Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily.

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.

Affinity purification and chemical analysis of the interleukin-1 receptor.

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.

Similarity between the interleukin 1 receptors on a murine T-lymphoma cell line and on a murine fibroblast cell line.

Interleukin 1 beta (IL-1 beta), one of two different polypeptide hormones with interleukin 1 (IL-1) biological activity, produced by activated human monocytes, is a 17.5-kDa protein. IL-1 beta binds specifically to a variety of cells; the cellular distribution of binding is consistent with reported biological responsiveness. In this report we show that two unrelated, but IL-1-responsive, cell lines, LBRM-33-1A5, a T-lymphoma line, and BALB/3T3, a fibroblast line, bind 125I-labeled IL-1 beta via similar plasma membrane receptor molecules. The T-lymphoma cells possess 238 +/- 16 plasma membrane receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 3.6 +/- 0.9 X 10(9) M-1. The IL-1 receptor has a molecular size of approximately equal to 79.5 kDa, as estimated by affinity cross-linking. The fibroblasts possess 4.8 +/- 0.5 X 10(3) IL-1 receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 2.6 +/- 0.5 X 10(9) M-1. The molecular size of the receptor molecule on the fibroblasts is approximately equal to 78 kDa. Despite the similarity in the characteristics of the ligand-receptor system on the two different cell types, the biological responses of the two cell types to IL-1 beta occur at IL-1 beta concentrations that differ by four orders of magnitude.