title: Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness.
abstract: The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity.
authors.affiliation: Laboratory of Host Defenses, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
authors.forename: Sergio D
authors.initials: SD
authors.lastname: Rosenzweig
authors.forename: Owen M
authors.initials: OM
authors.lastname: Schwartz
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Brown
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Leto
authors.forename: Steven M
authors.initials: SM
authors.lastname: Holland
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D002449:Cell Aggregation / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D007424:Intracellular Fluid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008361:Mannosidases / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D004151:Dipeptides; C086185:IFNGR2 protein, human; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase