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An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
Published on: March 14, 2016
High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells.
1Institut National de la Santé et de la Recherche Médicale (INSERM) U211, Unité de Recherche sur les Effecteurs Lympocytaires T Plateau Technique du CHR, Nantes, France.
Insights
This study identified specific receptor sites for human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) on various human cells. High-affinity receptors are found on tumor cells, while low-affinity receptors suggest a complex binding structure.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) is crucial in cellular processes.
- Identifying specific receptor sites is key to understanding HILDA/LIF function.
- Previous studies have not fully elucidated the receptor characteristics of HILDA/LIF.
Purpose of the Study:
- To identify and characterize specific receptor sites for HILDA/LIF on human cell types.
- To investigate the binding kinetics and affinity of HILDA/LIF to its receptors.
- To determine the molecular structure of the HILDA/LIF receptor complex.
Main Methods:
- Radioiodination of recombinant HILDA/LIF for receptor binding assays.
- Use of varying ligand concentrations and specific radioactivities to differentiate receptor affinities.
- Affinity cross-linking studies to identify receptor subunits.
- Kinetic analysis of HILDA/LIF binding to cell lines.
Main Results:
- High-affinity HILDA/LIF receptors (Kd 30-100 pM) were detected on monocytes and various non-lymphoid tumor cell lines (melanomas, neuroblastomas, carcinomas).
- Low-affinity receptors (Kd 1-4 nM) were also identified on several cell lines.
- Affinity cross-linking revealed two receptor species (120 and 250 kDa), with the 250-kDa subunit potentially mediating low-affinity binding and both subunits forming the high-affinity receptor.
Conclusions:
- HILDA/LIF exhibits distinct high- and low-affinity binding components on human cells.
- Tumor cell lines express significantly higher levels of high-affinity HILDA/LIF receptors compared to normal blood cells.
- The HILDA/LIF receptor is likely a multi-subunit complex involving 120- and 250-kDa proteins.
Abstract:
Radioiodinated recombinant human interleukin DA (HILDA)/leukemia inhibitory factor (LIF) purified from conditioned medium of Chinese hamster ovary transfected cells enabled the identification of specific receptor sites on a variety of human cell types. Using low concentrations (up to 500 pM) of the ligand iodinated at a high specific radioactivity, high affinity receptors (equilibrium dissociation constant Kd in the range of 30-100 pM) were first demonstrated. They were expressed at low levels by human peripheral blood monocytes but not by lymphocytes, NK cells, granulocytes, and platelets. The myelomonocytic cell line THP1 as well as the T lymphoma cell line HSB2 and the lymphoblastoid B cell line DAB were also receptor-negative. In contrast, most of the non-lymphoid tumoral cell lines tested, including melanomas, neuroblastomas, and carcinomas, expressed high affinity HILDA/LIF receptors at variable levels (Bmax from 20 to 600 sites/cell). The kinetics of HILDA/LIF high affinity binding to the choriocarcinoma JAR cell line were characterized at 4 degrees C with association and dissociation rate constants of k1 = 2.2 10(9) M-1 min-1 and k-1 = 0.0084 min-1, respectively, corresponding to a steady-state dissociation constant k1/k-1 = 3.8 pM. The subsequent use of higher concentrations of HILDA/LIF labeled at a lower specific radioactivity enabled the identification of a low affinity component on several cell lines (Kd in the range of 1-4 nM; Bmax from 1,000 to 5,000 sites/cell). On JAR cells, this low affinity component was characterized by association and dissociation rate constants at 4 degrees C of k1 = 7.3 10(7) M-1 min-1 and k-1 = 0.19 min-1, respectively (k-1/k1 = 2.6 nM). Affinity cross-linking of HILDA/LIF to JAR cells showed two cross-linked species under both reducing and nonreducing conditions corresponding to receptor species of 120 and 250 kDa, respectively. Whereas both bands had similar intensities under high affinity conditions, the higher band predominated under low affinity conditions. Our data suggest that the 250-kDa chain could constitute the low affinity binding component whereas the association of both 250- and 120-Da subunits would form the high affinity structure.
