Characterization of the receptor for insulin‐like growth factor II in bone cells

We have previously shown that insulin‐like growth factor II (IGF‐II) is produced by bone cells and that IGF‐II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF‐II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [¹²⁵]I IGF‐II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [¹²⁵]I IGF‐II binding to chick calvaria cells showed an apparent K_d of 1.4 × 10¹⁰ M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF‐II was significantly more potent than IGF‐I or insulin in displacing IGF‐II tracer. Competition for binding of [¹²⁵]I IGF‐II by unlabeled IGF‐II showed a dose‐dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [¹²⁵]I IGF‐II binding to chick and mouse calvarial cells was achieved at 1‐2 ng/ml; 90% of specific binding of [¹²⁵]I IGF‐II was displaceable in the presence of 125 ng/ml of unlabeled IGF‐II. IGF‐I showed less than 5% cross reactivity for displacement of [¹²⁵]I IGF‐II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R‐II‐PAB1 inhibited the binding of [¹²⁵]I IGF‐II to mouse bone cells and H‐35 rat hepatoma cells (which contain type II but not type I receptors) in a dose‐dependent manner. R‐II‐PAB1 also inhibited basal cell proliferation as well as IGF‐II‐, IGF‐I‐, and fibroblast growth factor (FGF)‐induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R‐II‐PAB1 inhibited neither binding of [¹²⁵]I IGF‐II nor IGF‐ll‐induced cell proliferation. These results together with our findings that IGF‐II increased chick bone cell proliferation in the presence of maximal doses of IGF‐I suggest that at least part of the mitogenic action of IGF‐II is mediated through type II rather than type I receptors in bone cells.