Differential activation of phosphotyrosine protein phosphatase activity in a murine T cell hybridoma by monoclonal antibodies to CD45.
The effect of two anti-CD45 (T200, LCA, Ly5) antibodies on the activation of the murine T-cell hybridoma 13.13 has been evaluated. These studies have been carried out in a system that did not require cross-linking or coclustering of antibodies. Activation of 13.13 cells with the anti-CD3 monoclonal antibody, 145.2C11, gave rise to rapid increases in intracellular calcium and interleukin-2 production. Additionally, within 1 min, phosphorylation on tyrosine of four major proteins of about 130,000, 110,000, 80,000, and 37,000 daltons could be seen. Pretreatment of the cells with the anti-CD45 mAb M1/89.18.7.HK markedly inhibited all three biological responses, while an alternate anti-CD45 antibody, M1/9.3.4.HL.2, had little effect. The two antibodies bound to CD45 with similar affinities, and no differences in the lateral mobility of antibody-CD45 complexes in the cell membrane were observed. The inhibition of activation of the cells by M1/89.18.7.HK was abrogated significantly both by the phosphotyrosine protein phosphatase inhibitor orthovanadate and by excess M1/9.3.4.HL.2. If M1/89.18.7.HK was added to the 13.13 cells after they had already been activated with anti-CD3, it very effectively stimulated dephosphorylation of substrates that had been phosphorylated on tyrosines prior to adding the anti-CD45 antibody. These results indicate that the phosphotyrosine protein phosphatase activity of CD45 is critical to its biological function and that bivalent (i.e. uncross-linked) anti-CD45 antibodies can give rise to markedly different responses. One of the antibodies, M1/89.18.7.HK, appears to behave much like a receptor ligand and is able to activate the enzymatic activity associated with the CD45 transmembrane protein.
Goldman
S J
SJ
Division of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts.
Uniyal
S
S
Ferguson
L M
LM
Golan
D E
DE
Burakoff
S J
SJ
Kiener
P A
PA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D015703:Antigens, CD / Q000276:immunology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate; D006649:Histocompatibility Antigens / Q000276:immunology / Q000502:physiology*; D006825:Hybridomas; D007376:Interleukin-2 / Q000096:biosynthesis; D007700:Kinetics; D017493:Leukocyte Common Antigens; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D006649:Histocompatibility Antigens; D007376:Interleukin-2; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium