Membrane Receptor Complex Research Articles

Biochemical analysis of MHC and non-MHC lymphocyte membrane antigens

Basic biochemical techniques have been skillfully used to characterized MHC-bound peptides and to examine the changes that occur in MHC molecules and lymphocyte membrane receptor complexes upon ligand binding. The fine tuning of these experimental approaches was possible because of the availability of a variety of biochemical and biological reagents.

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated.

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.

Function by association? MHC antigens and membrane receptor complexes

Function by association? MHC antigens and membrane receptor complexes

Internalization of interleukin 2 is mediated by the beta chain of the high-affinity interleukin 2 receptor.

High-affinity IL-2-R correspond to a membrane receptor complex composed of two different IL-2-binding proteins, the Tac antigen (alpha chain) and a 70-75 kD beta chain. Using cell lines that express either the alpha or the beta protein, we demonstrate that IL-2 internalization occurs when ligand is bound to the isolated beta chain, but not when it is bound to the isolated alpha chain. The kinetics of IL-2 internalization mediated by the intermediate-affinity beta chain were nearly identical to those of the high-affinity alpha/beta heterodimer (t1/2 of 10-15 min), and each type of receptor targeted the bound IL-2 for intracellular degradation in lysosomes. The beta chain thus appeared to provide the essential element necessary for ligand internalization by both types of IL-2-R.

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Purification of a HeLa cell receptor protein for group B coxsackieviruses.

Coxsackievirus B3 (CB3) firmly attaches to HeLa cells, forming a specific complex between the virus and its receptor on the cell surface. We extracted this virus-receptor complex (VRC) with the detergents sodium deoxycholate and Triton X-100. The VRC was identified by its sedimentation coefficient (140S), which was less than that of virions (155S). Formation of the VRC from cell lysates and 3H-CB3 occurred with the same specificity as did attachment of virions to cells, in that its formation was blocked by unlabeled CB3 but not by poliovirus. The VRC was purified 30,000-fold by differential and sucrose gradient centrifugation. Iodination with Na125I revealed that the purified VRC consisted of the normal CB3 proteins and one additional protein (RP-a) with an approximate molecular weight of 49,500. RP-a was eluted from the VRC and was shown to rebind with CB3 and CB1 virions but not with poliovirus type 1. We propose that Rp-a is a protein in the plasma membrane receptor complex which is responsible for the specific recognition and binding of the group B coxsackieviruses.

The membrane receptor complex regulating induction and clonal expansion of normal murine B lymphocytes.

Induction of resting B lymphocytes results from the interaction of competent ligands or helper cells with "triggering receptors." Subsequent clonal expansion and performance are thought to be regulated by the interaction of selective growth or maturation factors with specific receptors on induced B cells. A set of membrane molecules of B lymphocytes, including IgM, IgD, IA, IE, lipopolysaccharide receptors, receptors for Fc and C3b, and other non-immunoglobulin structures recognized by some antiidiotypic antibodies, display ligand-induced relationships. Functional studies also indicate that this group of molecules functions as a complex that regulates delivery of inductive signals, the expression of growth and maturation receptors, and/or the outcome of the interactions of these receptors with the corresponding factors.

Induction of cytolytic T lymphocytes by mitogenic lectins is specifically inhibited by anti-Lyt-2 antibodies.

The effects of anti-Lyt-2 antibodies at different levels of lectin-induced cytotoxic T lymphocyte (CTL) responses have been analyzed. Induction of interleukin 2 (T cell growth factor) reactivity ("step 1") by three different lectins was profoundly inhibited by anti-Lyt-2 antibodies. Also lectin-dependent cytotoxicity of polyclonally induced CTL was significantly inhibited by anti-Lyt-2 antibodies, whereas the interjacent IL2-dependent proliferation of "step 1"-induced CTL precursors was unaffected by these antibodies. The anti-Lyt-2-mediated inhibition was selective and specific to the target cell determinants and limited to the events in CTL responses which require interactions with the lectins. These results parallel those obtained in antigen-induced responses and indicate that antigens and lectins induce CTL responses by interacting with the same type of membrane receptor complex containing Lyt-2 molecules and antigen-specific receptors. It follows that T cell mitogenic lectins do not appear to deliver triggering signals through "mitogen" receptors.

Evidence for the presence of follicle-stimulating hormone receptor antibody in human serum

A male patient presented with polyostotic fibrous dysplasia, elevated serum gonadotropin levels, primary gonadal failure, and an immunoglobulin M (IgM) monoclonal gammopathy. When the patient's serum was added to radioiodinated human follicle-stimulating hormone (125I-hFSH)-bovine testis membrane-receptor complex solubilized by detergent, followed by the addition of antihuman immunoglobulin G ((anti-hIgG), the preformed complex was precipitated. No such precipitation was seen when normal human or rabbit serum, serum from a patient with polyostotic fibrous dysplasia, or serum from other patients with polyclonal gammopathies were utilized. The patient's serum did not precipitate free 125I-hFSH. Detergent-solubilized testis receptor not complexed to hFSH, as well as highly purified radioiodinated receptor were also precipitated when this serum was added followed by anti-hIgG. Our results indicate that serum from the patient contained antibodies to FSH testis receptor. It is possible that antibodies to gonadal receptors may exist in other patients, although it is not yet possible to assume a causal relationship between the presence of receptor antibodies and gonadal failure.

Binding sites of iron transferrin on rat reticulocytes inhibition by specific antibodies

1. 1. In the process of iron uptake by precursors of the erythrocytes probably more than one membrane component is involved; besides the specific transferrin receptor, another membrane component with a high iron activity after incubation with 59Fe can be isolated. 2. 2. A striking resemblance exists between rat and human reticulocyte components which are involved in the process of iron uptake. 3. 3. Incubation of reticulocytes with F ab fragments of an antibody against the membrane receptor for transferrin causes a concentration-dependent decrease in transferrin binding and iron uptake. 4. 4. The membrane receptor complex isolated is still heterogeneous; analytical ultracentrifugation studies suggest a molecular weight lower than 230 000. 5. 5. Intact immature red cells are necessary for specific binding of transferrin with the receptor followed by iron uptake. This is the only mechanism for iron uptake. 6. 6. Immunofluorescence studies showed that the receptor for transferrin is localized at the outside of the cell membrane.

Metiamide: More than an H2-receptor antagonist

The effect of metiamide on acid secretion and calcium uptake by the frog gastric mucosa was tested alone and in combination with histamine, pentagastrin, aminophylline, and dibutyrl cyclic AMP (db cAMP). Metiamide inhibited all stimulants including db cAMP which presumably acts intracellularly beyond the H2 receptor. Metiamide, therefore, actively inhibits at a point within the cell beyond the adenylated cyclase membrane receptor complexes. Metiamide also increases calcium influx into the parietal cell, suggesting yet another possible mechanism of inhibition.