To understand the role of membrane proteins in drug resistance to antimetabolites and alkylating agents, a series of drug resistant cell lines were analyzed for changes in membrane antigens and alterations in patterns of protein synthesis. A new 46 kD membrane protein (SQM1) was found to be decreased in MTX and CDDP resistant cells but not in cells resistant to anthracyclines or VP16. Revertant cells which had regained sensitivity to methotrexate (MTX) and cis-platinum (CDDP) also regained high expression of SQM1 membrane antigen and protein. Alterations in SQM1 expression appears to be associated with a new class of multi-drug resistance which is distinct from the alkaloid-anthracycline group. The role of SQM1 as regulator of membrane transport will now be investigated by observing its effects on drug influx and cytotoxicity. The genetic structure and regulation of this new membrane protein will be evaluated in drug-sensitive and drug-resistant cells. Since the SQM1 cDNA sequence has domains homologous to dihydrofolate reductase and epidermal growth factor receptor the applicant will analyze the function of these domains by transfection into human cells using a new mammalian expression vector. Interactions of MTX carrier protein with SQM1, possibly through a unique alpha-helical SQM1 domain with heptadic repeats of arginines, will be studied using affinity labelling of carrier proteins and immunoprecipitation with anti-SQM1 antibodies. The relationship between SQM1 gene expression induced by differentiation and hormones and the development of sensitivity to MTX and CDDP will be determined by immunoassays with anti-SQM1 antibodies and in-situ hybridization using SQM1 cDNA probes.