The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). 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 alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer 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 dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

Placentas from streptozotocin-diabetic rats have previously been shown to be morphologically and biochemically immature when compared with those of control rats. The binding of epidermal growth factor (EGF) to plasma membranes prepared from placentas of control and streptozotocin-diabetic fetuses has been characterized on days 17 and 21 of gestation. Results from competitive binding data analyzed by Scatchard analysis indicate the presence of a single class of receptors on day 17 (KD = 5.4 X 10-10) and the appearance of a second class of binding sites for`zI-EGF by day 21 (KD= 3.5 X 10'9) in membranes from control fetuses. Placental membranes from diabetic fetuses show decreased specific binding (-30%) on both days and the absence of a second class of binding sites on day 21 of gestation. Results from a radioreceptor assay indicate that the quantity of EGF in the serum of fetuses removed from control rats on day 21 is twofold greater than the quantity in sermm offetuses from diabetic rats. These data reveal a developmental increase in EGF-binding sites in the placenta of normal near-term fetal rats, largely because of the appearance of a second class of binding sites with a lower affinity for WGF. The failure (or delay) of this second class to develop in the diabetic may be important for the control of maturation and growth of this tissue.

To explore the utility of the baculovirus/insect-cell system for the expression of a soluble secreted human insulin-receptor (hIR) extracellular ligand-binding domain, we have engineered a recombinant virus encoding an hIR deletion mutant which is truncated eight residues from the beginning of the predicted transmembrane domain (i.e. 921 residues). Within 24 h after infection of Sf9 cells with virus, insulin-binding activity begins to accumulate in the culture medium, and reaches a maximum between 48 and 72 h. The intracellular transit and processing of this secreted receptor, designated 'AchIR01', is quite slow. After 24 h in pulse-chase experiments approximately 50% of the metabolically labelled protein is still inside the cell. This protein accumulates as a non-cleaved hIR precursor which is glycosylated, but the carbohydrate is entirely endoglycosidase H (endoH)-sensitive (i.e. high mannose). Approximately one-half of the receptor in the culture medium (i.e. approximately 25% of the total) is in the form of non-cleaved precursor, and about one half of its carbohydrate chains are now endoH-resistant. The remainder of the protein is proteolytically processed hIR (alpha-plus truncated beta-subunits). None of these hIR species exhibit O-linked carbohydrate. Only the processed form of the receptor in the medium binds insulin. This insulin-binding protein is secreted as a dimer (alpha beta)2, and binds insulin with an affinity which is comparable with that of both the wild-type hIR as well as the secreted form of the hIR expressed in mammalian cells. Despite the rather inefficient processing and altered glycosylation of the AchIR01 protein in insect cells, this high-affinity insulin-binding protein accumulates in the medium at levels (mg/litre) of about 100 times that achieved in a mammalian-cell system.

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