Figure 7.

Internalization of P-selectin and P-selectin mutants in NRK cells. NRK cells expressing the indicated P-selectin cDNAs were labeled at 0°C with disulfide-linked biotin and were warmed to 37°C for the indicated intervals before removal of the biotin remaining on the cell surface by reduction with glutathione at 0°C. P-selectin was immunoprecipitated from detergent lysates, separated on nonreducing SDS-PAGE, and transferred to Immobilon membranes. Biotin was detected with ¹²⁵I-streptavidin and quantitated using the PhosphorImager. The D763A, P767A, and L768A mutants were internalized more slowly than native P-selectin (wt) but significantly faster than the same mutations expressed in the context of the LLP chimeric protein (Figure 2) (Setiadi et al., 1995).