Analysis of Fc gamma RIII (CD16) membrane expression and association with CD3 zeta and Fc epsilon RI-gamma by site-directed mutation.
Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.
Lanier
L L
LL
Becton Dickinson Immunocytometry Systems, San Jose, CA 95131.
Yu
G
G
Phillips
J H
JH
D015703:Antigens, CD / Q000096:biosynthesis*; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011961:Receptors, Fc / Q000096:biosynthesis* / Q000235:genetics; D017452:Receptors, IgG; D013329:Structure-Activity Relationship; D014162:Transfection
D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011961:Receptors, Fc; D017452:Receptors, IgG; C054979:antigen T cell receptor, zeta chain