Retention in the endoplasmic reticulum is the underlying mechanism of some hereditary haemorrhagic telangiectasia type 2 ALK1 missense mutations

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterised by vascular dysplasia and increased bleeding that affect 1 in 5,000 people world-wide. Pathology is linked to mutations in genes encoding components of the heteromeric transforming growth factor-beta receptor (TGF-beta) and SMAD signalling pathway. Indeed HHT1 and HHT2 result from mutations in the genes encoding endoglin and activin-like kinase 1 (ALK1), TGF-beta receptor components. However, the fundamental cellular defects underlying HHT is poorly understood. Previously using confocal microscopy and N-glycosylation analysis, we found evidence that defective trafficking of endoglin from the endoplasmic reticulum (ER) to the plasma membrane is a mechanism underlying HHT1 in some patients. In this study, we used confocal microscopy to investigate whether a similar mechanism contributes to HHT2 pathology. To do this we expressed wild-type ALK1 and a number of HHT2 patient mutant variants as C-terminally tagged EGFP fusion proteins and tested their localisation in HeLa cells. We found that wild-type ALK1-EGFP was targeted predominantly to the plasma membrane, as evidenced by its colocalisation with the co-expressed HA-tagged endoglin. However, we found that in the majority of cases analysed the HHT2 patient mutant protein was retained within the ER as indicated by their colocalisation with the ER resident marker (calnexin) and lack of colocalisation with cell surface associated HA-endoglin. We conclude that defective trafficking and retention in the ER of mutant ALK1 protein is a possible mechanism of HHT2 in some patients.