can be activated without stimulating endogenous RTK including other FGFR, induces neurite outgrowth in a PDGF-dependent manner. By altering the protocol for preparing the retroviral vectors, cells with a wide range of expression levels can be obtained. The amount of these chimeric receptors seems to correlate with the time and the intensity of response as observed in neurite outgrowth assays. Analysis of proteins implicated in FGFR1 signalling indicates that upon stimulation, a tyrosine phosphorylated protein designated FRS2 associates with SOS, Grb2 and the receptor. The chimeric receptor appears entirely similar to that observed for the stimulation of native PC12 cells with FGF2. These results support the view that FRS2 is the dominant FGFR1 signalling entity in PC12 cells. Key words: chimeric receptor, ®broblast growth factor, FRS2, neuronal dierentiation, PC12 cells, signal transduction, tyrosine kinase. Introduction The PC12 (rat phaeochromocytoma) cell, a rat tumour cell line, is an excellent model for studying the signalling mechanisms involved in growth factor-stimulated dieren- tiation. 1 They can be reversibly dierentiated by a number of agents that activate dierent signalling pathways. Two growth factors, nerve growth factor (NGF) and ®broblast growth factor (FGF), produce this response by the stimu- lation of receptor tyrosine kinases. 2 Although the pathways utilized are not completely de®ned, they are clearly not identical. However, the cessation of cell division, extension and maintenance of neurites and sustained activation of the ERK (p42 and p44) are characteristic of their responses. 3 Interestingly, other growth factors, most notably epidermal growth factor (EGF), only stimulate PC12 cells to divide and are unable to produce a sustained activation of ERK under normal conditions. 4 The receptor tyrosine kinases utilized by these factors consist of an N-terminal extracellular ligand binding do- main, a single pass transmembrane-spanning region and an intracellular domain mainly composed of a tyrosine kinase eector. 5 Upon ligand binding, these receptors become autophosphorylated, producing phosphotyrosine residues at intracellular sites. Intermediate signalling molecules in- teract with the activated receptor, mainly through phos- phorylated tyrosine residues via Src homology (SH2) or phosphotyrosine binding (PTB) domains, to initiate bio- logical eects such as division and dierentiation. 6 For example, Shc and Grb2, complex with the guanine nucle- otide exchange factor SOS, to activate the small guanosine triphosphate (GTP) binding protein, Ras, and thereby link receptor tyrosine kinase activation to a cytosolic protein kinase cascade. A critical downstream event in signal transduction from the receptor is the activation of ERK. Four distinct genes encode the known FGF receptor (FGFR) family members. 7 One feature of some of the FGFR is the enormous repertoire of variants that result from alternative splicing. The FGFR are found in a wide range of tissues and cell lines. During development and in the adult animal, the FGFR1 and FGFR2 genes typically exhibit broad but distinct patterns of expression. On the other hand, the FGFR3 and FGFR4 genes appear to have more restricted patterns of expression. In PC12 cells all FGFR genes are apparently expressed, with FGFR1 being the most abundant. All the FGFR bind FGF1 (acidic FGF) and each receptor, including FGFR1, can bind several of Correspondence: RA Bradshaw, Department of Anatomy and Neurobiology, College of Medicine, University of California, Ir- vine, CA 92697, USA. Received 30 June 1998; accepted 30 June 1998. Immunology and Cell Biology (1998) 76, 406±413