Identification of the Cellular Mechanisms That Modulate Trafficking of Frizzled Family Receptor 4 (FZD4) Missense Mutants Associated With Familial Exudative Vitreoretinopathy

Citation: Milhem RM, Ben-Salem S, AlGazali L, Ali BR. Identification of the cellular mechanisms that modulate trafficking of frizzled family receptor 4 (FZD4) missense mutants associated with familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2014;55:3423-3431. DOI:10.1167/ iovs.14-13885 PURPOSE. Fifteen missense mutations in the frizzled family receptor 4 (FZD4) reported to cause familial exudative vitreoretinopathy (FEVR) were evaluated to establish the pathological cellular mechanism of disease and to explore novel therapeutic strategies. METHODS. The mutations were generated by site-directed mutagenesis and expressed in HeLa and COS-7 cell lines. Confocal fluorescence microscopy and N-glycosylation profiling were used to observe the subcellular localization of the mutant proteins relative to wild-type (WT). Polyubiquitination studies were used to establish the involvement of the proteasome. Culturing at reduced temperatures and incubation in the presence of chemical compounds were used to enhance mutant protein processing and exit out of the endoplasmic reticulum (ER). RESULTS. Confocal fluorescence microscopy of the mutants showed three distinct subcellular localizations, namely, a plasma membrane pattern, an ER pattern, and a mixed pattern to both compartments. Confocal fluorescence microscopy and N-glycosylation profiling established the predominant ER localization of P33S, G36N, H69Y, M105T, M105V, C181R, C204R, C204Y, and G488D mutants. Coexpression of these mutants with WT FZD4 showed the inability of the mutants to trap WT FZD4. Culturing the expressing cells at reduced temperatures or in the presence of chemical agents directed at ameliorating protein misfolding resulted in partial rescue of trafficking defects observed for M105T and C204Y mutants. CONCLUSIONS. Defective trafficking resulting in haploinsufficiency is a major cellular mechanism for several missense FEVR-causing FZD4 mutants. Our findings indicate that this trafficking defect might be correctable for some mutants, which may offer opportunities for the development of novel therapeutics approaches for this condition

Endoplasmic Reticulum Quality Control Is Involved in the Mechanism of Endoglin-Mediated Hereditary Haemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER) quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D) out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W) out of thirteen mutants in the Zona Pellucida (ZP) domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional residues are likely to be the cause of the mutant proteins' loss of function

The three-dimensional structure of endoglin monomer showing the locations of the twenty five missense mutants studied in this aricle.

<p>Endoglin consists of a small c-terminal intracellular domain and three extracellular domains that include the ZP-C (green), ZP-N (Orange) and orphan (yellow) domains. The endoglin model structure (Llorca et al. 2007) file was provided by Dr Carmelo Bernabeu and then was manipulated using RasMol 2.7 (<a href="http://www.RasMol.org" target="_blank">www.RasMol.org</a>). The ball and stick represntation of the ER-retained (back) and the predominantly plasma membrane (purple) mutants are indicated on the strucure. It is clear that the majority of the mutants affecting the orphan domain resulted in the retention of the protein in the ER whereas those affecting the ZP domains retained their plasma membrane localization.</p

Comparison of the subcellular localization of wild type Endoglin with two (L32R and C53R) orphan domain HHT1-causing mutants.

<p>HeLa cells were transiently transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids (panels A-C, G-I and M-O) or co-transfected with the same plasmids and the EGFP-tagged H-Ras plasmid (Panels D–F, J–L and P–R) and processed for fluorescence confocal microscopy as described in the methods. The HA tagged proteins were detected with Anti-HA monoclonal antibodies (red panels A, D, G, J. M and P) and the ER marker calnexin was detected with anti-calnexin polyclonal antibodies (panels B, H and N). GFP-H-Ras staining is shown in panels E, K and Q. The wild type predominantly showed plasma membrane localization as evidenced by its co-localization with Ras (D–F). On the other hand the two mutants (L32R and C53R) showed ER localization (G–R).</p

The majority of the missense mutants in the ZP and intraceullar domains traffic normally to the plasma membrane.

<p>HeLa cells were transiently co-transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids and the EGFP-tagged H-Ras plasmid and 24 hours later the cells were processed for fluorescence confocal microscopy as described in the methods. The following missense mutanst were shown to traffic predominantly to the plama membrane as eviednced by their co-localization with GFP-H-Ras: C382G ( data not shoiwn due to space limitations), F403S (panles A–C), S407N (not shown), C412S (not shown), G413V (not shown), N423S (panels D–F), R437W (not shown), A452G (not shown), Q476H (not shown), V504M (panels J–L), R571H (panels M–O ) and P615L (panels P–R).</p

Endoglycosidase H (EndoH) analysis of the expressed missense mutants.

<p>The pCMV5 HA-tagged endoglin plasmids were transfected into HEK293 cells and were allowed to express the proteins as described in the methods. This was followed by lysis of the cells and the immuoprecipitation of the proteins with anti-HA monoclonal antibodies. Each immunoprecipitate was divided into two sample with one of them was treated (+) with EndoH or left untreated (−). Both samples were then electrophorese and western blotted using anti-HA monoclonal antibodies as described in the methods section. The majority of the WT protein did not change mobility upon EndoH treatment indicating its maturation and acquisition of complex N-glycan moieties in the post ER compartments. The missense mutants L32R, V49F, C53R, V125D, A160D, P165L, I271N, A308D, C363Y and C382W showed a single band indicating an exclusively ER premature forms. The other mutants either showed two bands or a simple high molecular weight band indicating that at least a significant proportion of the expressed protein matured in post ER compartments. The EndoH treatment data are in agreement with the confocal microscopy data.</p