Insulin-like growth factor-I (IGF-I), a GH-dependent growth factor, suppresses GH secretion by pituitary cells. To clarify the role of ligand-mediated receptor internalization for IGF-I signaling to GH, human IGF-I receptor (IGF-IR) cDNAs mutated in the beta-subunit were stably transfected into GC rat pituitary cells. Overexpression of wild-type IGF-IR markedly enhanced IGF-I suppression of GH 4-fold (P < 0.005) compared to that of untransfected cells. A mutant IGF-IR with a 943Tyr-->Ala substitution in the IGF-IR submembrane domain only partially suppressed GH (73% of IGF-IR wild type), while replacement of 957Tyr-->Ala or 940Gly-->Ala produced IGF-IRs that retained enhanced IGF-I signaling to GH. Substitution of 950Tyr-->Ala or 1003Lys-->Ala in the human IGF-IR beta-subunit failed to enhance IGF-I signaling to GH above that of untransfected cells. Intracellular phosphorylation of insulin-responsive substrate-I by these mutant IGF-IRs paralleled the observed IGF-I suppression of GH, with no phosphorylation of IRS-I by 950Tyr-->Ala. Ligand-mediated receptor internalization, however, was not reduced by substitution of either 943Tyr-->Ala or 950Tyr-->Ala. In contrast, substitution of 957Tyr-->Ala reduced the internalization of labeled IGF-I to 35% that of wild-type IGF-IR. Substitution of 1003Lys-->Ala abolished IGF-IR internalization, as expected. These results demonstrate that both 950Tyr and 943Tyr are important for IGF-I signaling to GH and that IGF-IR internalization is discordant for IGF-I signaling to the GH gene.