Abdalla S A, Pece-Barbara N, Vera S, Tapia E, Paez E, Bernabeu C, Letarte M
Cancer and Blood Research Programme, The Hospital for Sick Children, and Department of Immunology, University of Toronto, Toronto M5G 1X8, Canada.
Hum Mol Genet. 2000 May 1;9(8):1227-37. doi: 10.1093/hmg/9.8.1227.
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.
ALK-1（激活素受体样激酶-1）是转化生长因子（TGF）-β超家族的I型受体，是2型遗传性出血性毛细血管扩张症（HHT2）中发生突变的基因，而HHT1中发生突变的是内皮糖蛋白。我们使用一种针对ALK-1的新型多克隆抗体，检测了来自HHT家族新生儿的人脐静脉内皮细胞（HUVEC）上的ALK-1表达，这些家族中受影响成员的内皮糖蛋白水平正常。在三个带有ALK-1错义突变密码子的HUVEC中，ALK-1水平特异性降低，而在两个未携带临床受影响亲属中存在的错义突变的新生儿中，ALK-1水平正常。在一个ALK-1的ATP结合位点缺失S232的HUVEC中，水平也正常。因此HHT2似乎与突变等位基因的功能丧失有关，这是由于蛋白质水平或活性的降低。我们还报告了三个新的ALK-1错义突变，导致G48E/A49P、C344Y和E407D替代。在COS-1转染细胞中，发现ALK-1存在于与内皮糖蛋白和TβRII相关的TGF-β1和-β3受体复合物中，但不存在于含有内皮糖蛋白的激活素受体复合物中。在HUVEC中，在TGF-β1或-β3受体复合物中未检测到ALK-1。然而，在没有配体的情况下，通过免疫沉淀/蛋白质印迹在正常以及HHT1和HHT2患者的HUVEC中观察到ALK-1和内皮糖蛋白的相互作用。我们的数据表明，TGF-β超家族的这两种蛋白质之间存在短暂关联，两者在关键水平上都是确保血管壁完整性所必需的。
