Proximity measurements between H-2 antigens and the insulin receptor by fluorescence energy transfer: evidence that a close association does not influence insulin binding.
Reports based on coprecipitation experiments have suggested that major histocompatibility complex class I products are complexed with the insulin receptor on the cell surface. Using an independent method that avoids the creation of immunoprecipitation artifacts during membrane solubilization, we have studied insulin receptor-class I product associations by determining the proximity between these class I products and the insulin receptor on intact cells with the use of fluorescence energy transfer. Significant energy transfer was seen between the insulin receptor and both murine H-2K- as well as H-2D-end products, indicating close proximity (e.g., within 10 nm). This cell-surface association is not from the relatively high class I density in that no significant energy transfer was measured between H-2K- vs. H-2D-end proteins. To extend these observations, we also tested whether class I products influence insulin-receptor binding and postbinding events as a result of their physical association. Using related cell lines positive and negative for class I expression, we found no correlation between insulin-receptor density or binding affinity with H-2 product expression. The class I-null variant, however, demonstrated an increase in insulin-mediated insulin-receptor internalization to suggest that if major histocompatibility complex class I products directly affect insulin-receptor function through specific cell-surface interactions, they may do so after ligand binding.
Liegler
T
T
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Szollosi
J
J
Hyun
W
W
Goodenow
R S
RS
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004735:Energy Transfer; D006183:H-2 Antigens / Q000032:analysis* / Q000502:physiology; D006239:Haplotypes; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014162:Transfection
D006183:H-2 Antigens; D007328:Insulin; D011994:Recombinant Proteins; D011972:Receptor, Insulin