The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein

The MIC2 locus is located in the pseudoautosomal (pairing) region of human X and Y chromosomes (Goodfellow et al., Science 234, 740-743, 1986). Despite extensive molecular analysis of MIC2 (see Darling et al., Cold Spring Harb. Symp. quant. Biol. 51, 205-211, 1986), study of the gene product has been limited (Banting et al., EMBO J. 41, 1967-1972, 1985). Here we report the combined use of monoclonal antibodies, plasmid expression vectors and structural prediction analysis to define the MIC2 gene product as an integral membrane protein. Random overlapping fragments of a cDNA, corresponding to the MIC2 locus, were cloned into the plasmid expression vector pEX1 (Stanley and Luzio, EMBO J. 3, 1429-1434, 1984) to produce "epitope libraries". Six different monoclonal antibodies, known to recognize the extracellular region of the MIC2 gene product, were used to screen these libraries. Clones recognized by these antibodies were sequenced and their sequences aligned with one another and with the complete MIC2 cDNA sequence. All antibodies tested recognized adjacent and/or overlapping epitopes in the same region of the molecule. These results complement data from a hydropathy plot of a conceptual translation of the MIC2 sequence, which demonstrated the presence of a single long hydrophobic region in the mature protein. Since the antibodies recognize the extracellular portion of the molecule, we were able to determine the orientation in the plasma membrane. This method of analysis is generally applicable where antibodies and cloned cDNAs are available.