Functional Dissection of an AP-2 β2 Appendage-binding Sequence within the Autosomal Recessive Hypercholesterolemia Protein

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 ␤2 subunit, we have characterized the ARH ␤2 appendagebinding sequence in detail. Unlike the known ␣ appendage-binding motifs, ARH requires an extensive sequence tract to bind the ␤ appendage with comparably high affinity. A minimal 16-residue sequence functions autonomously and depends upon ARH residues Asp 253 , Phe 259 , Leu 262 , and Arg 266 . We suggested that biased ␤ subunit engagement by ARH and the only other ␤2 appendage selective adaptor, ␤-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.Clathrin-coated vesicles are short lived transport intermediates fabricated solely for the preferential sorting and intracellular trafficking of select transmembrane proteins, bound ligands, and lipids (1-3). Clathrin coats assemble at several discrete intracellular sites where, typically, different cargo molecules are sorted. As the two major clathrin-associated heterotetrameric adaptor proteins (AP-1 and AP-2) are generally restricted to particular sorting stations within the cell, a long standing model has been that adaptors direct the process of cargo selection (4). Different subunits of the adaptor complex directly engage different classes of sorting sequences. The 2 subunit binds to YXXØ-type (where Ø is a bulky hydrophobic amino acid) sorting signals in a phosphorylation-dependent manner (3,5,6). In the basal state, the orientation of the 2 subunit within AP-2 makes the YXXØ interaction surface inaccessible. Phosphorylation on 2 residue Thr 156 is believed to alter the conformation of 2 to allow productive engagement of the YXXØ sequence (7-9). Another sorting sequence termed the (DE)XXXL(LI) motif is recognized by AP-1, AP-2, and AP-3 (6). Recent evidence suggests that the (DE)XXXL(LI) sequence is bound by a hemicomplex of the ␥ and 1 subunits in AP-1 or the ␦ and 3 subunits in AP-3 (10), although the precise location of the interaction site remains to be det...

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

“…Previously identified AP-2 interaction motifs range from small tripeptide (DPF/W) sequences (17) to as many as 16 residues found in ARH or arrestins (29). None of the previously characterized motifs are present in the regions flanking the BRAG2 clathrin boxes, and it is therefore likely that other, unidentified motifs mediate interactions with AP-2.…”

Atomic structure of the autosomal recessive hypercholesterolemia phosphotyrosine-binding domain in complex with the LDL-receptor tail

“…Together, this set of interactions enables ARH to function as an endocytic adaptor for the clathrin-mediated endocytosis of LDLR in the liver. Accordingly, ARH has been classified as a clathrin-associated sorting protein (CLASP) (12), a group of proteins that serve as a molecular bridge between receptors and the clathrin machinery for their endocytic internalization.…”

Correcting Estimators of θ and Tajima's D for Ascertainment Biases Caused by the Single-Nucleotide Polymorphism Discovery Process

“…The former is a transmembrane protein and the second a mannosidase. The latter encodes for a cytosolic protein that interacts with the LDL receptor, and mutations in it have cause hypercholesterolemia, an autosomal recessive disorder (Mishra et al 2005;Quagliarini et al 2007). Negative Tajima's D values $ À2 were found in windows containing HIST2H*, FCGR1A, and PPIAL4, a histone cluster, a fragment of the IgG receptor, and the peptidylprolyl isomerase A, respectively.…”