Endoglin is an auxiliary component of the transforming growth factor-␤ (TGF-␤) receptor system, able to associate with the signaling receptor types I (T␤RI) and II (T␤RII) in the presence of ligand and to modulate the cellular responses to TGF-␤1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and T␤RI or T␤RII. This study shows that full-length endoglin interacts with both T␤RI and T␤RII, independently of their kinase activation state or the presence of exogenous TGF-␤1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/T␤RI interaction was different from that of endoglin/T␤RII. T␤RII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas T␤RI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both T␤RI and T␤RII interact with the cytoplasmic domain of endoglin, but T␤RI only interacts when the kinase domain is inactive, whereas T␤RII remains associated in its active and inactive forms. Upon association, T␤RI and T␤RII phosphorylate the endoglin cytoplasmic domain, and then T␤RI, but not T␤RII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of T␤RII, T␤RI, and downstream Smad proteins as well as a modulation of TGF-␤ signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-␤ responses.

Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-␤ (TGF-␤) signaling receptors to bind TGF-␤ family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-␤, TGF-␤ signaling receptors, and TGF-␤-stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglin promoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated the endoglin promoter and those of collagen ␣1(I), urokinase-type plasminogen activator, TGF-␤1, and TGF-␤ receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the Cterminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-␤ signaling complex in vascular repair. IntroductionCoordinated gene expression is a crucial requirement in the response to tissue injury. Extracellular matrix proteins, 1-3 growth factors such as transforming growth factor-␤ (TGF-␤), 1,4,5 and proteases such as urokinase-type plasminogen activator (uPA) 6,7 are jointly regulated. In particular, the TGF-␤ family plays a central role in the injury response based on the following: (1) TGF-␤1 expression is up-regulated after injury; 8,9 (2) infusion of TGF-␤ polypeptide or transfection of cDNA into injured arteries increases extracellular matrix production; 1,10 and (3) antibodies to TGF-␤ reduce intimal hyperplasia. 11 Members of the TGF-␤ superfamily exert their biologic functions through membrane receptors known as type 1 (T␤RI) and type 2 (T␤RII) serine/threonine kinases. After ligand binding, T␤RII recruits and phosphorylates T␤RI, which initiates the signaling pathway by phosphorylating the Smad family of proteins. 12,13 Endoglin is a homodimeric membrane glycoprotein that functions, in association with T␤RI and T␤RII, as an auxiliary receptor for TGF-␤1, TGF-␤3, activin, bone morphogenetic protein 2 (BMP-2), and BMP-7. [14][15][16] It is highly expressed by endothelial cells 17,18 and, at lower levels, by activated monocytes/ macrophages 19 and by mesenchymal cells, including...

Endoglin is a component of the transforming growth factor-␤ receptor complex abundantly expressed at the surface of endothelial cells and plays an important role in cardiovascular development and vascular remodeling. By using the cytoplasmic domain of endoglin as a bait for screening protein interactors, we have identified ZRP-1 (zyxin-related protein 1), a 476-amino acid member that belongs to a family of LIM containing proteins that includes zyxin and lipoma-preferred partner. The endoglin interacting region was mapped within the three double zinc finger LIM domains of the ZRP-1 C terminus. Analysis of the subcellular distribution of ZRP-1 demonstrated that in the absence of endoglin, ZRP-1 mainly localizes to focal adhesion sites, whereas in the presence of endoglin ZRP-1 is found along actin stress fibers. Because the LIM family of proteins has been shown to associate with the actin cytoskeleton, we investigated the possibility of a regulatory role for endoglin with regard to this structure. Expression of endoglin resulted in a dramatic reorganization of the actin cytoskeleton. In the absence of endoglin, F-actin was localized to dense aggregates of bundles, whereas in the presence of endoglin, expressed in endothelial cells, F-actin was in stress fibers and colocalized with ZRP-1. Furthermore, small interfering RNA-mediated suppression of endoglin or ZRP-1, or clustering of endoglin in endothelial cells, led to mislocalization of Factin fibers. These results suggest a regulatory role for endoglin, via its interaction with ZRP-1, in the actin cytoskeletal organization.

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The integrin ␣ 4 ␤ 7 is the cell adhesion receptor for the mucosal vascular addressin MAdCAM-1, and this interaction is dominant in lymphocyte homing to Peyer's patch high endothelial venules, and plays key roles in lymphocyte recruitment at sites of inflammation. To identify ␣ 4 subunit amino acids important for ␣ 4 ␤ 7 /MAd-CAM-1 interaction, we expressed mutant ␣ 4 and wild type ␤ 7 chains in K562 cells and analyzed the effect of the mutations on cell adhesion to a soluble MAdCAM-1 (sMAdCAM-1-Ig

The N-terminal region of the K K integrin subunits is predicted to fold into a L L-propeller domain. Using K562 K K4 transfectants we show that mutations at K K4 subunit residues Gly IQH and Gly IWH affect the conformation of this domain causing a reduction in the recognition of K K4 by anti-K K4 antibodies which map to the L L-propeller. The improper K K4 conformation also led to an altered association with the L L1 subunit, and to a lack of K K4L L1 adhesion to VCAM-1 and CS-1/fibronectin, as well as an abolishment of anti-K K4-and anti-L L1-dependent homotypic aggregation. The total conservation of Gly IQH and Gly IWH among integrin K K subunits suggests their importance in the correct folding of their respective L L-propeller domains, and thus, in the adhesive activity of the integrins.z 1998 Federation of European Biochemical Societies.