Abstract
We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.
Highlights
Miinchen 70, the slnstitut fiir Diabetesforschung, Kijlner Platz 1, 8000 Miinchen 40, and the TlGesellschuft fiir Strahlen- und Umweltforschung, Institut fiir Klinische Htimatologie, Marchioninistrasse
The leukemic promyelocytic cell line HL-60 can be continuously grown in serum-free medium provided it contains insulin [2] or IGF-I [4], respectively, and transferrin
When we compared the growth stimulatory effects of various concentrations of IGF-I and insulin in the presence of a constant amount of transferrin we found IGF-I to be a more potent stimulator than insulin (Fig. 1)
Summary
Materials-Porcine insulin was purchased from Novo Industries (Denmark). ATP (2900 Ci/mmol) was from New England Nuclear (Federal Republic of Germany), and 3-[“‘I]iodotyrosyl. 5 pg of WGA-purified protein was preincubated at 22 “C for 30 min with insulin or IGF-I in different concentrations (O-1000 nM/liter), followed by an incubation with [-r-32P]ATP (10 &i/FM) in elution buffer containing 10. The incubation was terminated by the addition of Laemmli buffer containing 100 mM/liter dithiothreitol and subsequent boiling for 25 min at 95 “C. The 105-kDa phosphoprotein was purified from the incubation and stop buffer by a lofold dilution with deionized water and subsequent concentration of the phosphoprotein with a Centricon 10 column (Amicon) until the original volume was regained (180 ~1). 9 min. 50 fractions of 130 ~1 were collected and Cerenkov radiation measured using an LKB liquid scintillation counter
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