Mutational analysis of the FXNPXY motif within LDL receptor-related protein 1 (LRP1) reveals the functional importance of the tyrosine residues in cell growth regulation and signal transduction.

LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.