J. Biol. Chem., Vol. 266, Issue 23, 14970-14977, 08, 1991

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity

M Fountoulakis, HW Lahm, A Maris, A Friedlein, M Manneberg, D Stueber and G Garotta
Hoffmann-La Roche Ltd., Pharma Research New Technologies, Basel, Switzerland.

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti- interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Najarro, P. Traktman, and J. A. Lewis Vaccinia Virus Blocks Gamma Interferon Signal Transduction: Viral VH1 Phosphatase Reverses Stat1 Activation J. Virol., April 1, 2001; 75(7): 3185 - 3196. [Abstract] [Full Text]

				P. Bongioanni, F. Lombardo, G. Moscato, S. Mosti, and G. Meucci T-Cell Interferon Gamma Receptor Binding in Interferon Beta-1b-Treated Patients With Multiple Sclerosis Arch Neurol, February 1, 1999; 56(2): 217 - 222. [Abstract] [Full Text] [PDF]

				G. F. Seelig, W. W. Prosise, J. C. Hawkins, and M. M. Senior Development of a Receptor Peptide Antagonist to Human [IMAGE]-Interferon and Characterization of Its Ligand-bound Conformation Using Transferred Nuclear Overhauser Effect Spectroscopy J. Biol. Chem., April 21, 1995; 270(16): 9241 - 9249. [Abstract] [Full Text] [PDF]

				S Huang, W Hendriks, A Althage, S Hemmi, H Bluethmann, R Kamijo, J Vilcek, R. Zinkernagel, and M Aguet Immune response in mice that lack the interferon-gamma receptor Science, March 19, 1993; 259(5102): 1742 - 1745. [Abstract] [PDF]