Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Abstract

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

GRP78 expression inhibits insulin and ER stress–induced SREBP-1c activation and reduces hepatic steatosis in mice

“…The IR is synthesized as a 190-kDa precursor in the ER and processed in the Golgi to yield 2 subunits of 135 and 95 kDa (the IR-α and IR-β subunits, respectively; ref. 40), and its half-life has been estimated to be about 2 h. We found that the IR precursor did not accumulate in Ad GRP78-treated hepatocytes and that the concentration of the β subunit did not decrease, indicating that there is no general impairment of the ER to Golgi transport ( Figure 12A). …”

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits

“…These data identify and characterize the N-linked carbohydrate attached to the insulin pro-receptor, all of which we have previously shown to be of the high-mannose endoglycosidase H-sensitive type [2,3]. The pattern of transfer of the N-linked high-mannose oligosaccharides from the dolichol intermediate to the protein backbone is thought to occur via a general mechanism in animal cells (reviewed in [7]), with the exception of some minor variations in certain cell types during glucose deprivation [7][8][9][10][11].…”

“…1). Both subunits are known to contain high-mannose-type (endoglycosidase H-sensitive) and complex-type (endoglycosidase H-resistant) oligosaccharide chains [2,3]. The bands corresponding to the a-and f-subunits were excised, and digested with endoglycosidase H in a similar manner to that followed with the pro-receptor.…”

“…The insulin receptor is a membrane glycoprotein composed of two major subunits, each of which contains N-linked high-mannose-type and complex-type carbohydrate side chains [1,2]. Both mature subunits are processed from a single polypeptide chain pro-receptor ofapparent Mr 190000 that contains only high-mannosetype carbohydrate side chains [2,3] and is found predominantly in the endoplasmic reticulum and Golgi apparatus [3]. Thus processing of the pro-receptor involves: peptide cleavage, trimming of some mannose residues from high-mannose side chains, addition of the distal sugars (N-acetylglucosamine, galactose, sialic acid) to form complex carbohydrate side chains, and insertion of the mature receptor into the plasma membrane.…”

“…This sequence contains 21 potential N-glycosylation sites. The structure of the actual N-linked carbohydrate chains has been characterized so far only in terms of their endoglycosidase H sensitivity and their differential labelling by radioactive monosaccharides [2,3].…”