The murine Fc receptor for immunoglobulin: Purification, partial amino acid sequence, and isolation of cDNA clones

Abstract

The murine Fc receptor for IgG (FcγR) was purified to homogeneity by immunoaffinity chromatography from detergent lysates of the macrophage cell line J774. Microsequencing of intact protein yielded a single aminoterminal sequence, which was confirmed and extended to 20 residues by the isolation of an overlapping peptide. The isolation of additional proteolytic fragments obtained by using Staphylococcus aureus V8 protease, cyanogen bromide, and lysine C proteinase, facilitated sequence analysis of a total of 119 amino acid residues. Codon usage charts were used to construct oligonucleotide probes based on the amino acid sequences of three nonoverlapping peptides. These probes were used to screen a cDNA library derived from the WEHI-3B myelomonocytic cell line, and a single cDNA clone (pFc24) to which all three probes hybridized was isolated. This clone, containing a 1.02-kilobase cDNA insert, has been characterized by restriction mapping and partial DNA sequencing, and it has been shown to encode the FcγR. The sequence at the 5' end of the clone contained the coding information for the aminoterminal sequence of the FcγR as well as a putative 13-amino acid signal sequence. The 3' end of the clone encoded a peptide identified in purified receptor preparations. Thus, the presence of coding information at the 5' and 3' ends of this clone suggests that full-length Fc receptor cDNA spans >1 kilobase.