Analysis of ALK-1 and endoglin in newborns from families with hereditary hemorrhagic telangiectasia type 2

ALK-1 (activin receptor-like kinase-1), a type I receptor of the transforming growth factor (TGF)-beta superfamily, is the gene mutated in hereditary hemorrhagic telangiectasia type 2 (HHT2) while endoglin is mutated in HHT1. Using a novel polyclonal antibody to ALK-1, we measured ALK-1 expression on human umbilical vein endothelial cells (HUVEC) of newborns from HHT families whose affected members had normal endoglin levels. ALK-1 levels were specifically reduced in three HUVEC with ALK-1 missense mutant codons, and normal in two newborns not carrying the missense mutations present in the clinically affected relatives. Levels were also normal in a HUVEC with deletion of S232 in the ATP binding site of ALK-1. Thus HHT2 appears to be associated with a loss of function of the mutant allele due to a reduction in either protein level or activity. We also report three new ALK-1 missense mutations leading to G48E/A49P, C344Y and E407D substitutions. In COS-1 transfected cells, ALK-1 was found in the TGF-beta1 and -beta3 receptor complexes in association with endoglin and TbetaRII, but not in activin receptor complexes containing endoglin. In HUVEC, ALK-1 was not detectable in the TGF-beta1 or -beta3 receptor complexes. However, in the absence of ligand, ALK-1 and endoglin interactions were observed by immunoprecipitation/western blot in HUVEC from normal as well as HHT1 and HHT2 patients. Our data suggest a transient association between these two proteins of the TGF-beta superfamily, both required at a critical level to ensure vessel wall integrity.