Functional Properties of Two Naturally Occurring Isoforms of the Human Insulin Receptor in Chinese Hamster Ovary Cells

We and others have previously demonstrated that the human insulin receptor messenger RNA (mRNA) is alternatively spliced such that the 36-nucleotide sequence encoded by exon 11 of the receptor gene is included (Ex11+) or excluded (Ex11-). Although both Ex11- and Ex11+ insulin receptors which differ in the presence or absence of 12 amino acids in the carboxy-terminal alpha-subunit have been demonstrated to function as insulin receptors when independently overexpressed and studied, the possibility that subtle functional differences between the two isoforms exist has received limited attention. Given that the relative abundance of the two mRNA transcripts is highly regulated in a tissue-specific manner, differences in the functional properties of the two receptor variants might contribute to tissue-specific differences in insulin receptor function and insulin action that are known to exist. To address this hypothesis, we transfected cDNAs encoding the two receptor isoforms into Chinese hamster ovary (CHO) cells and prepared several stable CHO cell lines expressing high numbers of Ex11- or Ex11+ receptors. Several functional properties of the expressed insulin receptors were compared in parallel with the following results: 1) steady state binding of insulin to cells expressing the Ex11- isoform exhibited higher (approximately 2-fold) affinity; 2) using two different methods, a significant difference in receptor-mediated insulin internalization was noted such that the Ex11- isoform displayed a higher (approximately 25% increase in the rate constant, Ke) rate of internalization; 3) partially purified Ex11- and Ex11+ receptors displayed similar maximal and insulin dose-response characteristics for receptor autophosphorylation and kinase activity toward an exogenous substrate (poly Glu-Tyr, 4:1); 4) the ability of expressed Ex11- and Ex11+ receptors to couple to a metabolic (glucose incorporation into glycogen) and mitogenic (thymidine incorporation into DNA) action of insulin was not discernibly different. Thus, when expressed in CHO cells, the two alternatively spliced isoforms of the insulin receptor have subtle differences in insulin binding affinity and the kinetics of ligand-stimulated internalization that would be expected to influence the pattern of insulin receptor expression and signaling in vivo in a tissue-specific manner.