Abstract We studied the binding of 125 I‐labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO‐K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (≤23,000 cells per cm 2 for CHO‐K1 cells; ≤80,000 cells per cm 2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low‐ and high‐density cells was 33‐fold for CHO‐K1 cells and 19‐fold for Vero cells. We estimated the maximum number of DTX receptors on low‐density CHO‐K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO‐K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm 2 , respectively). Vero cells grown on a surface that had been conditioned by high‐density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low‐density cells were more sensitive to DTX than high‐density cells, suggesting that low‐density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO‐K1 and Vero cells were equally protected by SITS (4‐Acetamido‐4prime;‐Isothiocyano‐Stilbene‐2,2′‐disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO‐K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.
Characterization of an endoprotease-deficient mutant Chinese hamster ovary (CHO) cell, designated RPE.40, revealed that it bound less than 10% as much insulin as did its parent, CHO-K1. We examined processing of the endogenous insulin receptor in CHO-K1 and RPE.40 cells, and processing of the human insulin receptor expressed in these cells. RPE.40 cells did not process the endogenous insulin proreceptor to its subunit forms, and processed the human insulin proreceptor inefficiently. Accumulation of the proreceptor form of the insulin receptor was seen in both cases. Furin is a mammalian endoprotease that cleaves proproteins at a consensus sequence of basic amino acids found in the insulin proreceptor. Expression of mouse furin in RPE.40 cells restored normal processing of the endogenous and the human insulin receptor in these cells. In addition, expression of mouse furin corrected the reduced binding of insulin in RPE.40 cells, indicating that receptor function as well as processing was restored.
Abstract We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB‐R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6: 487–492). Both cell lines bind 125 I‐EGF and internalize the cell‐bound hormone at the same rate. However, when the degradation of internalized 125 I‐EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin‐resistant KB‐R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell‐bound 125 I‐EGF was degraded and released by 80 min in the KB and KB‐R2A cells, respectively. To investigate the fate of cell‐associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125 I‐EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125 I‐EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB‐R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB‐R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB‐R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor‐mediated endocytosis and that this defect is located beyond the initial endocytic step.
The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species.
PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.
