Receptor-mediated endocytosis of insect lipoprotein : insight into LDL receptor functioning

The extracellular transport of water-insoluble lipids through the aqueous circulatory system of animals is mediated by lipoproteins. The lipoprotein of insects, lipophorin (Lp), is homologous to that of mammalian low-density lipoprotein (LDL). Moreover, an endocytic receptor for Lp has been identified (insect lipophorin receptor, LpR) that is homologous to the LDL receptor (LDLR). LpR was cloned from mRNA isolated from insect fat body, an organ that combines the functions of mammalian liver and adipose tissue. We transfected LDLR-expressing mammalian cells with LpR cDNA to study the endocytic uptake and intracellular pathways of LDL and Lp simultaneously. Our studies provide evidence that LDL and Lp follow distinct intracellular routes after receptor-mediated endocytosis. Multicolor imaging and immunofluorescence showed that upon internalization, LDL and Lp share endocytic vesicles. Subsequently, however, Lp evacuates the LDL-containing endosomes. In contrast to LDL, that is completely degraded in lysosomes after dissociating from LDLR, both Lp and LpR converge in the endocytic recycling compartment (ERC), as confirmed by colocalization of Lp with transferrin (Tf), a ligand that is transported to the ERC upon endocytosis by its receptor (TfR). Eventually, Lp is resecreted from the ERC with a t½ of ~13 min. Insect fat body cells internalize fluorescently-labeled Lp only when LpR is expressed. Expression of LpR is down-regulated on the fourth day after an ecdysis. Consequently, Lp is no longer internalized. Starvation experiments revealed that expression of LpR is regulated by the demand of fat body tissue for lipids. In Drosophila S2 transfectants, LDLR and TfR specifically mediate endocytosis of LDL and Tf, respectively. Both ligands colocalize in endosomes immediately after endocytic uptake, as observed in mammalian cells. However, in S2 cells, the ligands also colocalize after a chase, indicating that Tf is not recycled. Similar results were obtained with Lp internalized by LpR-transfected S2 cells. In contrast, fat body tissue that was subjected to similar conditions showed a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand. Class 5 familial hypercholesterolemia (FH) mutations in the LDLR gene impair ligand uncoupling and result in degradation of the receptor-ligand complex. To investigate whether the intracellular tail of LDLR induces degradation of LpR when Lp remains attached, the intracellular C-terminus of insect LpR was replaced by that of human LDLR. This LpR1-790LDLR791-839 hybrid receptor internalized and recycled Lp when expressed in mammalian cells. Apparently, the intracellular tail of LDLR does not elicit degradation of a ligand dissociation-deficient lipoprotein receptor. The hybrid approach was extended by generating a receptor harboring the ligand binding domain of LpR and the region from EGF domain to intracellular tail of LDLR. Although ligand endocytosis by this LpR1-342LDLR293-839 was unaffected, the receptor did not recycle. These findings suggest that latter hybrid follows an intracellular route that is similar to ligand dissociation-deficient LDLR. We studied the naturally occurring LDLR H562Y (LDLRH562Y) FH mutation in vitro, which showed constitutive recycling; the receptor, however, was degraded after LDL internalization. His562 in LDLR corresponds to Asn643 in LpR; yet, LDLRH562N did not recycle in complex with ligand, but behaves like LDLRH562Y. Substitution of the ligand binding domain of LpR by that of LDLR generates a hybrid receptor (LDLR1-292LpR343-850) that is able to bind LDL, and recycles constitutively, however, is degraded after LDL internalization. The data suggest that class 5 mutations can be divided in two distinct subclasses: (1) mutations that impair ligand dissociation (e.g. LDLRH562Y), and (2) those that prevent receptor recycling (e.g. LDLRDEGF).