Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1).
The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.
Pal
R
R
Department of Cell Biology, Advanced BioScience Laboratories, Inc., Kensington, Maryland 20895.
Nair
B C
BC
Hoke
G M
GM
Sarngadharan
M G
MG
Edidin
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology / Q000378:metabolism*; D004058:Diffusion; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005452:Fluoresceins; D015726:Giant Cells / Q000166:cytology; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D016399:Lymphoma, T-Cell; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D005452:Fluoresceins; D015699:HIV Envelope Protein gp120; C069153:6-(4,6-dichlorotriazinyl)aminofluorescein