The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

Endoglin is an accessory receptor for transforming growth factor ␤ (TGF␤) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng ؊/؊ ) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover Endoglin (CD105) is a homodimeric transmembrane glycoprotein expressed on all types of endothelial cells (1) and increased in cells in culture and during angiogenesis in vivo (2-7). Endoglin expression is also enhanced in vascular smooth muscle cells during injury and inflammation (8 -10). Endoglin is critically important in the cardiovascular system as revealed by a lethal phenotype in endoglin null (Eng Ϫ/Ϫ ) murine embryos at gestational day E10.5 because of defects in vessel and heart development (11-13). Vasculogenesis in the Eng Ϫ/Ϫ mice is normal, but angiogenesis is impaired along with remodeling of the primary vascular plexus. Mice exhibit poor vascular smooth muscle development that results in dilatation and rupture of the vascular channels. Heart development is arrested in Eng Ϫ/Ϫ mice at E9.0. The atrioventricular canal endocardium fails to undergo mesenchymal transformation and to generate the cushion tissue essential for valve formation and heart septation (11). Transient expression of endoglin is also striking during human development, as it is up-regulated during heart valve formation but subsequently reduced as the valves mature (14). In the adult vasculature, endoglin haploinsufficiency causes the vascular dysplasia hereditary hemorrhagic telangiectasia type 1 (HHT1) 1 associated with dilated vessels and arteriovenous malformations (15,16).Endoglin associates with transforming growth factor ␤ (TGF␤) receptors (17). TGF␤ is a multifunctional cytokine that controls proliferation, migration, adhesion, and apoptosis of diverse cell types (18,19). TGF␤ signals through a heteromeric complex of type I and type II transmembrane serine/threonine kinase receptors (20). Receptor activation occurs upon binding of ligand to the type II receptor (T␤RII), which recruits and phosphorylates type I receptors and then propagates the signal to downstream target receptor-regulated Smads (21,22). The specificity of cellular responses to TGF␤ is mediated by the type I receptors. Most cells utilize the type I receptor ALK5, which phosphorylates Smad2 and Smad3. However, endothelial cells have an additional type I receptor, ALK1, which phosphorylates Smad1 and Smad 5 (23,24). Of note, TGF␤-dependent activation of ALK1 requires ALK5, such that both are present with T␤RII in a composite receptor complex that acts via Smad1 and Smad5. Once phosphorylated, one of these receptor-regulated Smads combines with the common Smad4,

ENDOGLIN codes for a homodimeric membrane glycoprotein that interacts with receptors for members of the TGF-beta superfamily and is the gene mutated in the autosomal dominant vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We recently demonstrated that functional endoglin was expressed at half levels on human umbilical vein endothelial cells (HUVECs) and peripheral blood activated monocytes from HHT1 patients. Two types of mutant protein were previously analyzed, the product of an exon 3 skip which was expressed as a transient intracellular species and prematurely truncated proteins that were undetectable in patient samples. Here we report the analysis of four proteins resulting from point mutations, with missense codons G52V and C53R in exon 2, W149C in exon 4 and L221P in exon 5. Metabolic labeling of activated monocytes from confirmed, clinically affected patients revealed reduced expression of fully processed normal endoglin in all cases. Pulse-chase analysis with HUVECs from a newborn with the C53R substitution indicated that mutant endoglin remained intracellular as a precursor form and did not impair processing of the normal protein. Biotinylation of cell surface proteins, metabolic labeling and pulse-chase analysis revealed that none of the engineered missense mutants was significantly expressed at the surface of COS-1 transfectants. Thus, these four HHT1 missense mutations lead to transient intracellular species which cannot interfere with normal endoglin function. These data suggest that haploinsufficiency, leading to reduced levels of one of the major surface glyco-proteins of vascular endothelium, is the predominant mechanism underlying the HHT1 phenotype.