Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors.
The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 x10(-9)cm(2)/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D=3.1 x10(-9)cm(2)/s) with a marked reduction after 30 min of TNF treatment (D=0.9 x 10(-9)cm(2)/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.
Gerken
Margarita
M
Institute of Physics, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
Krippner-Heidenreich
Anja
A
Steinert
Steffen
S
Willi
Sylvia
S
Neugart
Felix
F
Zappe
Andrea
A
Wrachtrup
Jörg
J
Tietz
Carsten
C
Scheurich
Peter
P
D000818:Animals; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D017434:Protein Structure, Tertiary; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism*; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D008024:Ligands; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins