AbstractThe Insulin receptor is an integral transmembrane glycoprotein comprised of two α‐(∼ 135kDa) and two β‐(∼ 95kDa) subunits, which is synthesized as a single polypeptide chain precursor (αβ). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into α‐and truncated β‐subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (αβ)2dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain, binds insulin with an apparent dissociation constant comparable to that of the wild‐type hIR. However, the secreted dimmer displays a linear Scatchard plot, while that of the wild‐type membrane‐associated hIR curvilinear. These results, together with previous results with a soluble derivative of the hIR cytoplasmic protein/tyrosine kinase domain, are consistent with the deduced topology. The IR is indeed comprised of two large soluble domains connected by a single membrane spanning domain, which on the one hand act concert upon ligand binding (insulin‐dependent activation of the protein/tyrosine kinase) to initiate the insulin response in cells, but on the other hand are each capable of autonomous function (ligand binding and protein/tyrosine kin