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 activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.
Gardin
A
A
INSERM U. 338, 67084 Strasbourg, France INSERM U. 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.
Auzan
C
C
Clauser
E
E
Malherbe
T
T
Aunis
D
D
Crémel
G
G
Hubert
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006021:Glycophorins / Q000235:genetics*; D007328:Insulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D006021:Glycophorins; D007328:Insulin; D011994:Recombinant Proteins; D011972:Receptor, Insulin