The Expression of Transforming Growth Factor Alpha Receptor Protein and its Activation in Chicken Ovarian Granulosa Cells of Maturing Follicles

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-α. In a previous study, we localized EGF/TGF-α receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF-α binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF-α receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF-α followed by crosslinking with bis(sulphosuccinimidyl)suberate showed that TGF-α binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF-α to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF-α-stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF=TGF-α receptor protein, which dimerizes on binding to TGF-α, suggesting that the receptor protein may be involved in the signal transduction of TGF-α actions in the chicken granulosa cells.