Unique features of SHIP, SHP-1 and SHP-2 binding to FcgammaRIIb revealed by surface plasmon resonance analysis.
A growing family of inhibitory receptors characterized by content of one or more immunoreceptor tyrosine-based inhibitor motif (ITIM), I/V xYxxL/V, has been shown to regulate activation and effector function of immune system cells. The inhibitory activity of these receptors is mediated in large part by tyrosyl phosphorylated ITIM (pITIM) interactions with cytoplasmic effectors. Interestingly, different members of the family utilize partially distinct subsets of effectors from a group that includes SHP-1, SHP-2 and SHIP, an inositol 5' phosphatase. For example, while killer inhibitory receptors bind only SHP-1 and SHP-2, FcgammaRIIB bind SHIP, SHP-1 and SHP-2. The basis of selectivity of ITIMs for effectors is unclear. In this study surface plasmon resonance has been used to characterize the binding of phosphorylated FcgammaRIIB ITIM peptides to SHP-1, SHP-2 and SHIP derived Src-homology 2 (SH2) domains. SHIP was found to bind with highest affinity with intermediate on and off rates. SHP-1 bound with lowest affinity with slow on and slow off kinetics, and only its C-terminal SH2 domain exhibited binding activity. Both C- and N-terminal SH-2 domains of SHP-2 bound the pITIM. The affinity of these interactions were similar, however, they exhibited relatively fast on fast off and slow on slow off kinetics respectively. Interestingly, removal of the Ala-Glu-Asn sequence which lies immediately N-terminal from the ITIM in FcR ablated binding to SHP-1 and SHP-2 but not to SHIP. These results reveal a previously unrecognized level of complexity of effector binding to pITIM, including dependence of optimal SHP-1 and SHP-2 binding on residues N-terminal from the ITIM.
Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.
S J
SJ
Famiglietti
K
K
Nakamura
J C
JC
Cambier
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism; D007700:Kinetics; D016393:Lymphoma, B-Cell; D051379:Mice; D008969:Molecular Sequence Data; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases; D017452:Receptors, IgG / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000502:physiology
D015703:Antigens, CD; C080571:Fc gamma receptor IIB; D017452:Receptors, IgG; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases