Functional dissection of an AP-2 β2 appendage-binding sequence within the autosomal recessive hypercholesterolemia protein

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 carbosyl-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 β2 subunit, we have characterized the ARH β2 appendage-binding sequence in detail. Unlike the known α appendage-binding motifs, ARH requires an extensive sequence tract to bind the β 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 β subunit engagement by ARH and the only other β2 appendage selective adaptor, β-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.