Abstract. Diphtheria toxin (DT) receptor associateswith a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A humanmouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/ CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.IPHTHERIA toxin (DT), ~ secreted by Corynebacterium diphtheriae, is a cytotoxic protein (Mr = 58,342) that inhibits cellular protein synthesis in eukaryotes by inactivating elongation factor 2 through ADP ribosylation (for reviews, see references 18 and 48). Entry of the toxin, or at least the A fragment, into the cytoplasm is required for the cytotoxic action to occur (65). The toxin binds to a specific receptor on the cell surface (15, 37) and is then internalized by receptor-mediated endocytosis (42,45). A conformational change of the toxin molecule takes place in an acidic compartment, resulting in the interaction of hydrophobic domains of the toxin molecule with the lipid bilayer of the compartment (10, 11, 14, 52). Finally, the enzymatically active A fragment is translocated to the cytosol (43, 44), where it inactivates elongation factor 2. The sensitivity of cells to DT varies among species. Cells from many mammals, including humans and monkeys, are sensitive to 1. Abbreviations used in this paper: DRAP27, 27-kD diphtheria toxin receptor-associated protein; DT, diphtheria toxin; RDS, retinal degeneration slow proteins.DT, but those from rats and mice are not (40). The difference in DT sensitivity among cells is primarily determined by the number of DT-specific receptors on the surface (31,36,41).The Vero cell line, derived from monkey kidney, is one of the cell lines most sensitive to DT (40). The use of Vero cells has facilitated biochemical studies of the DT receptor. Chemical cross-linking of the protein(s) on the cell surface with DT fo...