Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action

To study the role of transmembrane (TM) domains interactions in the activat ion of the insulin receptor, we have replaced the insulin receptor TM domai n with that of glycophorin A (GpA), an erythrocyte protein that spontaneous ly forms detergent-resistant dimers through TM-TM interactions. Insulin rec eptor cDNA sequences with the TM domain replaced by that of GpA were constr ucted and stably transfected in CHO cells, Insulin binding to cells and sol ubilized receptors was not modified. Electrophoresis after partial reductio n of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interac tions between the beta subunits of the receptor. Insulin signaling was mark edly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced rece ptor kinase activity was observed for solubilized chimeric receptors, In co nclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to tran smit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in confo rmation necessary for activation of the receptor kinase. Other mutants modi fying the structure or the membrane orientation of the glycophorin A TM dom ain are required to better understand these constraints.