Functional dissection of an AP-2 beta2 appendage-binding sequence within the autosomal recessive hypercholesterolemia protein

J Biol Chem. 2005 May 13;280(19):19270-80. doi: 10.1074/jbc.M501029200. Epub 2005 Feb 22.

Abstract

The autosomal recessive hypercholesterolemia (ARH) protein plays a critical role in regulating plasma low density lipoprotein (LDL) levels. Inherited defects in ARH lead to a hypercholesterolemia that closely phenocopies that caused by a defective LDL receptor. The elevated serum LDL-cholesterol levels typical of ARH patients and the pronounced accumulation of the LDL receptor at the cell surface of hepatocytes in ARH-null mice argue that ARH operates by promoting the internalization of the LDL receptor within clathrin-coated vesicles. ARH contains an amino-terminal phosphotyrosine-binding domain that associates physically with the LDL receptor internalization sequence and with phosphoinositides. The carboxyl-terminal half of ARH contains a clathrin-binding sequence and a separate AP-2 adaptor binding region providing a plausible mechanism for how ARH can act as an endocytic adaptor or CLASP (clathrin-associated sorting protein) to couple LDL receptors with the clathrin machinery. Because the interaction with AP-2 is highly selective for the independently folded appendage domain of the beta2 subunit, we have characterized the ARH beta2 appendage-binding sequence in detail. Unlike the known alpha appendage-binding motifs, ARH requires an extensive sequence tract to bind the beta appendage with comparably high affinity. A minimal 16-residue sequence functions autonomously and depends upon ARH residues Asp253, Phe259, Leu262, and Arg266. We suggested that biased beta subunit engagement by ARH and the only other beta2 appendage selective adaptor, beta-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics*
  • Adaptor Proteins, Signal Transducing / physiology*
  • Alanine / chemistry
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Arginine / chemistry
  • Arrestins / metabolism
  • Calorimetry
  • Carbocyanines / pharmacology
  • Cholesterol, LDL / blood
  • Clathrin / metabolism
  • Cytosol / metabolism
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / physiology*
  • Endocytosis
  • Fluorescent Dyes / pharmacology
  • Genes, Recessive
  • Glutathione Transferase / metabolism
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Hypercholesterolemia / genetics*
  • Kinetics
  • Lipoproteins, LDL / metabolism
  • Mice
  • Microtubule-Associated Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Peptides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, LDL
  • Sequence Homology, Amino Acid
  • Transcription Factor AP-2
  • Transcription Factors / chemistry*
  • Transcription Factors / physiology*
  • Two-Hybrid System Techniques
  • beta-Arrestins

Substances

  • Adaptor Proteins, Signal Transducing
  • Arrestins
  • CLASP1 protein, human
  • Carbocyanines
  • Cholesterol, LDL
  • Clathrin
  • DNA-Binding Proteins
  • Fluorescent Dyes
  • LDLRAP1 protein, human
  • Lipoproteins, LDL
  • Microtubule-Associated Proteins
  • Peptides
  • Receptors, LDL
  • Transcription Factor AP-2
  • Transcription Factors
  • beta-Arrestins
  • Green Fluorescent Proteins
  • 3,3'-dioctadecylindocarbocyanine
  • DNA
  • Arginine
  • Glutathione Transferase
  • Alanine