We have performed additional RNA extractions from tissues in order to perform a statistical comparison of IGF-II/mannose 6-phosphate mRNA levels between fetal (20 day and postnatal (20 day) tissues. mRNA levels were measured with a nuclease protection assay. With the exception of lung, the postnatal decline in receptor mRNA was statistically significant in all of the tissues examined. These additional experiments support our earlier conclusion that the control of expression of the IGF- II/M6P receptor is primarily at the level of mRNA in most tissues. In addition, by using a sense strand standard which we transcribed from a pGem 4 plasmid containing a 0.5 kbIGF-II/M6P receptor insert, we have measured the number of receptor mRNA copies in fetal heart, the tissue with the highest level of receptor (10(8) molecules/heart). We have examined the role of G proteins in signaling by the IGF-I receptor by examining the effect of pertussis toxin on stimulation of DNA synthesis by IGF-I in the human osteosarcoma cell line MG63. In six experiments, pertussis toxin (0.01 pg/ml to 1 ng/ml) failed to inhibit IGF-I induced DNA synthesis in MG63 cells. Lack of G protein involvement in the IGF-I receptor signaling pathway was also supported by the inability of GTP's to inhibit the binding of (125)I-IGF-I to MG63 membrane preparations. We have begun to characterize the IGF-I dependent phosphorylation of the IGF-I receptor in MG63 cells as a first step in investigating signaling by the IGF-I receptor in this cell line. After metabolic labeling with ortho[32P]phosphate and addition of IGF-I, cell lysates were purified by affinity chromatography on anti-phosphotyrosine Agarose and/or immunoprecipitation by anti-IGF-I receptor monoclonal antibodies. Because of phosphorylated bands which migrated similarly to the IGF-I receptor on SDS-PAGE, a combination of both anti-phosphotyrosine affinity chromatography and immunoprecipitation was required in order to demonstrate IGF-I dependent phosphorylation of the IGF-I receptor.
