Lateral diffusion of nerve growth factor receptor: modulation by ligand-binding and cell-associated factors.
We compared the properties in human melanoma cell line A875 and rat pheochromocytoma cell line PC12 of nerve growth factor receptor (NGFr). We also analyzed NGFr and a truncated NGFR lacking the cytoplasmic domain, which were transiently expressed in COS cells. The full-length NGFR expressed in COS cells bound nerve growth factor (NGF) with positive cooperativity, but A875 NGFr and truncated NGFr in COS cells did not display positive cooperativity. The anti-human NGFr monoclonal antibody NGFR5 was characterized and found not to compete with NGF for binding to NGFr. Fabs were prepared from NGFR5 and 192, an anti-rat NGFR monoclonal antibody that was previously shown not to compete with NGF for binding. Fluorescein-labeled Fabs were used to measure the distribution and lateral diffusion of the NGFr. NGFr expressed on COS and A875 cells are diffusely distributed, but NGFr on the surface of PC12 cells appeared, for some cells, to be patched. In A875 cells, 51% of the NGFr was free to diffuse with diffusion coefficient (D) approximately 7 X 10(-10) cm2/s. In COS cells, 43% diffused with D approximately 5 X 10(-10) cm2/s. There was no significant difference in diffusibility between the full-length NGFr and the truncated NGFr. We compared NGFr diffusion on PC12 cells in suspension or adherent to collagen-coated coverslips. For suspension cells, we obtained 32% recovery with D approximately 2.5 X 10(-9) cm2/s. On adherent cells, we obtained 17% recovery with 6 X 10(-9) cm2/s. Binding of NGF enhanced lateral diffusion of NGFr in A875 cells and in PC12 cells in suspension but did not alter lateral diffusion of NGFr in COS cells or in adherent PC12 cells. NGF had no effect on the diffusing fraction or the distribution of NGFR for any cell line.
Venkatakrishnan
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Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
McKinnon
C A
CA
Ross
A H
AH
Wolf
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DE
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D050356:Lipid Metabolism; D009414:Nerve Growth Factors / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D008024:Ligands; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor