A single lysine of the two-lysine recognition motif of the D3 domain of receptor-associated protein is sufficient to mediate endocytosis by low-density lipoprotein receptor-related protein

Int J Biochem Cell Biol. 2011 Mar;43(3):431-40. doi: 10.1016/j.biocel.2010.11.017. Epub 2010 Dec 7.

Abstract

Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by complement-type repeats (CR) each comprising a binding pocket for a single basic amino acid residue. It has been proposed that at least two CRs are required for high-affinity interaction by utilising two spatially distinct lysine residues on the ligand surface. LDL receptor-related protein (LRP) mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We now addressed the role of the two critical lysines not only in LRP binding but also in LRP-dependent endocytosis. Variants of the third domain (D3) of receptor-associated protein (RAP) were created carrying lysine to alanine or arginine replacements at the putative contact residues K253, K256 and K270. Surface plasmon resonance revealed that replacement of K253 did not affect high-affinity LRP binding at all, whereas replacement of either K256 or K270 markedly reduced the affinity by approximately 10-fold. Binding was abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding. This suggests that despite their positive charge, arginine residues do not support receptor binding at all. Confocal microscopy and flow cytometry studies surprisingly revealed that the single mutants were still taken up and still competed for the uptake of full length RAP despite their receptor binding defect. We therefore propose that the presence of only one of the two critical lysines is sufficient to drive endocytosis.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Substitution / genetics
  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism*
  • Endocytosis*
  • Fluorescein-5-isothiocyanate
  • Humans
  • LDL-Receptor Related Proteins / metabolism*
  • Lysine / metabolism*
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Rats
  • Structure-Activity Relationship
  • Surface Plasmon Resonance

Substances

  • Carrier Proteins
  • LDL-Receptor Related Proteins
  • Mutant Proteins
  • Fluorescein-5-isothiocyanate
  • Lysine