Abstract
Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT.
Highlights
The released heparin-binding EGF-like growth factor (HB-EGF) is able to act as a mitogen by binding to the EGF receptor [9], while the remaining cell surface-bound proHBEGF is able to function as a juxtacrine growth factor [14] as well as a diphtheria toxin (DT) receptor in toxin-sensitive cells [8, 15]
It is possible that Mk CD9 increases DT sensitivity by (i) increasing the number of DT receptors through a chaperone-like function and/or protecting them from proteolytic cleavage and/or (ii) increasing DT receptor affinity by a physical interaction on the cell surface that changes the conformation of the receptor
Increasing the Levels of Cell Surface Mk proHB-EGF in LCD9 Cells Results in Cells with Increasing DT Sensitivity but with Decreasing Toxin Receptor Affinity—We investigated the effect of cell surface expression of Mk proHB-EGF on the DT sensitivity and DT receptor affinity of LCD9 cells
Summary
Materials—The plasmid vectors pcDNA3 and pCDM8, the Escherichia coli host strains TOP10FЈ and DH5␣, and the cDNA Cycle and Original TA Cloning (pCR2.1) kits were purchased from Invitrogen. This new host cell line was constructed by transfection of pMkHB-EGF into the normally DT-resistant mouse L-M(TKϪ) cell line and cloned as described previously [24]. A DNA fragment (designated as Ms1–34) encoding Ms CD9 amino acid residues 1–34 was PCR-amplified from pMsCD9 with primers MsCD9(5Ј) and ExI-1-down. A third DNA fragment (designated as Ms55– 226) encoding Ms CD9 amino residues 55–226 was PCR-amplified from pMsCD9 with primers ExI-2-up and MsCD9(3Ј). A DNA fragment (designated as Ms1–110) encoding Ms CD9 amino acid residues 1–110 was PCR-amplified from pMsCD9 with primers MsCD9(5Ј) and ExII-1-down. Two italic letters in the sequence of MsCD9(5Ј) and MsCD9(3Ј) primers indicate the extra sequence for the XbaI and BamHI digestions
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