The very-low-density-lipoprotein receptor (VLDLR) is a transmembrane lipoprotein receptor of the low-density-lipoprotein (LDL) receptor family. VLDLR shows considerable homology with the members of this lineage. Discovered in 1992 by Tokuo Yamamoto and Sadao Takahashi, VLDLR is widely distributed throughout the tissues of the body, including the heart, skeletal muscle, adipose tissue, and the brain, but is absent from the liver. This receptor has an important role in cholesterol uptake, metabolism of apolipoprotein E-containing triacylglycerol-rich lipoproteins, and neuronal migration in the developing brain. In humans, VLDLR is encoded by the VLDLR gene. Mutations of this gene may lead to a variety of symptoms and diseases, which include type I lissencephaly, cerebellar hypoplasia, and atherosclerosis.
Protein structure VLDLR is a member of the low-density-lipoprotein (LDL) receptor family, which is entirely composed of type I transmembrane lipoprotein receptors. All members of this family share five highly conserved structural domains: an extracellular N-terminal ligand-binding domain with cysteine-rich repeats (also called ligand-binding repeats), an epidermal growth factor (EGF), an O-linked glycosylation sugar domain, a single transmembrane sequence, and a cytoplasmic domain which contains an NPxY sequence. The NPxY motif functions in signal transduction and the targeting of receptors to coated pits and consists of the sequence Asparagine-Proline-X-Tyrosine, where X can be any amino acid. Mimicking this general structure, VLDLR has eight, 40 amino acid long cysteine-rich repeats in its extracellular N-terminal ligand-binding domain. This is the main difference from the main member of the LDL receptor family, LDLR, which has only seven cysteine-rich repeats which are also 40 amino acids long. Each of these cysteine-rich repeats, in both VLDLR and LDLR, has three disulfide bonds and a coordinated Ca2+ ion. The N-terminus also consists of a glycine residue followed by 27 hydrophobic residues that constitute the signal peptide. Following this region is an EGF repeat, a β-propeller segment that plays a role in the pH-dependent dissociation of the ligand-receptor complex, and two more EGF repeats. The VLDLR O-linked glycosylation domain, next in the sequence, has many threonine and serine residues and totals 46 amino acids. The transmembrane domain, which functions in anchoring the receptors to the membrane, is 22 amino acids long. Final in the sequence is the 54 amino acid cytoplasmic domain, which contains the NPxY motif.
Isoforms The full-length human VLDLR genome is located on locus 9p24 on chromosome 9. It consists of a 40 kb segment that includes 19 exon-coding sequences, which is one more exon than encoded by LDLR. This extra exon in the VLDLR gene accounts for the extra cysteine-binding repeat not found in LDLR. Together, the exons making up the VLDLR gene encode a protein that is 873 amino acid residues long. VLDLR is known to exist as four different protein isoforms: type I, II, III, and IV. These different isoforms result from variations in alternative splicing. The transcript of type I VLDLR (VLDLR-I) is composed of all 19 exons. VLDLR-II, on the other hand, lacks exon 16, which encodes for the O-glycosylation domain between sugar regions. VLDLR-III lacks exon 4 that encodes the third ligand-binding repeat. Finally, VLDLR-IV transcripts lack both exon 16 and exon 4. It has been shown that 75% of VLDLR transcripts exist as isoform type II in mouse brain models. This shows that most VLDLRs in the brain are not glycosylated, as type II lacks exon 16 which encodes the O-glycosylation domain. Isoform type IV is known to be the second most prominent.
Evolutionary conservation There is a high level of conservation within the LDL receptor family. In particular, there is 50% overall sequence homology between VLDLR and ApoER2, another lipoprotein receptor of this family. Comparing LDLR and VLDLR, it was found that their primary structures are 55% identical within their ligand-binding regions. The modular structures of these two proteins are almost superimposable, with the only difference being the additional cysteine-rich repeat in VLDLR. This is demonstrated through the alignment of the two receptors according to their linker region; in LDLR, the linker region is located between cysteine-rich repeats four and five of its seven repeats while in VLDLR, the linker region appears to be between repeats five and six of its eight repeats.
VLDLR also shows high homology among various species. VLDLR of humans, mice, rats, and rabbits have been identified as 95% identical. Furthermore, there is approximately 84% conservation with the respective protein in chickens. This level of homology between species is much higher than that found for LDLR. Hence, these gene comparisons suggest that VLDLR and LDLR diverged before the LDLRs did among vertebrates.
Ligand binding VLDLR binds compounds containing apolipoprotein E (apoE). These ligands attach to the cysteine binding repeats in the N-terminus end. The difference in cysteine-rich repeats between the members of the LDL receptor family lead to the differences in binding affinity. VLDLR, in particular, binds VLDL and intermediate-density lipoprotein (IDL), but not LDL. This inability to bind LDL is due to VLDLR's incapability to bind apolipoprotein B (apoB), which is present in LDL.
Inhibitors Receptor-associated protein (RAP) and thrombospondin-1 (THBS1) have been identified as compounds that bind VLDLR. In many cases, these compounds exhibit inhibitory effects. THBS1 binds VLDLR and blocks ligand binding. This plays an important role in the reelin pathway, as THBS1 can block the attachment of reelin, while simultaneously stimulating the transcription factors normally activated by reelin. This binding of THBS1, however, does not induce the subsequent degradation of these transcription factors, as reelin does, and can thus lead to greatly amplified effects. The RAP protein acts similarly by blocking reelin from binding VLDLR. However, in this case phosphorylation of transcription factors, usually performed by reelin, is also blocked.
Tissue distribution and expression VLDLR is found throughout the body, with particularly high expression in fatty acid tissues due to their high level of triglycerides, VLDLR’s primary ligand. These tissues include those of the heart, skeletal muscle, and adipose layer. In addition, the receptor is found in macrophages, endothelial cells of capillaries, and in the brain, where it has a very different function from that found in the rest of the body. There is a preferred expression for VLDLR type I in the heart, skeletal muscle and brain, as opposed to type II, which is mainly expressed in non-muscular tissues including the cerebrum, cerebellum, kidney, spleen, and aortic endothelial cells. The highest expression of VLDLR is found in the brain. Although VLDLR is found in almost all regions of the brain, its highest expression is restricted to the cortex and cerebellum. Here, the receptor can be found on resting or activated microglia that are associated with senile plaques and cortical neurons, neuroblasts, matrix cells, Cajal-Retzius cells, glioblasts, astrocytes, oligodendrocytes, and region-specific pyramidal neurons. Despite its major role in cholesterol and fatty acid metabolism, VLDLR is not found in the liver. This phenomenon is mainly attributed to the very high levels of LDLR in these areas. In addition, it has been uncovered that this receptor is found, sub-cellularly, in the non-lipid raft sections of cell membranes.
Regulation Unlike LDLR, VLDLR does not exhibit any feedback mechanism, and hence intracellular lipoproteins are incapable of regulating it. This phenomenon is due to a difference in the sterol regulatory element-1 (SRE-1) of VLDLR. Normal SRE-1 sequences, like those found in LDLR, are characterized by two repeats of the codon CAC separated by two intervening C nucleotides (5’-CACCCCAC-3’). The sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor, targets the CAC repeats of SRE-1 to regulate the protein’s transcription. However, the VLDLR gene is encoded by two SRE-1-like sequences that contain single nucleotide polymorphisms. These polymorphisms disrupt the SREBP-1 binding to the CAC repeats, and hence eliminate the feedback mechanism seen in other proteins.
VLDLR expression is regulated by peroxisome proliferator-activated receptor-gamma (PPAR-γ). A 2010 study showed that the prescription drug Pioglitazone, an agonist of PPAR-γ, increases VLDLR mRNA expression and protein levels in experiments using mouse fibroblasts. The Pioglitazone treated mice exhibited a higher conversion rate of plasma triglycerides into epididymal fats. As expected, mice deficient in VLDLR did not show this same response. These results suggest that VLDLR is important in fat accumulation.
Many other hormones and dietary factors also regulate VLDLR expression. Thyroid hormone positively regulates VLDLR expression in skeletal muscles of rats, but not in adipose or heart tissues. In rabbits, VLDLR expression in heart muscle is up-regulated by estrogen and down-regulated by granulocyte-macrophage colony-stimulating factor. In trophoblast-derived cell lines, up-regulated VLDLR expression occurs when cells are incubated with hypolipidemic agents such as insulin and clofibrate. In contrast, 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) down-regulates VLDLR expression. Finally, VLDLR is affected by the presence of apoE and LDLR. The presence of apoE is required for VLDLR expression regulation, while the absence of LDLR alters the sterol-regulatory-element-1-like sequences of VLDLR to make them functional in only heart and skeletal muscle.
Function
Beyond the nervous system VLDLR is a peripheral lipoprotein receptor that functions in lipoprotein metabolism, cardiac fatty acid metabolism, and fat deposition. In effect, VLDLR will allow cholesterol to reach tissues from the bloodstream, where it may be used in cellular membranes. In addition, it will allow fatty acids to get into cells where they may be used as an energy source. Overall, VLDLR primarily modulates the extra-hepatic metabolism of triglyceride-rich lipoproteins.
Lipoprotein uptake VLDLR only plays a discrete role in lipid metabolism, but is more significant in stressed situations. Mice with double knockouts in VLDLR and LDLR have higher serum triglyceride levels than those with only a knockout in the LDLR gene. In addition, LDLR knockout mice overexpressing VLDLR have decreased serum triglyceride levels. Although fat deposition is close to normal without VLDLR, its role gains importance when LDLR is deficient. Despite this knowledge on its role in lipoprotein uptake, the complete mechanism of lipid metabolism performed by VLDLR is not fully understood.
Endocytosis VLDLR is known to employ endocytosis, although the exact mechanism of this process is unknown for this protein. Endocytosis is mediated through NPxY sequences known to signal for receptor internalization through clathrin-coated pits. The presence of this sequence in the cytoplasmic tail of VLDLR makes endocytosis possible. In general, lipoprotein receptors undergo a process by which they are endocytosed with their ligand into clathrin-coated pits. From here, they are together transported to early and late endosomes until reaching the lysosome. At this point, hydrolysis occurs and lipoprotein is released into the cytoplasm while the receptors are recycled back to the cell surface. It is not yet confirmed if VLDLR follows this exact mechanism, but one closely related to it is likely.
In the nervous system