Abstract
Fc receptor-antibody interactions are key mechanisms through which antibody effector functions are mediated. The low affinity receptor for IgG, Fc gamma RII, is expressed on most hematopoietic cells, and through the binding of immune complexes mediates a large spectrum of biological responses vital for resistance to infection and the regulation of immunity. In this study the key residues of human Fc gamma RII involved in the interaction with IgG1 have been identified. Chimeric receptors composed of extracellular regions of Fc gamma RII and the Fc epsilon RI alpha chain have been used to localize the IgG1 binding site of Fc gamma RII to an 8-residue stretch in the second extracellular domain, Asn154 to Ser161. Site-directed mutagenesis of this region revealed that substitution of Ile155 or Gly156 with alanine ablated the binding of human and mouse IgG1, whereas replacement of Leu159, Phe160, or Ser161 with alanine enhanced binding. Molecular modeling has been used to generate a putative 3-dimensional model structure of the second extracellular domain of Fc gamma RII, suggesting that the binding site lies in an exposed loop region at the interface of domains 1 and 2.
Highlights
Fc receptor-antibody interactions are key mecha- Val'69, was identified as necessary for the binding of IgG, but nisms through which antibody effector functions are the precise location and identity of the critical Ig interactive mediated
The chimeric FcR were generated as follows: chimera or Serlslwith alanine enhancedbinding.Molecular mod- y, ~ " " ~ ' used oligonucleotides NR1 and EGA2 with EGAl and EG5; eling has been used to generate a putative 3-dimensional model structure of the second extracellular domain of FcyRII, suggesting that the binding site lies in an exposed loop region at the interface of domains 1and 2
(e,y154-161), wherebyA ~ n ' ~ ~ - S e orf' ~F'cyRIIwas inserted into an expressible formof the FceRI a chain (FCERICX-AC).OS-7 cells transfected with this chimeric receptor bound IgG-coated erythrocytes (Fig. lA)as did wild type FcyRII (Fig. 1B),but not FceRIa-A(Fig.lC,Table I)
Summary
TCTCATCG;PM10, GATGAGAACAGAGCGTAGCCTATG; GPM11, with FcR expression constructs were incubated with EAcomplexes, GGCTACACGGC'LTTCTCATCCAAG, GPM1C2,TTGGATGAGAAA-. Levelsof cell surface FcyRII expression were determined using the anti-FcyRII mAb 8.2, shown to bind distantly to the binding site [20], and used to correct for variable cell surface receptor expression between the mutant FcyRII COS-7 cell transfectants. (e,y154-161), wherebyA ~ n ' ~ ~ - S e orf' ~F'cyRIIwas inserted into an expressible formof the FceRI a chain (FCERICX-AC).OS-7 cells transfected with this chimeric receptor bound IgG-coated erythrocytes (Fig. lA)as did wild type FcyRII (Fig. 1B),but not FceRIa-A(Fig.lC,Table I). Tially no detectable bindingto the low af€inity FcyRIIa [1,2,3].In. It shouldalso benoted that residue 131(i.e. or His131) addition, the levels of cell surface FcyRII expression werdeeof polymorphic FcyRIIahas been shownto influence the bind- termined using the anti-FcyRII mAb, 8.2, and used to staning of some IgG classes (2931). Substitution of IlelS6or GlylB6with of the FcyRIIa-derivedsequence Ser'%136
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