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

We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found 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. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour 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 two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 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 185000-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 molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.