Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

Abstract

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 having properties consistent with those of a membrane glycoprotein. MATERIALS AND METHODSPreparation of Soluble Insulin Receptor. Membranes were prepared from male CD-1 albino rats (100-140 g) as described (9) and were extracted,(5 mg of membrane protein per ml of buffer) for 1 hr at 40 with 2% (vol/vol) Triton X-100 in a calcium-free phosphate buffer, pH 7.5, containing: 130 mM NaCl, 5 mM KCI, 1.3 mM MgSO4-7 H20, and 8.6 mM Na2HPO4.After dialysis for 18 hr at 40 against the same buffer containing 0.1% (vol/vol) Triton X-100 (PT buffer), extracts were centrifuged at 150,000 X g for 60 min and the resulting supernatant was used as the source of the soluble insulin receptor. Subsequent experiments with soluble receptor were routinely done in PT buffer. Further purification of the insulin receptor was accomplished by affinity chromatography on insulin/succinyldiaminodipropylamino-Sepharose 4B (0.28 mg of insulin per ml of packed gel) prepared as described (derivative C in ref. 10) and washed with eluting buffer before regeneration with PT buffer. Receptor was eluted with an eluting buffer (0.05 M sodium acetate, pH 6.0/4 M urea/0.1% Triton X-100 in 1 ml fractions which were usually diluted immediately with 1 ml of 0.1 M sodium phosphate, pH 7.0. Insulin-binding fractions were pooled and immediately dialyzed against PT buffer for 18 hrs at 4°. The measurement of the specific binding by both soluble and particulate material of 125I-labeled insulin ug) as well as the preparation of 125I-insulin have been described in detail (1,11). Soluble receptor was measured by using polyethylene glycol [carbowax 6000; firnal conc. 10.4% (wt/vol)] to precipitate the hormone-receptor complex, as described (1). Calci...

The structure of the hepatic insulin receptor and insulin binding

“…They concluded that the 450 kDa insulin receptor contained a component that did not bind insulin, but could regulate on insulin binding. Maturo & Hollenberg (1978) demonstrated that the affinity of the purified insulin receptor was altered by its association with uncharacterized membrane glycoproteins which could not bind insulin themselves. Ginsberg et al (1976) and Krupp & Livingston (1979) suggested that the affinity of the insulin-binding subunit of the receptor was altered by its association with other receptor components.…”

alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

“…Thus, both uncoated and coated vesicles are involved in this process. Receptor molecules for protein hormones exist on the cell surface, some of which are probably glycoproteins or associated with glycoproteins (7,12,18). We speculate that Con A may cross-link these receptors and induce them to cluster in coated pits .…”

Insulin Receptor Regulation and Desensitization in Rat Hepatoma Cells: Concomitant Changes in Receptor Number and in Binding Affinity

“…Second, we 4344 and others 45 have presented evidence that in the membrane, the insulin receptor may be noncovalently associated with another protein that modulates receptor affinity. This affinity regulator can be demonstrated in radiation inactivation experiments 4344 and in other situations in which affinity is altered, including after acute insulin exposure, its interaction with the receptor appears to be altered.…”