Ligands for murine NKG2D display heterogeneous binding behavior.
NKG2D transmits stimulatory signals to natural killer cells and other hematopoietic cells, leading to enhanced proliferation, cytokine secretion and target killing. Murine and human NKG2D each recognize five known class I-related molecules with distinct primary structures. Here, we used surface plasmon resonance to examine the binding of murine NKG2D to its cognate ligands: RAE-1B6 (a newly described C57BL/6J variant of RAE-1), RAE-1 delta (common to BALB and C57BL6/J), and H60 (expressed in BALB, but not C57BL/6J). While RAE-1B6 and H60 display relatively high affinities for NKG2D with K(D) in the 20-30 nM range and k(off )in the 0.03s(-1) to 0.06s(-1) range (t(1/2) approximately 10-20s); the RAE-1 delta variant binds with a lower affinity: K(D) of approximately 750 nM. Furthermore, RAE-1 delta displays biphasic kinetics with dominant k(off) of approximately 0.2s(-1) (t(1/2) approximately 3s), partially explaining the lower affinity. Thus, H60 and RAE-1B6 bind NKG2D with almost identical kinetics while sharing only 20% amino acid sequence identity; whereas other RAE-1 molecules demonstrate faster dissociation and lower affinities than RAE-1B6 despite sharing 90% sequence identity. C57BL/6J mice, although not expressing the H60 gene product, retain a high-affinity ligand for NKG2D in the form of RAE-1B6.
Carayannopoulos
Leonidas N
LN
Barnes-Jewish Hospital, Division of Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Naidenko
Olga V
OV
Kinder
Jeremy
J
Ho
Emily L
EL
Fremont
Daved H
DH
Yokoyama
Wayne  M
W
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D015778:Minor Histocompatibility Antigens / Q000378:metabolism*; D008969:Molecular Sequence Data; D055655:NK Cell Lectin-Like Receptor Subfamily K; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*; D055607:Receptors, Natural Killer Cell; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology
C527085:KLRK1 protein, human; C527086:Klrk1 protein, mouse; D008024:Ligands; D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D055655:NK Cell Lectin-Like Receptor Subfamily K; C491220:Rae-1B6 protein, mouse; C491219:Raet1a protein, mouse; D011971:Receptors, Immunologic; D055607:Receptors, Natural Killer Cell; D011993:Recombinant Fusion Proteins; C114735:minor H antigen H60