Reoxidation of the class I disulfides of the rat adipocyte insulin receptor is dependent upon the presence of insulin: the class I disulfide of the insulin receptor is extracellular

Abstract

Elements of the quaternary structure of the native and dithiothreitol- (DTT) reduced rat adipocyte insulin receptor have been elucidated by vectorial probing and subunit cross-linking. The charged reducing agents glutathione and beta-mercaptoethylamine were used to reduce the class I disulfides of the receptor in intact adipocytes, demonstrating the extracellular location of the disulfide directly. This interpretation was confirmed by use of DTT as a reducing agent and the nonpermeant sulfhydryl blocking reagent Thiolyte MQ to prevent the reoxidation of the class I sulfhydryl groups which occurred when they were not blocked. It was found that the above reoxidation of the receptor is dependent on the concentration of insulin in the nanomolar range, not occurring measurably at 4 degrees C in its absence. Cross-linking studies with ethylene glycol bis(succinimidyl succinate) demonstrated that the alpha subunits could not be cross-linked to each other after reduction of the class I disulfides, suggesting that the interaction between the receptor heterodimers may be due primarily to the disulfide bonds.

Identification of the cysteine residues involved in the class I disulfide bonds of the human insulin receptor: properties of insulin receptor monomers.

“…When cells expressing the mutant receptor were incubated with 10 nM insulin before solubilization, most of the receptor became dimeric ( Figure 4C). Figure This result supports the view that insulin induces dimerization of the IR (Boni-Schnetzler et al, 1988;Chiacchia, 1988;Morrison et al, 1988). Surprisingly, however, at the highest concentration of insulin a substantial fraction of the IR is still present as monomers, suggesting that the value of the dimer-monomer dissociation constant for the liganded receptors is close to 20 ,tM, the equivalent receptor concentration in the plasma membrane.…”

“…One possibility is that the interactions with other proteins either for signal transduction or for receptor internalization are facilitated by having two copies of the monomers close together. It does seem apparent, however, that in the case of the insulin receptor, the interactions between monomers are sufficiently weak, as suggested by Chiacchia (1988), so that insulin binding cannot promote dimerization at a normal receptor concentration in the membrane. Hence, the necessity to use the device of covalent dimer formation.…”

Sulphydryl agents modulate insulin- and epidermal growth factor (EGF)-receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors

“…However, oxidizing agents inhibit the isolated insulin-receptor tyrosine kinase, in direct contrast with their stimulatory effects on insulin action (Wilden and Pessin, 1987). The reductant dithiothreitol (DTT) has been shown by many groups to lead to progressive dissociation of the dimeric holoreceptor (Fujita-Yamaguchi and Kathuria, 1985;Sweet et al, 1987;Chiacchia, 1988). Initially, the Class I disulphides between two a-subunits are cleaved, resulting in receptor monomers.…”

Construction and Characterization of a Monomeric Insulin Receptor

“…The membranes from cells expressing dimeric or monomeric insulin receptors were incubated with 125 I-insulin overnight in the absence or presence of excess unlabeled insulin. The membranes were then exposed to EGS to bring about insulin-␣ chain and ␣ chain-␣ chain cross-linking (13). The membranes were then subjected to SDS-PAGE under reducing conditions to obtain ␣ chain monomers and dimers, and the gel was analyzed with a Bio-Rad imager.…”