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-1δ (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 0.03 s^-1 to 0.06 s^-1 range (t_1/2 approximately 10-20 s); the RAE-1δ variant binds with a lower affinity: K_D of approximately 750 nM. Furthermore, RAE-1δ displays biphasic kinetics with dominant k_off of approximately 0.2 s^-1 (t_1/2 approximately 3 s), 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.