Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats.
Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.
Fiedler
Ulrike
U
Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, 79106 Freiburg, Germany.
Krissl
Tanja
T
Koidl
Stefanie
S
Weiss
Cornelia
C
Koblizek
Thomas
T
Deutsch
Urban
U
Martiny-Baron
Georg
G
Marmé
Dieter
D
Augustin
Hellmut G
HG
D043925:Angiogenesis Inducing Agents / Q000302:isolation & purification / Q000378:metabolism*; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D007136:Immunoglobulins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins; D042787:Receptor, TIE-2; D020133:Reverse Transcriptase Polymerase Chain Reaction
D043925:Angiogenesis Inducing Agents; D042683:Angiopoietin-1; D042702:Angiopoietin-2; C467628:Angpt1 protein, mouse; D017931:DNA Primers; D007136:Immunoglobulins; C105373:MEN1 protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D042787:Receptor, TIE-2