Phenotypic Mutation 'r26' (pdf version)
Alleler26
Mutation Type missense
Chromosome19
Coordinate27,223,054 bp (GRCm39)
Base Change C ⇒ T (forward strand)
Gene Vldlr
Gene Name very low density lipoprotein receptor
Chromosomal Location 27,193,884-27,231,631 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The low density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. This gene encodes a lipoprotein receptor that is a member of the LDLR family and plays important roles in VLDL-triglyceride metabolism and the reelin signaling pathway. Mutations in this gene cause VLDLR-associated cerebellar hypoplasia. Alternative splicing generates multiple transcript variants encoding distinct isoforms for this gene. [provided by RefSeq, Aug 2009]
PHENOTYPE: Homozygous null mutants exhibit modest reductions in body weight and adiposity. In behavioral tests, mutants display deficits in contextual fear conditioning and long term potentiation. [provided by MGI curators]
Accession Number

NCBI RefSeq: isoform 1 (NM_013703), isoform 2 (NM_001161420); MGI: 98935

MappedYes 
Amino Acid Change Leucine changed to Phenylalanine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P98156
SMART Domains Protein: ENSMUSP00000025866
Gene: ENSMUSG00000024924

DomainStartEndE-ValueType
signal peptide 1 23 N/A INTRINSIC
EGF_like 32 68 7.38e1 SMART
LDLa 32 69 1.69e-16 SMART
LDLa 71 110 5.81e-15 SMART
LDLa 112 151 1.96e-12 SMART
LDLa 153 190 7.15e-15 SMART
LDLa 192 231 1.23e-13 SMART
LDLa 238 275 1.1e-15 SMART
LDLa 277 314 1.13e-12 SMART
LDLa 317 357 3.86e-11 SMART
EGF_CA 356 395 1e-5 SMART
EGF_CA 396 435 6.1e-10 SMART
Blast:LY 461 495 4e-15 BLAST
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000049145
Gene: ENSMUSG00000024924
AA Change: L701F

DomainStartEndE-ValueType
signal peptide 1 23 N/A INTRINSIC
EGF_like 32 68 7.38e1 SMART
LDLa 32 69 1.69e-16 SMART
LDLa 71 110 1.25e-14 SMART
LDLa 112 149 7.15e-15 SMART
LDLa 151 190 1.23e-13 SMART
LDLa 197 234 1.1e-15 SMART
LDLa 236 273 1.13e-12 SMART
LDLa 276 316 3.86e-11 SMART
EGF_CA 315 354 1e-5 SMART
EGF_CA 355 394 6.1e-10 SMART
LY 420 462 2.16e-1 SMART
LY 464 506 9.54e-12 SMART
LY 507 550 2.22e-12 SMART
LY 551 593 1.66e-11 SMART
LY 594 637 5.97e-4 SMART
EGF 664 709 2.16e-1 SMART
transmembrane domain 728 750 N/A INTRINSIC
Predicted Effect possibly damaging

PolyPhen 2 Score 0.662 (Sensitivity: 0.86; Specificity: 0.91)
(Using ENSMUST00000047645)
SMART Domains Protein: ENSMUSP00000130382
Gene: ENSMUSG00000024924

DomainStartEndE-ValueType
LDLa 1 26 1.58e0 SMART
EGF 28 64 4e-5 SMART
LY 88 130 2.16e-1 SMART
LY 132 174 9.54e-12 SMART
LY 175 218 2.22e-12 SMART
LY 219 258 3.25e-5 SMART
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000127329
Gene: ENSMUSG00000024924
AA Change: L742F

DomainStartEndE-ValueType
signal peptide 1 23 N/A INTRINSIC
EGF_like 32 68 7.38e1 SMART
LDLa 32 69 1.69e-16 SMART
LDLa 71 110 5.81e-15 SMART
LDLa 112 151 1.96e-12 SMART
LDLa 153 190 7.15e-15 SMART
LDLa 192 231 1.23e-13 SMART
LDLa 238 275 1.1e-15 SMART
LDLa 277 314 1.13e-12 SMART
LDLa 317 357 3.86e-11 SMART
EGF_CA 356 395 1e-5 SMART
EGF_CA 396 435 6.1e-10 SMART
LY 461 503 2.16e-1 SMART
LY 505 547 9.54e-12 SMART
LY 548 591 2.22e-12 SMART
LY 592 634 1.66e-11 SMART
LY 635 678 5.97e-4 SMART
EGF 705 750 2.16e-1 SMART
transmembrane domain 797 819 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.450 (Sensitivity: 0.89; Specificity: 0.90)
(Using ENSMUST00000167487)
SMART Domains Protein: ENSMUSP00000126730
Gene: ENSMUSG00000024924
AA Change: L742F

DomainStartEndE-ValueType
signal peptide 1 23 N/A INTRINSIC
EGF_like 32 68 7.38e1 SMART
LDLa 32 69 1.69e-16 SMART
LDLa 71 110 5.81e-15 SMART
LDLa 112 151 1.96e-12 SMART
LDLa 153 190 7.15e-15 SMART
LDLa 192 231 1.23e-13 SMART
LDLa 238 275 1.1e-15 SMART
LDLa 277 314 1.13e-12 SMART
LDLa 317 357 3.86e-11 SMART
EGF_CA 356 395 1e-5 SMART
EGF_CA 396 435 6.1e-10 SMART
LY 461 503 2.16e-1 SMART
LY 505 547 9.54e-12 SMART
LY 548 591 2.22e-12 SMART
LY 592 634 1.66e-11 SMART
LY 635 678 5.97e-4 SMART
EGF 705 750 2.16e-1 SMART
transmembrane domain 769 791 N/A INTRINSIC
Predicted Effect probably damaging

PolyPhen 2 Score 0.989 (Sensitivity: 0.72; Specificity: 0.97)
(Using ENSMUST00000172302)
Meta Mutation Damage Score Not available question?
Is this an essential gene? Possibly nonessential (E-score: 0.285) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(2) : Targeted, knock-out(1) Targeted, other(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01346:Vldlr APN 19 27217081 missense possibly damaging 0.93
IGL01575:Vldlr APN 19 27224031 missense probably benign
IGL01626:Vldlr APN 19 27221173 missense probably damaging 1.00
IGL02213:Vldlr APN 19 27218726 missense probably benign 0.09
IGL02365:Vldlr APN 19 27223025 missense probably damaging 1.00
IGL02488:Vldlr APN 19 27215675 missense probably damaging 1.00
IGL02708:Vldlr APN 19 27215485 missense possibly damaging 0.92
IGL02947:Vldlr APN 19 27217120 missense probably benign 0.03
disturbed UTSW 19 27216204 nonsense probably null
spotty UTSW 19 27216192 missense probably damaging 1.00
PIT4142001:Vldlr UTSW 19 27212269 missense probably benign 0.05
R0195:Vldlr UTSW 19 27215786 missense probably damaging 1.00
R0288:Vldlr UTSW 19 27218051 splice site probably benign
R0536:Vldlr UTSW 19 27217364 missense probably damaging 1.00
R0537:Vldlr UTSW 19 27225318 missense probably damaging 1.00
R0542:Vldlr UTSW 19 27213655 missense probably benign 0.01
R0594:Vldlr UTSW 19 27212219 missense probably damaging 1.00
R0624:Vldlr UTSW 19 27215663 missense possibly damaging 0.91
R0726:Vldlr UTSW 19 27215786 missense probably damaging 1.00
R1017:Vldlr UTSW 19 27218733 missense probably damaging 1.00
R1148:Vldlr UTSW 19 27218691 missense probably benign 0.01
R1148:Vldlr UTSW 19 27218691 missense probably benign 0.01
R1443:Vldlr UTSW 19 27217121 missense possibly damaging 0.91
R1493:Vldlr UTSW 19 27218691 missense probably benign 0.01
R1520:Vldlr UTSW 19 27224466 missense possibly damaging 0.96
R1520:Vldlr UTSW 19 27217943 missense probably damaging 0.99
R1657:Vldlr UTSW 19 27223070 missense probably benign 0.00
R1901:Vldlr UTSW 19 27218709 missense probably damaging 1.00
R2047:Vldlr UTSW 19 27212238 missense probably damaging 1.00
R2258:Vldlr UTSW 19 27215786 missense probably damaging 1.00
R2273:Vldlr UTSW 19 27225415 missense probably damaging 1.00
R2423:Vldlr UTSW 19 27213688 missense possibly damaging 0.49
R3196:Vldlr UTSW 19 27220554 missense probably damaging 0.98
R3752:Vldlr UTSW 19 27215731 missense probably damaging 1.00
R3801:Vldlr UTSW 19 27195021 missense probably damaging 0.99
R3835:Vldlr UTSW 19 27212214 missense probably damaging 1.00
R4027:Vldlr UTSW 19 27215713 missense probably benign
R4301:Vldlr UTSW 19 27215802 missense possibly damaging 0.80
R4470:Vldlr UTSW 19 27212219 missense probably damaging 0.96
R4541:Vldlr UTSW 19 27216192 missense probably damaging 1.00
R4765:Vldlr UTSW 19 27217947 missense probably damaging 1.00
R4771:Vldlr UTSW 19 27217290 missense probably damaging 0.97
R4795:Vldlr UTSW 19 27216252 splice site probably null
R4839:Vldlr UTSW 19 27215465 missense probably damaging 1.00
R5074:Vldlr UTSW 19 27215677 missense probably damaging 1.00
R5134:Vldlr UTSW 19 27216212 nonsense probably null
R5281:Vldlr UTSW 19 27221631 missense probably benign 0.44
R5466:Vldlr UTSW 19 27217243 critical splice acceptor site probably null
R5514:Vldlr UTSW 19 27221624 missense probably damaging 0.97
R5886:Vldlr UTSW 19 27221171 missense probably benign 0.03
R5889:Vldlr UTSW 19 27217064 missense probably damaging 1.00
R6110:Vldlr UTSW 19 27215477 missense possibly damaging 0.92
R6343:Vldlr UTSW 19 27223049 missense probably damaging 0.99
R6833:Vldlr UTSW 19 27217974 missense probably damaging 1.00
R6838:Vldlr UTSW 19 27225370 missense probably damaging 1.00
R7169:Vldlr UTSW 19 27221728 missense probably benign
R7197:Vldlr UTSW 19 27212241 missense probably benign 0.36
R7304:Vldlr UTSW 19 27216004 missense possibly damaging 0.93
R7403:Vldlr UTSW 19 27213674 nonsense probably null
R7658:Vldlr UTSW 19 27220536 missense probably benign 0.33
R7754:Vldlr UTSW 19 27195015 start codon destroyed probably benign 0.01
R8105:Vldlr UTSW 19 27216204 nonsense probably null
R8377:Vldlr UTSW 19 27212258 missense probably damaging 1.00
R8529:Vldlr UTSW 19 27207656 missense probably benign 0.03
R8777:Vldlr UTSW 19 27217946 missense probably benign 0.00
R8777-TAIL:Vldlr UTSW 19 27217946 missense probably benign 0.00
R9380:Vldlr UTSW 19 27216192 missense possibly damaging 0.63
R9400:Vldlr UTSW 19 27216175 missense probably damaging 0.99
R9483:Vldlr UTSW 19 27224031 missense probably benign 0.00
R9502:Vldlr UTSW 19 27218742 missense probably damaging 1.00
R9509:Vldlr UTSW 19 27221687 missense probably benign 0.44
R9630:Vldlr UTSW 19 27207623 missense probably damaging 1.00
R9767:Vldlr UTSW 19 27212274 missense probably damaging 1.00
R9768:Vldlr UTSW 19 27218720 missense possibly damaging 0.47
Mode of Inheritance Autosomal Recessive
Local Stock None
Repository

Dr. Xiaohua Gong, University of California at Berkeley

Last Updated 2016-09-20 12:20 PM by Katherine Timer
Record Created unknown
Record Posted 2011-10-25
Phenotypic Description

Figure 1: Fundus photographs show depigmented patches in the r26 homozygous mutant mice. In comparison to the fundus image from a 3-week-old heterozygous r26/+ control mouse (A), the homozygous r26/r26 mutant mouse at the same age displayed depigmented patches (B). A homozygous r26/r26 mutant mouse at 4 weeks of age showed depigmented patches (arrows) in the fundus photograph (C) that match the hyperfluorescent spots (arrows) in the angiogram (D).

The r26 phenotype was identified in ENU-mutagenized G3 mice.  Homozygous r26 mutant mice in the C57BL/6J genetic background were mated to wild-type C3H/HeN mice to generate heterozygous hybrid mice.  Backcrossed mice were generated by crossing the heterozygous hybrid mice with homozygous r26 mutant mice; these mice were used for phenotyping.  Indirect ophthalmoscopy revealed that the retinas in r26 homozygous male mice presented with depigmented patches in the retina (Figure 1B); heterozygous r26 mice had normal retinal pigmentation (Figure 1A) (1).   In the r26 mutant, fluorescein angiography showed that the depigmented patches overlapped with hyperfluorescent spots (Figure 1C, 1D).

Figure 2: Histology of r26 mutant retinas. Compared with the 4-week-old wild-type (+/+) control (A), the homozygous r26/r26 mutant at the same age showed a relatively normal number of photoreceptor nuclear layers (B). However, some areas of the ONL were distorted, and abnormal retinal vessels were growing from the inner retinal layers into the subretinalspace (B, arrows). (C) Enlarged view of a region in (B) shows retinal vessels in the subretinalspace, growing from the OPL toward the disorganized RPE layer (arrows). The photoreceptor cells were distorted along the overgrown vessels. (D) A 2-month-old r26/r26 retinal section displays a region of retinal ONL that grew to the RPE. (E, F) Fourteen-month-old r26/r26 retinal sections show degenerated photoreceptor layers with four to seven remaining rows of ONL. The subretinalspace and RPE layer were disorganized, although the Bruch's membrane seemed to be intact (E). Arrows: retinal vessel in the ONL (F). Scale bars: (A, B) 50 μm; (C–F) 20 μm.

Histology revealed that at four weeks of age, there were structural abnormalities in the homozygous retina (Figure 2B, 2C), including distortions and vacuoles in the outer nuclear layer (ONL), the subretinal space and the retinal pigment epithelium (RPE), compared to the wild-type cohort (Figure 2A).  At two months, photoreceptor cell loss, including the loss of inner and outer segments, became apparent (Figure 2D).  Retinal degeneration was prominent by fourteen months with vessellike structures appearing in the near the retinal pigment epithelium (RPE) (Figure 2E, 2F) (1)

Figure 3: Abnormal retinal vessel growth in the r26 mutant retina. (A) Immunostainingof 3-week-old wild-type and r26 mutant retinas. In the wild-type control retina, vascular endothelial cells stained by an anti-CD31 antibody were observed in the OPL, IPL, and GL. However, in the homozygous r26mutant retina, CD31-positive signals were expressed not only in the OPL, IPL and GL, but also in the photoreceptor ONL. (B) 3D images of retinal vasculature labeled with a lipophilic fluorescent dye in 8-week-old r26 littermates. Top: 3D front views of retinal vasculature; bottom: the same images rotated 90°. The heterozygous r26 control (r26/+) shows a normal three-layer vessel network in the GL, IPL, and OPL. No apparent difference is visible between the front views of the homozygous r26 mutant (r26/r26) retinal vasculature and the r26/+ control; bottom: overgrown vessels that extend beyond the OPL toward the subretinal space in the r26/r26 retina (arrow).

Further studies found that the r26 homozygous mouse presented with uncontrolled retinal blood vessel growth in the photoreceptor cell layer and subretinal space (1). The presence of vascular endothelial cells throughout the outer plexiform layer (OPL), inner plexiform layer (IPL), ganglion cell layer (GCL), ONL and subretinal space was detected in the homozygous r26 retina by immunohistochemistry using an endothelial cell-specific antibody (to CD31) (1).  At as early as three weeks postnatal, CD31 positive vascular endothelial cells were detected in the outer nuclear layer and subretinal space, a unique finding to the wild-type or r26 heterozygote cohort (Figure 3A).  The neovascularization was also demonstrated using 3D reconstruction of retinal vasculature in a retinal whole-mount after perfusion with a fluorescent dye (Figure 3B) (1).  The r26 homozygous retina had abnormal vessels that extended beyond the outer plexiform layer (OPL) toward the subretinal space (Figure 3B, right, arrow).

The subretinal neovascularization observed in the r26 homozygous mouse is similar to that observed in the Vldlr-/- mouse.

Nature of Mutation

The r26 locus was identified on chromosome 19 by genome-wide linkage analysis (LOD = 4.8) (1). Previous studies characterizing the retinal phenotype of a very low density lipoprotein receptor (Vldlr) knockout mouse (Vldlr-/-) reported a retinal phenotype similar to that of the r26 mutant (2-8).  Also, the Vldlr gene resides on Chromosome 19.  To determine if the r26 mutation was in Vldlr, retinal cDNA was synthesized from the r26 homozygous mouse and Vldlr was directly sequenced.  Sequence analysis determined that there was a C to T mutation at nucleotide 2834 (in exon 15 (of 19)) of the Vldlr gene.

variant 1:

2819 CTCGAAGAAAATGGACGAGAGTGTCAAAGTACT

742  -L--E--E--N--G--R--E--C--Q--S--T-

variant 2:

2819 CTCGAAGAAAATGGACGAGAGTGTCAAAGGATC

742  -L--E--E--N--G--R--E--C--Q--R--I-

The C2239T mutation codes a premature stop codon in both Vldlr transcript variant 1 (NM_013703) and 2 (NM_001161420).  The mutated nucleotide is indicated in red lettering (above), and results in conversion of codon 747 (Arg) to a premature stop codon in the VLDLR protein.  The r26 mutation is a loss-of-function mutation.

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 4. Domain structure of VDLR. The r26 mutation results in conversion of Arginine to a stop codon at amino acid 747 of the encoded protein. SP, Signal peptide; R, LDLR class A domain; EGFL, EGF-like; B, LDLR class B domain; OD, O-linked sugar domain; TM, transmembrane domain; CT, cytoplasmic tail

VLDLR is a highly conserved 873 amino acid, single-pass (type 1) transmembrane receptor from the LDL receptor family. Other members of the LDLR receptor family include ApoER2/LDL receptor-related protein 8 (LRP8), LDL receptor-related protein 1 (LRP1), LRP1B, Megalin/GP330, SorLA/LR11, LDL receptor-related protein (LRP5/6, see r18), α2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (α2MR/LRP) and LDL receptor-related protein 4 (LRP4) (9-11)
 
Structural characterization of the VLDLR protein has revealed 5 highly conserved domains (Figure 4):
  1. VLDLR’s N-terminus is an extracellular ligand-binding domain with cysteine-rich repeats, the first 27 amino acids of which serve as the signal peptide.  The cysteine-rich repeats are a part of LDLR class A domains that consist of approximately 40 amino acids.  Each cysteine-rich repeat contains two loops connected by three disulfide bonds.  VLDLR’s N-terminus distinguishes it from the other LDLR family members in that VLDLR has a single exon (exon 5) that encodes the first cysteine-rich class A domain (12).  VLDLR has eight class A domains whereas LDLR contains seven, a difference that may account for its unique ligand binding specificity. The class A domain is similar to cysteine-rich repetitive sequences found in complement proteins C8 and C9 (13).
  2. VLDLR has an extracellular epidermal growth factor (EGF) domain encoded by amino acids 356-750.  This region of VLDLR is similar to a region of the epidermal growth factor (EGF) precursor, and encodes three calcium-binding EGF-like repeats separated by six LDLR class B repeats (also named YWTD motifs after the most conserved region of the repeat).  In each EGF-like repeat are six conserved core cysteines that form three disulfide bridges.  The LDLR class B repeats of VLDLR are predicted to form a six-bladed β-propeller structure, as in LDLR (14;15).
  3. An extracellular O-linked glycosylation domain is a 40 amino acid segment (aa 751-790) that is rich in serine and threonine resides.
  4. VLDLR’s single transmembrane domain is encoded by amino acids 798-819.
  5. VLDLR’s 54 amino acid cytoplasmic C-terminus contains an NPxY motif.  The NPxY motif is essential for binding of disabled-1 (Dab1), a protein required for the activation of the Reelin signaling pathway (11;16;17)as well as for binding of the receptor to coated pits (18).
Figure 5. 3D structure of the extracellular domain of the LDL receptor. UCSF Chimera model is based on PDB 1N7D, Rudenko et al. Science. 298, 2353-2358 (2002). This image is interactive. Click on the 3D structure to view it rotate.
The three dimensional structure of VLDLR has not been determined, but the structure of LDLR’s extracellular domain (19) provides insight into how ligand binding and release are likely to work for both LDLR and VLDLR (Figure 5). LDLR binds cholesterol-carrying lipoproteins from plasma circulation by receptor-mediated endocytosis (20).  At neutral pH, the extracellular domain of LDLR binds to LDL via apoB-100 (21), triggering endocytosis.  The acidic environment of the endosome causes release of the ligand, leading to its subsequent lysosomal degradation, while the receptor is recycled back to the cell membrane.  In the crystal structure at pH 5.3, the ligand binding domain (class A repeats R2 to R7) folds back as an arc over the EGF precursor homology domain (19).  Class A repeats R4 and R5, which are critical for lipoprotein binding (22), associate with the β propeller of the EGF precursor homology domain.  Thus, within the endosome the β propeller may function as an alternate substrate for the ligand binding domain, displacing and thereby releasing the bound lipoprotein.  In the case of VLDLR, binding to lipoproteins is via apoE.
 
Cells transfected with a mutant VLDL receptor lacking both the EGF precursor homology domain and the O-linked glycosylation domain provide information on the mechanism of ligand binding and release by VLDLR. The mutants were able to bind and internalize receptor associated protein (RAP), an LDLR-associated receptor antagonist and molecular chaperone in the endoplasmic reticulum that prevents premature receptor-ligand interactions (23-25). However, RAP failed to dissociate from the mutant receptor and consequently was not degraded, implicating the EGF precursor domain and O-linked glycosylation domain in ligand release within endosomes.
 
The mouse Vldlr gene has nine splice variants, six of which are protein coding.  Two of the protein coding variants have been validated, Vldlr-1 (NM_013703.2) and Vldlr-2 (NM_001161420.1). The 2622 bp Vldlr-1 transcript encodes an 873 aa protein from 19 exons; the 3855 bp Vldlr-2 transcript encodes an 845 aa protein variant from 18 exons (this variant lacks an in-frame exon in the 3’ coding region).  Human Vldlr codes several transcripts by alternative splicing, four of which have been characterized (11;26)Vldlr-a is derived from all of Vldlr’s 19 exons; Vldlr-b lacks the exon 16-derived sequence which encodes the O-linked glycosylation domain; Vldlr-c lacks the third ligand-binding repeat encoded by exon 4 and Vldlr-d lacks exon 4 or 16 (11)
 
The r26 mutation results in truncation of the protein following amino acid 746, close to the boundary of the EGF precursor homology domain and the O-linked sugar domain.  The mutation leaves the EGF precursor homology domain essentially intact, and affects both variant 1 and variant 2 sequences in the same way because it occurs 13 nucleotides 5’ to the alternatively spliced exon.  The premature stop codon created at position 747 results in nonsense-mediated decay of the Vldlr mRNA and reduced expression of the VLDLR protein.
 
Expression/Localization

Vldlr was initially isolated from rabbit heart cDNA, but it has been subsequently cloned in chicken, mouse, cattle, monkey and human (16;17;27). Vldlr has been documented in most tissues, with highest expression in the brain, heart, skeletal muscle and kidney (11;16;27). Vldlr is expressed in nearly all regions of the brain by several cell types (11;16;26;28;29).  Lesser amounts of Vldlr are expressed in ovary, lung, testis, spleen, the adrenal gland, adipose and very little, if any, was detectable in liver and small intestine (16). Subsequent studies demonstrated that Vldlr is expressed in endothelial cells, such as in the endothelium of capillaries and small arterioles of skeletal muscle, heart, ovary, and brain, and in smooth muscle cells of the heart (30-32). Vldlr is also predominantly expressed in the eye within the RPE, GCL, optic nerve, retinal epithelium cells (RECs) and around the outer limiting membrane (OLM) (6).  Northern blot analysis demonstrated that the expression pattern of the Vldlr transcripts is tissue-specific:  full length Vldlr (Vldlr-1) is mainly expressed in the heart, muscles and brain; Vldlr-2 is expressed mainly in the kidney, spleen, adrenal gland and testis (33)

The VLDLR protein is localized on the cell surface, traverses through endosomes upon endocytosis, and is subsequently recycled to the cell surface (20).

Background

Lipoproteins are complexes of lipids and proteins that enable insoluble fats and cholesterol to be transported through the bloodstream to various tissues.  In the post-absorptive (fasted) condition, lipoproteins are secreted by the liver in the form of VLDL and contain lipids originating from adipose tissue.  There are five types of lipoproteins classified based on their density (Table 1).  Cell surface receptors regulate the internalization of lipoproteins into tissues, predominantly adipose and muscle tissue, where the transported fatty acids are either stored or used for fatty acid oxidation.

Table 1. Lipoproteins

Class

Density (g/mL)

Function

high density lipoprotein (HDL)

>1.063

carry cholesterol from tissues to liver

low density lipoprotein (LDL)

1.019-1.063

carry cholesterol from liver to tissues

intermediate density lipoprotein (IDL)

1.006-1.019

remnants of VLDL produced by lipolysis

very low density lipoprotein (VLDL)

0.95-1.006

carry triglycerides from liver to tissues; source of HDL and LDL

chylomicrons

<0.95

carry triglycerides from intestine to tissues; source of HDL and LDL

 

Lipid raft-sorting lipoprotein receptors are associated with clathrin-coated pits, indicating a role for the receptors in ligand endocytosis and degradation. In general, neuronal signaling-activated LDLRs promote an increased activation of SFKs and a decreased activation of Jnk (11).  In conjunction with activation of SFKs and a decrease in Jnk signaling, there is also an indirect activation of extracellular signal-regulated kinases 1 & 2 (ERK1 & 2) through calcium-medicated signal transduction pathways (11)

VLDLR was first discovered by screening an LDL receptor-subtracted rabbit heart cDNA library for clones that would cross-hybridize to an LDL receptor probe under low stringency conditions (13).  It was shown to recognize and mediate the uptake, via endocytosis, of apoE-containing VLDL, β-migrating VLDL (VLDL in which the proportions of triglycerides and cholesterol are altered by dietary challenge with cholesterol), and IDL (remnants of VLDL produced by lipolysis) (13). VLDLR is involved in tissue delivery of VLDL-triglyceride (TG)-derived FFA by facilitating the expression of lipoprotein lipase (LPL) (34).  Changes in serum triglyceride levels upon the absence or overexpression of VLDLR suggest that VLDLR affects the peripheral uptake of VLDL triglycerides

VLDLR acts as a peripheral lipoprotein receptor for VLDL triglycerides (13).  Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides (35).  VLDLR-III exhibited the highest capacity in binding to apoE enriched beta-VLDL in vitro and was more effective in removing apoE containing lipoproteins from the circulation than other variants in vivo (26).

Figure 6.  VLDLR regulation of CNV through the Wnt-Frizzled/LRP5/6 pathway.  (left) Without the Wnt ligand, cytosolic ß-catenin is bound to the destruction complex (APC-axin-GSK3ß-CK1).  ß-catenin is phosphorylated by GSK3ß, ubiquitylated (Ub), and degraded by the proteasome.  The loss of ß-catenin expression prevents transcription of Wnt target genes. (right)  When Wnt is present, it binds the Frizzled/LRP5/6 co-receptor complex, which activates Disheveled (DVL/DSH).  By an unknown mechanism, casein kinase I (CK1/CK2) inactivates GSK3ß in the degradation complex allowing for the accumulation of a hypo-phosphorylated ß-catenin in the cytoplasm.  The ß-catenin translocates to the nucleus where it binds TCF/LEF, facilitating the transcription of Wnt target genes (i.e. VEGF).

VLDLR binds to and internalizes triacylglycerol-rich apolipoprotein (apo) E-containing lipoproteins including VLDL, β-migrating VLDL, and intermediate density lipoproteins (IDL) (13).  VLDLR interacts with several other proteins through its different domains.   These proteins activate a number of cell signaling pathways and are responsible for essential cellular functions.  Reelin, presenelin, apolipoprotein E (apoE), thrombospondin and F-spondin interact the VLDLR extracellular domain.  Reelin is secreted and promotes cortical layer formation through interactions with cytoskeleton-related proteins.  Reelin activates Src-family kinases (SFKs) and facilitates the phosphorylation of Dab-1 (36-40).  Once bound by VLDLR, Reelin is endocytosed via clathrin-coated vesicles and degraded. Presenilin-1 (Psen1) regulates the PI-3/Akt pathway through Psen1-associated gamma-secretase activity (41).  ApoE binds to VLDLR’s ligand binding repeats and is involved in phospholipid and cholesterol homeostasis.  ApoE binding to VLDLR leads to a release of VLDLR’s extracellular domains and accumulation of its C-terminal fragments (11).  Thrombospondin (TSP-1 and 2) and F-spondin binding to VLDLR is involved in neurite outgrowth, neuronal migration and synaptogenesis as well as in angiogenesis and cell cycle progression through an inhibition of PI3-K and MAPK pathways (11;42).  Furthermore, binding of VLDLR to F-spondin functions to regulate cell-cell and cell-matrix communications via the Dab-1-related signaling pathway (11;43).  VLDLR interacts with α- and γ-secretases through its membrane spanning domain facilitating the cleavage of VLDLR following ligand binding (11;44). Cleavage by γ-secretase generates soluble intracellular domains of VLDLR.  Interactions with disabled-1 (Dab-1), Pafah1b complex, and FE65 occur through the intracellular domain of VLDLR.  Dab-1 binds a 14-residue peptide surrounding VLDLR’s NPxY motif to facilitate neuronal migration and cell surface localization (11).  The alpha subunits of the Pafah1b complex bind to VLDLR at the NPxY sequence that is followed by a leucine.  This protein-protein interaction promotes clathrin-dependent receptor endocytosis and neuronal migration (11).  FE65 binds to VLDLR’s NPxY sequences at its PTB1 domain.  FE65 has a documented role as an amyloid precursor binding protein in Alzheimer’s Disease (11)

It is proposed that VLDLR binds and/or internalizes its ligands or transduces extracellular signals across the cell membrane (33).  VLDLR can interact with various ligands, contributing to its functional diversity, including lipoprotein lipase (LPL), receptor-associated protein (RAP), thrombospondin- 1, urokinase plasminogen activator/plasminogen activator inhibitor-1 complex, tissue factor pathway inhibitor (TFPI) (uPA/PAI-1) and several other proteinase-serpin complexes (33).  Along with its role in fatty acid and cholesterol metabolism,VLDLR is also essential for cell proliferation, migration and differentiation (11;16;17). VLDLR facilitates neural development, migration and other processes in the central nervous system through its interaction and activation of Reelin-associated signaling (1;11); Vldlris involved in cell migration through activation of the uPA pathway (33)

Age-related macular degeneration (AMD) is a retinal disease in which central and color vision are lost, with progression to complete vision loss (5).  There are several factors that are associated with the development and progression of AMD, including, but not limited to, oxidative stress and inflammation of the retina (2;4;45). In AMD, choroidal neovascularization initiates and progresses, leading to retinal scarring and vision loss (6).

Figure 7.  VLDLR regulation of CNV. VLDLR expression regulates the levels of the Wnt co-receptor LRP5/6 and the pro-angiogenic factor, VEGF.  The regulation of VEGF expression prevents choroidal neovascularization (CNV) from initiating in the eye.  Without VEGF expression (i.e. in the VEGF-/- mouse model), LRP5/6 expression increases, leading to an increase in VEGF expression.  The increase in the expression of VEGF leads to stimulation of CNV in the eye and subsequent vision loss.

VLDLR expression has been shown to be essential for maintaining vascular homeostasis during retinal development and in retinal pathologies such as diabetic retinopathy and retinal angiomatous proliferation, a subtype of AMD (1;4;5).  The Vldlr knock-out (Vldlr-/-) mouse retina displays choroidal neovascularization through activation of the Wnt signaling pathway and subsequent upregulation of vascular endothelial growth factor (Vegf), an angiogenesis stimulator (1;3;7;46) (Figure 6). Wnt signaling is initiated upon the binding of the Wnt ligand to the frizzled/LDL receptor-related protein 5/6 co-receptor complex (3).  When the Wnt ligand is not present, the transcription factor, ß-catenin is phosphorylated and subsequently degraded.  Upon binding of the Wnt ligand to the co-receptor complex, ß-catenin accumulates and translocates to the nucleus, regulating numerous target genes, including Vegf (3)

In the Vldlr-/- retina and RPE, there is activation of the Wnt signaling pathway through an upregulation of the co-receptor complex LRP5/6 (Figure 7) (3;46). VLDLR and LRP5/6 both belong to the LDL receptor gene family, but an interaction between them was only recently documented (3).  It is proposed that Vldlr regulates LRP5/6 at the transcription level, possibly through an intracellular signaling pathway through which VLDLR regulates expression of target genes such as LRP5/6 (3).  Subsequently, as a result of changes in the expression of LRP5/6, phosphorylation of Gsk-3β and β-catenin are altered, leading to overexpression of VEGF (3). The Vldlr-/- mouse develops the characteristic features of AMD as early as postnatal week two (7), including cone photoreceptor cell degeneration (and subsequent rod photoreceptor cell loss), retinal inflammation, vascular leakage and leukostasis in the vasculature of the retina (3).  As these pathological changes progress and the upregulation of VEGF persists, choroidal neovascularization (CNV) occurs leading to vision loss (3).

Mutations in Vldlr have been identified in human patients with disequilibrium syndrome or congenital ataxia, a form of cerebral palsy (OMIM: 224050)  (47-49).  In disequilibrium syndrome, patients present with cerebellar ataxia and mental retardation with delayed quadrupedal locomotion (47).  Some patients also have other pathologies including brain malformations and congenital infections (49).  In one study, a homozygous nonsense mutation in exon 10 of Vldlr (C1342T) led to the coding of a premature stop codon (R448X), truncation of the transmembrane domain and a portion of the receptor domain at the N-terminus; the mutant mRNA underwent nonsense-mediated decay (48).  In another study, there is a mutation that encodes a premature stop codon at amino acid 779 (N-terminal to the transmembrane domain) that leads to nonsense-mediated decay of the Vldlr mRNA (47).  It is proposed that the mutations affect VLDLR’s activity in the Reelin-associated signaling pathway which guides neuroblast migration in the cerebral cortex and cerebellum (cerebellogenesis) (47;49).

Putative Mechanism

The premature stop codon generated by the C2239T mutation generates a 746 amino acid truncated protein with a premature termination at amino acid residue 747 (R747X).  The premature stop codon results in the loss of 127 C-terminal amino acids including the transmembrane domain.  The generation of a premature stop codon leads to loss of a detectable protein in the retina (1).  Further characterization using an in vitro model found that the recombinant r26 mutant protein was expressed, but that it was mislocalized from the plasma membrane to the cytosol (1).  Reduced expression of the truncated VLDLR in the cytosol suggests that the r26 mutation is leading to nonsense-mediated decay of the Vldlr, similar to the mutations mentioned above.

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References
Science Writers Anne Murray
Illustrators Victoria Webster
AuthorsChun-hong Xia, Eric Lu, Haiquan Liu, Xin Du, Bruce Beutler, Xiaohua Gong