Expression and regulation of the insulin-like growth factor-1 receptor by growing and quiescent H4IIE hepatoma

Abstract

Recent evidence that insulin-like growth factor-1 (IGF-1) influences certain properties of H4IIE hepatoma cells independent of insulin led us to examine whether H4IIE cells express IGF-1 receptors. Competitive binding experiments demonstrated IGF-1, but not insulin or IGF-II, could compete with [ 125I]IGF-1. Chemical crosslinking detected a protein with an apparent mass of 175 kDa and its identity as the IGF-1 receptor α-subunit was confirmed by Western blotting. The apparent molecular mass of this protein decreased to 135 kDa following deglycosylation. Immunofluorescence microscopy verified that both insulin and IGF-1 receptors were present, although measurement of IGF-1 receptor quantity revealed they were less abundant than insulin receptors. Binding of IGF-1 was low in growing cells and higher in a quiescent cell population. Scatchard analysis confirmed that receptor density was increased in non-growing H4IIE cells while there was no apparent difference in receptor affinity. Western blot analysis and RT-PCR revealed that both protein and mRNA levels were elevated as cell growth ceased. Interestingly, addition of insulin to quiescent H4IIE cells, which stimulates cell proliferation, further increased IGF-1 receptor protein levels with a peak at 12–24 h. Distinct modes of regulating IGF-1 receptor expression are indicated.