Abstract
The structure of receptors for insulin-like growth factors in rat liver plasma membranes and the BRL 3A2 rat liver cell line has been examined by chemical cross-linking with disuccinimidyl suberate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Two receptor subtypes have been identified: (i) 125I multiplication-stimulating activity cross-linked to liver membranes or intact cells appeared in a complex of Mr = 260,000 (reduced) and 220,000 (nonreduced) and (ii) 125I-insulin-like growth factor I cross-linked to BRL 3A2 cells appeared predominantly in two bands of Mr greater than 300,000 without disulfide reduction and in a Mr = 130,000 complex following reduction. The two subtypes of insulin-like growth factor receptors identified by structural analysis correspond to previously observed differences in their specificity for insulin and insulin-like growth factors.
Highlights
In competitive binding studies, IGF receptors of different specificity have been demonstrated in different tissues and
In the present paper we have examined the structure of two types of IGF receptors by affinity labeling them with 1251-MSAor '251-IGF-I using disuccinimidyl suberate: (i) the MSA receptors of rat liver plasma membranes and cultured rat liver cells,receptors that do not interact with insulin, and (ii) the IGF-I receptor of cultured rat liver cells, a receptor that does interact with
(reduced) and220,000 and(ii) '2SI-insulin- 11-1was used for iodination; Sephadex G-75peak I1 MSA was usedas like growth factor I cross-linkedto BRL 3A2 cells ap- unlabeled peptide
Summary
Der reducing and nonreducing conditTiownos.receptor Porcine insulin (lot 1JM95AN) was purchased from Elanco (Indisubtypes have been identified: (i) 12'1 multiplication- anapolis, IN). The binding reaction mixture (0.2 m l ) included '"I-MSA ( ~ng3/ml) or '251-insulin( ~nM1),purified liver membranes (0.1mg), and unlabeled proteins as indicated in Krebs-Ringer phosphate buffer, pH 7.4, containing 8.25 mg/ml of bovine albumin. M, = 260,000 band with a specificity consistent with it representing a Iz5I-MSAMSAreceptor complex[10].Unlabeled MSA decreased the radioactivity associated with this band in a dose-dependent manner (Fig. 1,A-D),whereas high concentrations of unlabeled insulin (Fig. 1E) or an IgG preparation containing antibodies to insulin receptors (Fig. 1F)had little the cell monolayer washed once with cold phosphate-buffered saline. To cross-link the bound radioligand, disuccinimidylsuberate (0.1 mM final concentration) was added in 0.8 ml of pH 7.4 Hepes binding buffer (without albumin) and incubated for 15 min a t 15 "C.
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