Purification and identification of a growth factor that sustains the growth of fibroblasts null for the IGF-I receptor

Shi-Qiong Xu, Thomas Jefferson University


The R$\sp-$ cell line is a 3T3-like cell line with a homozygous disruption of the IGF-IR genes. R$\sp-$ cells cannot grow at all in serum-free medium (SFM) supplemented by known growth factors that are sufficient for the stimulation of their wild-type counterparts and other 3T3 cells, either singly or in combination. They are resistant to a transformation by different oncoproteins or tyrosine kinase receptors, which readily transform 3T3 cells expressing wild-type IGF-IR. Expression of the wild-type IGF-IR in R$\sp-$ cells restores growth in SFM supplemented by growth factors and the susceptibility to transformation. R$\sp-$ cells can grow in 10% serum and also in media conditioned (CM) by certain cell lines, suggesting that serum contains an unidentified growth factor that can promote cell growth in cells devoid of IGF-IRs. Using R$\sp-$ cells, we found that the BRL-3A CM contain an activity capable of stimulating DNA synthesis and cell proliferation. One of the active component(s) in the BRL-3A CM was purified to homogeneity by SDS-PAGE following chromatographies, and identified as the granulin/epithelin (Grn/Epn) precursor by an accurate determination of the masses of endoproteinase lys-C peptides using MALDI-TOF MS, followed by a database search. This precursor is a poorly characterized growth factor of 60-65 kDa, secreted by many epithelial and hemopoietic cells. At present, it is the only purified growth factor that can stimulate the growth of fibroblasts null for the IGF-IR. The Grn/Epn family is a family of cysteine-rich growth factors consisting of 7 1/2 Grn/Epn-like repeats, which share a novel structure consisting of a parallel stack of $\beta$-hairpins stapled together by six disulphide bonds. The human grn/epn gene is comprised of 12 exons and 11 introns at chromosome 17, widely conserved in species and expressed in different tissues. A putative promoter of the granulin gene is characterized by binding sites for many transcription factors and several inhibitory factors. All these findings suggest a possibly widespread growth regulatory function, although a receptor Grn/Epn is yet unknown.

Subject Area

Genetics|Molecular biology|Cellular biology

Recommended Citation

Xu, Shi-Qiong, "Purification and identification of a growth factor that sustains the growth of fibroblasts null for the IGF-I receptor" (1998). ProQuest ETD Collection - Thomas Jefferson University. AAI9829092.