Binding, surface mobility, internalization, and degradation of rhodamine-labeled .alpha.2-macroglobulin

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Abstract
Quantitative fluorescence methods were used to examine the fate of rhodamine-labeled .alpha.2-macroglobulin (R-.alpha.2M) after binding to cell-surface receptors on [rat kidney] NRK and [mouse embryo fibroblast] Swiss 3T3 cells. Measurements of fluorescence intensities in NRK cells fixed after incubation with R-.alpha.2M showed that uptake was saturable and that half-maximal uptake occurred at 130 nM R-.alpha.2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-.alpha.2M near 130 nM. It was estimated that NRK cells can take up 106 molecules of R-.alpha.2M/h via receptor-mediated endocytosis. The mobility of .alpha.2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The 2-dimensional effective diffusion coefficient of R-.alpha.2M receptors was .apprx. 8 .times. 10-10 cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-.alpha.2M by the cells was followed by using the loss of fluorescence from the 185,000-dalton band in sodium dodecyl sulfate-polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded .alpha.2M to low MW fragments with a t1/2 [half-time] of 15 min. Swiss 3T3 cells degraded about 75% of the .alpha.2M with a t1/2 of 1 h. The remaining 25% remained as the intact 185,000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.