Cell, Vol. 87, 1161±1169, December 27, 1996, Copyright 1996 by Cell Press Isolation of Angiopoietin-1, a Ligand for the TIE2 Receptor, by Secretion-Trap Expression Cloning Samuel Davis, Thomas H. Aldrich, Pamela F. Jones, Flt-4, and Flk-1/KDR, all of which are members of the Ann Acheson, Debra L. Compton, Vivek Jain, vascular endothelial growth factor (VEGF) receptor fam- Terence E. Ryan, Joanne Bruno, ily. The requisite roles of Flt-1 and Flk-1 during vascular Czeslaw Radziejewski, Peter C. Maisonpierre, development, as well as that of VEGF, have been con- and George D. Yancopoulos firmed by analysis of genetically engineered mice lack- Regeneron Pharmaceuticals, Inc. ing these proteins(Fong et al., 1995; Shalaby et al., 1995; 777 Old Saw Mill River Road Carmeliet et al., 1996; Ferrara et al., 1996). The more Tarrytown, New York 10591 recently discovered TIE receptor family (Dumont et al., 1992; Partanen et al., 1992; Iwama et al., 1993; Maison- pierre et al., 1993; Sato et al., 1993; Schnurch and Risau, Summary 1993; Ziegler et al., 1993), consisting of TIE1 and TIE2 (also termed Tek), also have been found to be critically TIE2 is a receptor-like tyrosine kinase expressed al- involved in the formation of vasculature (Dumont et al., most exclusively in endothelial cells and early hemo- 1994; Puri et al., 1995; Sato et al., 1995). Mice deficient poietic cells and required for the normal development in TIE1 die between embryonic day 13.5 (E13.5) and of vascular structures during embryogenesis. We re- birth and display edema and hemmorhage resulting from port the identification of a secreted ligand for TIE2, poor structural integrity of the endothelial cells (Puri et termed Angiopoietin-1, using a novel expression clon- al., 1995; Sato et al., 1995). In contrast, mice deficient ing technique that involves intracellular trapping and in TIE2 have an earlier lethal phenotype and die by E10.5 detection of the ligand in COS cells. The structure of (Dumont et al., 1994; Sato et al., 1995). The most promi- Angiopoietin-1 differs from that of known angiogenic nent defects observed in these mice include the failure factors or other ligands for receptor tyrosine kinases. of the endothelial lining of the heart to develop properly, the failure of remodelling of the primary capillary plexus Although Angiopoietin-1 binds and induces the tyro- into large and small vessels, and the lack of capillary sine phosphorylation of TIE2, it does not directly pro- sprouts into the neuroectoderm. In addition to their ex- mote the growth of cultured endothelial cells. How- pression by endothelial cells, the TIEs are also specifi- ever, its expression in close proximity with developing cally expressed in early hemopoietic stem cells (Iwama blood vessels implicates Angiopoietin-1 in endothelial et al., 1993; Batard et al., 1996; Hashiyama et al., 1996), developmental processes. perhaps reflecting the origin of both lineages from a common hemangioblast precursor (Shalaby et al.,1995); Introduction however, the early death of mice lacking the TIEs has limited the use of these mice in elucidating the precise Embryonic vascular development involves a complex roles of the TIEs in hemopoiesis (Rodewald and Sato, series of events during which endothelial cells differenti- 1996). Because the TIE receptor family is critically in- ate, proliferate, migrate, and undergo morphologic or- volved in angiogenesis and may play a role in hemopoie- ganization in the context of their surrounding tissues sis as well, we initiated a search for ligands that may (Risau, 1991, 1995). Vascular development is generally activate these receptors. Here we describe the use of a classified into two successive phases. The first, known novel expression cloning strategy to identify a secreted as vasculogenesis, refers to the process whereby newly ligand for the TIE2 receptor, which we designate Angio- differentiated endothelial cells spontaneously coassem- poietin-1 to reflect not only its requisite role in angiogen- ble into tubules that fuse to form a rather homogeneous esis (Suri et al., 1996 [this issue of Cell]) but also its primary vasculature in the embryo. Subsequent remod- potential actions during hemopoiesis. eling of this primary vascular network into large and Searches for the ligands for orphan receptors have small vessels bringsinto play a different process, termed traditionally proceeded by several routes, depending on angiogenesis. Angiogenesis in the embryo also leads to the type of ligand that is sought. In the case of secreted the sprouting of vessels into initially avascular organs, ligands, two major approaches have been used. The such as the brain. In the adult, angiogenesis accounts first uses soluble forms of the receptors to effect affinity for neovascularization that accompanies the normal purification of the ligands, followed by protein sequenc- processes of ovulation, placental development, and ing and cloning of cDNAs containing the desired pep- wound healing, as well as various clinically significant tides (e.g., Stitt et al., 1995). Alternatively, expression pathologic processes such as tumor growth and dia- cloning strategies involve the construction and screen- betic retinopathy (Ferrara, 1995; Folkman, 1995; Hana- ing of ªpooled expression librariesº (e.g., Lok et al., han and Folkman, 1996). 1994). In these strategies, many small pools of cDNAs Intercellular signaling mechanisms that govern the are individually transfected into cells, and conditioned formation of blood vessels have only recently begun to media from the individual transfections are then sep- be studied at the molecular level. Two families of recep- arately assayed for their ability to produce activities that tor tyrosine kinases have been identified whose expres- stimulate receptor-bearing reporter cells. A sensitive sion is largely restricted to endothelial cells and which and simple assay must be available, since tens of thou- are essential for normal development of blood vessels sands of pools often must be screened, particularly if (Mustonen and Alitalo, 1995). One family includes Flt-1, the desired cDNA is present only at low abundance. Cell 1162 Less labor-intensive approaches, such as those based on the yeast two-hybrid system for cloning interacting partners (Chien et al., 1991), cannot be used since growth factor±receptor binding depends on a large in- teracting surface between two essentiallyfull-length and correctly folded proteins (de Vos et al., 1992), the folding of which depends on correct disulfide pairing, which usually occurs efficiently only during secretion from mammalian cells. The yeast two-hybrid system takes advantage of interactions between short stretches of peptides that can occur within the reducing environment found within the yeast cell cytoplasm. The expression cloning of cDNAs encoding mem- brane-bound (as opposed to secreted) ligands for or- phan receptors has been far easier. Transfection of en- tire cDNA libraries into large numbers of cells can be used and still allow very rapid screening, since an indi- vidual cell containing the desired cDNA is uniquely marked on its surface by expression of the desired li- gand. This rare cell can be individually detected within a background of millions of other cells and thus allows isolation of the ligand-encoding cDNA (most efficiently by Davis et al., 1994) using soluble and epitope-tagged Figure 1. Identification of TIE2 Binding andPhosphorylating Activity forms of the orphan receptor for the detection by a in Conditioned Media of C2C12ras Cells and SHEP1±1 Cells process that is formally identical to methods previously (A) BIAcore assay of TIE2 binding activity. Conditioned media (CM) used to clone receptors using epitope-tagged ligands from each cell line was passed over a BIAcore sensor chip that had TIE2-Fc covalently coupled to it. Specificity of binding was assessed (Davis et al., 1991). Here we introduce an extension of by the inclusion of excess competing soluble TIE2-Fc or TrkB-Fc this method, termed ªsecretion-trap expression clon- during the measurement. Binding to the sensor chip is given as ing,º that allows rapid expression cloning of secreted resonance units (RUs). ligands based only on receptor±ligand binding and that (B) Phosphorylation of TIE2 receptor is induced by C2C12ras condi- we have exploited in the isolation of Angiopoietin-1 as tioned medium. ABAE cells were either unchallenged (MOCK) or a ligand for the TIE2 receptor. This approach should be challenged with 10-fold±concentrated conditioned medium from C2C12ras cells that had previously been depleted with either beads useful not only for isolating ligands for other orphan alone (CM) or with beads coated with TIE2-Fc (CM 1 TIE2-Fc). receptors but also for identifying interacting partners for Lysates of cells were immunoprecipitated (I.P.) with anti-TIE2 anti- the plethora of uncharacterized secreted proteins being bodies and analyzed for levels of tyrosine-phosphorylated TIE2 re- discovered in the attempt to sequence the mammalian ceptor. genome. Results TIE2-Fc (Figure 1B). Despite the detectable binding in their conditioned media, no binding of TIE2-Fc to the Identification of a Source for a TIE2 Binding surface of either SHEP1±1 or C2C12ras cells could be and Phosphorylating Activity detected, indicating that both cell lines were producing To identify potential sources for TIE2 ligands, we con- a secreted as opposed to a membrane-bound ligand structed a probe molecule consisting of the ectodomain for TIE2. of TIE2 fused to the Fc