The autosomal recessive hypercholesterolemia (ARH) protein interfaces directly with the clathrin-coat machinery

The PTB domain of ARH binds to the unphosphorylated FDNPVY internalization sequence in LDLR in a sequence-specific manner.

“In vitro studies with purified recombinant proteins indicate that the PTB domain of ARH binds to the unphosphorylated FDNPVY internalization sequence in LDLR in a sequence-specific manner (8,9).”

The clathrin box sequence in ARH can mediate high affinity binding to the heavy chain of clathrin.

“The clathrin box sequence in ARH can mediate high affinity binding to the heavy chain of clathrin (8,9).”

Some PTB domains, including the domain in ARH, bind phosphoinositides.

“some PTB domains, including the domain in ARH, bind phosphoinositides (8,11,12).”

ARH encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and clathrin and AP-2 in vitro.

“which encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and two components of the clathrin endocytic machinery, clathrin and adaptor protein-2 (AP-2) in vitro.”

In vitro pulldown studies defined sequences within ARH that interact with the LDLR, the terminal domain of the heavy chain of clathrin, and β2-adaptin.

“In vitro pulldown studies defined sequences within ARH that interact with the LDLR, the terminal domain of the heavy chain of clathrin, and ␤2-adaptin, a component of AP-2 (11, 13).”

Residues 212-216 of ARH comprise a clathrin box sequence (LLDLE) that mediates high affinity binding to the terminal domain of the heavy chain of clathrin.

“Residues 212-216 comprise a canonical clathrin box sequence (LLDLE) that mediates high affinity binding to the terminal domain of the heavy chain of clathrin (11,13,14).”

Data from in vitro pull-down experiments are consistent with ARH functioning as an endocytic adaptor that links the LDLR to structural components of the coated pit.

“Data from in vitro pull-down experiments are consistent with the hypothesis that ARH functions as an endocytic adaptor that links the LDLR to structural components of the coated pit (11,13).”

Amino acids 44-178 of ARH encode a PTB domain that binds the FDNPVY sequence in the LDLR cytoplasmic tail and phosphoinositides.

“Amino acids 44 -178 encode a phosphotyrosine binding (PTB) domain that binds the internalization sequence (FDNPVY) in the LDLR cytoplasmic tail (11,13) and phosphoinositides (11).”

Substitution of the tyrosine in the FDNPVY motif (Tyr807) with alanine resulted in a failure of ARH to promote clustering of LDLRs in WIF-B cells.

“Substitution of the tyrosine in the FDNPVY motif (Tyr 807 ) with alanine, which markedly reduced the interaction between ARH and the LDLR tail in vitro (11,13), also resulted in a failure of ARH to promote clustering of LDLRs in the WIF-B cells.”

The vast majority of LDLRs in lymphocytes are located in intracellular, EEA1-positive vesicles.

“In contrast the vast majority of LDLRs in lymphocytes are located in intracellular, EEA1-positive vesicles (10 -12).”

Following lipoprotein binding, the LDLR adaptor protein ARH binds to the intracellular NPxY motif in the cytoplasmic tail of LDLR.

“Following lipoprotein binding, the LDLR adaptor protein autosomal recessive hypercholesterolemia protein93,94 (ARH) binds to the intracellular NPxY motif in the cytoplasmic tail of LDLR (Fig. 6A).”

The phosphotyrosine-binding sequence also binds inositol phospholipids, which may anchor the protein to the plasma membrane.

“The phosphotyrosine-binding sequence also binds inositol phospholipids, which may anchor the protein to the plasma membrane (11).”

In non-confluent, non-polarized HepG2 cells, the ARH signal appears punctate and homogenously dispersed throughout the cytoplasm.

“As previously described for primary cultured fibroblasts and the HeLa epithelial cell line (11), we found that in non-confluent, non-polarized HepG2 cells the ARH signal appears punctate and homogenously dispersed throughout the cytoplasm (Fig. 1A).”

ARH is associated with β-adaptin and Dab2.

“We found that ARH is associated with ␤-adaptin and Dab2, in agreement with similar findings by Mishra et al. (11).”