Normal Sorting but Defective Endocytosis of the Low Density Lipoprotein Receptor in Mice with Autosomal Recessive Hypercholesterolemia

Coated pits contain many potential binding partners for LDLR including AP2, PTB domain-containing adaptor proteins such as Dab2, and the clathrin heavy chain.

“In accordance with this mechanism, coated pits contain many potential binding partners for LDLR including AP2 (34), PTB domain-containing adaptor proteins such as Dab2 (12,28) and the clathrin heavy chain (35).”

ARH is required for normal uptake and degradation of LDL in human lymphocytes and in mouse liver.

“We have shown previously that ARH is required for normal uptake and degradation of LDL in human lymphocytes and in mouse liver (17,28), but the specific role of ARH in LDLRmediated endocytosis has not been defined.”

In wild-type mice, approximately two-thirds of the hepatic LDLRs are intracellular, whereas in Arh-/- mice the majority of immunodetectable LDLRs are located on the sinusoidal membrane.

“In wild-type mice, approximately two-thirds of the hepatic LDLRs are intracellular, whereas in Arh Ϫ/Ϫ mice the majority of immunodetectable LDLRs are located on the sinusoidal membrane (9).”

Serum cholesterol is much higher in LDLR-null than in arh-deleted mice.

“serum cholesterol is much higher in LDLR-null than in arh-deleted mice”

The distribution of LDLR in ARH-/- lymphocytes mirrors the distribution observed previously in hepatocytes from ARH-/- mice.

“The distribution of LDLR in the ARH Ϫ/Ϫ lymphocytes mirrors the distribution observed previously in hepatocytes from ARH Ϫ/Ϫ mice (28).”

ARH is required for the normal transit of LDLR from the cell surface to endosomes.

“These observations indicate that ARH is required for the normal transit of LDLR from the cell surface to endosomes.”

ARH is not constitutively associated with the LDLR-clathrin complex in hepatocytes.

“clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex (15).”

Chow-fed Arh-/- mice are only mildly hypercholesterolemic.

“Because chow-fed Arh Ϫ/Ϫ mice are only mildly hypercholesterolemic (9)”

ARH is not constitutively associated with the LDLR-clathrin complex.

“clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex.”

Experiments performed in ARH-deficient mice demonstrated that ARH does not direct LDLR to the sinusoidal membrane.

“Experiments performed in ARH-deficient mice demonstrated that this was not the case.”

In control untreated cells, the majority of ARH co-sedimented with vesicles containing EEA1 and Rab-4.

“As previously reported (15), in control untreated cells the majority of ARH co-sedimented with vesicles containing EEA1 and Rab-4 (Fig. 4A).”

In arh-/- mouse livers, LDLR appears to be normally sorted to the sinusoidal surface.

“In arh Ϫ/Ϫ mouse livers, LDLR appears to be normally sorted to the sinusoidal surface (15).”

Immunoprecipitation experiments demonstrated that, at the steady state, the majority of ARH protein appears in fractions from which the LDLR receptor is absent.

“Moreover, immunoprecipitation experiments demonstrated that, at the steady state, the majority of ARH protein appears in fractions from which the LDLR receptor is absent (15), clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex.”

Hepatic clearance of LDL from plasma is markedly retarded in arh−/− mice as well as in ARH patients.

“On the other hand, our observations concur with the evidence that hepatic clearance of LDL from plasma is markedly retarded in arhϪ/Ϫ mice as well as in ARH patients (12,15).”

The increase in CCP size by Dab2 and ARH might reflect the need to accommodate the very large ligands bound by LDLR and LRP (LDL particles, d∼22 nm and α2-macroglobulin, d∼18.5 nm).

“might reflect the need to accommodate the very large ligands bound by LDLR and LRP (Jones et al., 2003) (i.e., LDL particles, d∼22 nm and α2-macroglobulin, d∼18.5 nm; Krimbou et al., 1998), respectively.”