Characterization of an interferon receptor on human lymphoblastoid cells.

Abstract

A cell-free assay was developed to measure the binding of iodinated human interferon-a2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0C and 30WC, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubiized receptor. An assay was developed to measure the binding of 'MIlabeled interferon ("'I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at OC. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H20 and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic (1)(2)(3)(4)(6)(7)(8)(9) and correlated with biological responses to IFN (6, 9, 10). These studies were carried out with intact cells, and the receptors were defined in terms of their affinity for IFN (1)(2)(3)(4) (pH 8.5) was allowed to react for 2 hr at 4°C with 1 mCi of Bolton-Hunter 125I reagent (New England Nuclear; 2,000 Ci/mmol; 1 Ci = 3.7 x 101' Bq). After addition of 0.5 ml of 0.2 M glycine/0.1 M sodium borate, pH 8.5, the reaction mixture was chromatographed on a Sephadex G-75 column (0. 7 x 26 cm) equilibrated with 0.25% gelatin in phosphate-buffered saline (PiNaCl) at pH 7. Fractions containing 25I-IFN were pooled as described (2). The initial specific activity of the 125I-IFN was 740 Ci/mmol; recovery of antiviral activity was >30%. For use in binding assays the 125I-IFN was diluted 1:10 with 20 mM Hepes KOH, pH 7.4/0.1% bovine serum albumin (Hepesalb.) and filtered through a Millex-GV filter (Millipore).Binding of 125I-IFN to Plasma Membranes. Plasma membranes were prepared from Daudi cells by the hypoosmotic borate method of Thom et al. (14). The purified membranes were suspended at 10 mg/ml in 10 mM Hepes buffer (pH 7.4) and stored at -70°C. For a standard binding assay, 1-3 ,ul of membrane suspension was incubated for 30 min at 30°C with 0.5 ng of 125I-IFN and Hepes-alb. in 50-,ul reaction mixtures.The incubations were diluted with 0.2 ml of cold Hepes-alb. and filtered through GVWP Durapore filters (Millipore). The filters were washed with 15 ml of Hepes-alb. and counted in a y-counter. An incubation without membranes was used as a blank; this corresponded to <2% of the input radioactivity. Nonspecific binding was measured in the presence of a 50-fold excess of unlabeled HuIFN-a2 at different time points and 125I-IFN concentrations. This nonspecific binding was consistently <15% of the total binding and was subtracted from the data reported.Abbreviations: IFN, interferon; HuIFN, human IF...

Differential human interferon alpha receptor expression on proliferating and non-proliferating cells

“…A number of studies using radiolabeled IFN-a, IFN-P and IFN-y suggest that IFN-a and IFN-P interact with a common cellular binding site, while IFN-y is recognized by a distinct receptor [2 -61. This conclusion is further supported by affinity-labeling experiments, which identify IFN-a-binding complexes of 140-160 kDa [4,7,81, and IFN-y-binding complexes of about 110 kDa [6, 91, in a variety of human cell types. Recent evidence suggests an indirect interaction between these two classes of IFN receptors, since IFN-y treatment of at least one human cell line reduces the affinity of subsequent IFN-a receptor binding [ 101.…”

“…A number of studies using radiolabeled IFN-a, IFN-P and IFN-y suggest that IFN-a and IFN-P interact with a common cellular binding site, while IFN-y is recognized by a distinct receptor [2 -61. This conclusion is further supported by affinity-labeling experiments, which identify IFN-a-binding complexes of 140-160 kDa [4,7, 81, and IFN-y-binding complexes of about 110 kDa [6, 91, Similarities are evident in a comparison of the receptor interactions of many polypeptide hormones and growth factors, and the interaction of IFNs with their receptors. These similarities include ligand-induced down-regulation of receptors [S, 12, Recently we described a regulatory feature of IFN-a binding to human lymphoblastoid cells, which is consistent with a model of negative cooperativity [13] as originally described by DeMeyts for insulin -receptor interaction [17].…”

“…Most of these studies have utilized probes that contain, at best, only 30% labeled IFN molecules [2,4,7,8,10,12,14,15,26,311. Since it remained likely that IFN receptor expression was incompletely described in these reports, we have characterized HuIFN-a2 receptor expression on human cells, using '251-labeled IFN-a2 iodinated to a mean incorporation of 90 -100%.…”

“…These agents have been successfully used to identify such components in a number of laboratories [4,7,81. Cell numbers and reaction volumes for each experiment are outlined in the appropriate figure legends.…”

“…The bifunctional cross-linking agents, DSS and DSP, have been succesfully employed in the identification of IFN-binding cell-surface components [4,7,81. In these experiments labeled IFN-cr is bound to cells at 4"C, after which the crosslinking agent is added to the cell suspension.…”