Design, synthesis, expression, and characterization of the genes for mouse FcγRIIb1 and FcγRIIb2 cytoplasmic regions

Abstract

The cytoplasmic regions of the mouse low-affhity FcyRII isoforms, mFcyRIIb1, and mFcyRIIb2, play a key role in signal transduction by mediating different cellular functions. mFcyRIIbl has a 94-residue cytoplasmic region, whereas mFcyRIIb2 has a 47-residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFcyRIIbl (bl-94) and mFcyRIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequencespecific protease, thrombin, was used to release the bl-94 peptide, which was purified by using HPLC. The b2-47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of bl-94 and b2-47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the bl-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant a-helical structure. , 1991;Fridman et al., 1992;Hulett & Hogarth, 1994;Indik et al., 1995). In mouse and human, there are at least three Fcy receptors, denoted FcyRI, FcyRII, and FcyRIII. The present work concerns mouse FcyRII (mFcyRII). This receptor is found on numerous cell types, including lymphocytes, neutrophils, macrophages, eosinophils, platelets, mast cells, and monocytes; and is thought to participate in a number of biological processes, including endocytosis, phagocytosis, Reprint requests to: Gary J. Pielak or Nancy L. Thompson, Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290; e-mail: gary-pielak@unc.edu or nlt@unc.edu.Abbreviarions: bl-94, 94-amino acid cytoplasmic region of mFcyRIIbl with a methionine and six histidine residues at the C-terminus; b2-47, 47-amino acid cytoplasmic region of mFcyRIIb2; DHFF, dihydrofolate reductase; IgG, immunoglobulin G; IF'TG, iso-propyl-/%D-thiogalactoside; ITAM, immune receptor tyrosine-based activation motif; ITIM, immune receptor tyrosine-based inhibitor motif; MBP, maltose-binding protein; MBPbl-94, MBP fusion with thrombin cleavage site and bl-94; MBP-DHFFb2-47, MBP-DHFR fusion with thrombin cleavage site, b2-47, and a methionine and six histidine residues at the C-terminus; NTA, nitrilotriacetic acid; pLC47, plasmid containing the MBP-DHFR-b2-47 gene; pLC94, plasmid containing the MBP-bl-94 gene; pMAL-c2, plasmid containing the MBP gene; TFE, trifluoroethanol; 1B4C14, monoclonal antibody against bl-94 and b2-47.

The Cytoplasmic Region of Mouse FcγRIIb1, but Not FcγRIIb2, Binds Phospholipid Membranes

“…Therefore, the cytoplasmic region of mFcγRIIb1 almost certainly interacts directly with the phospholipid surface. Previous work has demonstrated that the b1-94 peptide, a rather large ≈10 kDa peptide, is largely unstructured in aqueous solutions but that nonaqueous solvents such as trifluoroethanol and methanol induce significant structure (26). Therefore, the result that b1-94 binds to phospholipid membranes suggests that this nearby surface may be crucial to the in vivo structure of the cytoplasmic region of mFcγRIIb1.…”

“…Because these peptides are covalently attached to the plasma membrane in vivo, the results strongly suggest that the cytoplasmic region of mFcγRIIb1 is in the membranebound state in intact cells. Because this peptide has very little structure in aqueous solution (26), it is possible that the membrane surface confers structure on this peptide. The association of the b1-94 peptide with phospholipid vesicles is weakly dependent on the phospholipid composition and Ca 2+ concentration, raising the possibility that membrane binding by the mFcγRIIb1 cytoplasmic region might be modulated by these factors in vivo.…”

“…Peptides. The cytoplasmic regions of mFcγRIIb1 and mFcγRIIb2 (Figure 1) were generated as previously described (26). A gene was constructed that contains (from the N terminal to the C terminal) the coding regions for maltose binding protein, a thrombin cleavage site, the mFcγRIIb1 cytoplasmic region, a methionine residue, and a six-residue histidine tag.…”

“…Because the only structural difference between the two isoforms of mFcγRII occurs in the cytoplasmic region, it is reasonable to believe that this sequence difference plays an important mechanistic role. To investigate the relationship between the structure and function of the mFcγRIIb1 and mFcγRIIb2 cytoplasmic regions, we previously generated peptides corresponding to the cytoplasmic regions of mFcγRIIb1 and mFcγRIIb2 (26). The mFcγRIIb1 cytoplasmic region was produced by overexpression in E. coli as a fusion peptide with maltose binding protein, purification of the fusion protein by affinity chromatography, enzymatic cleavage with thrombin to release the mFcγRIIb1 cytoplasmic peptide, and HPLC purification.…”

“…The mFcγRIIb2 cytoplasmic region was chemically synthesized and purified by HPLC. The secondary structures of these two peptides in different solutions were examined with circular dichroism (26). Here, steady-state light scattering has been used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid membranes.…”