DAB389 interleukin-2 receptor binding domain mutations. Cytotoxic probes for studies of ligand-receptor interactions.
Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.
vanderSpek
J C
JC
Evans Department of Clinical Research, Boston University Medical Center Hospital, Massachusetts 02118 USA.
Sutherland
J A
JA
Ratnarathorn
M
M
Howland
K
K
Ciardelli
T L
TL
Murphy
J R
JR
D002460:Cell Line; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D004167:Diphtheria Toxin; D007376:Interleukin-2; D008024:Ligands; D015335:Molecular Probes; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; C078456:denileukin diftitox