Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2.
Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2'). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structure-function relationships of Ang1 with Tie2.
Kim
Kyung-Tae
KT
Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon.
Choi
Han-Ho
HH
Steinmetz
Michel O
MO
Maco
Bohumil
B
Kammerer
Richard A
RA
Ahn
So Young
SY
Kim
Hak-Zoo
HZ
Lee
Gyun Min
GM
Koh
Gou Young
GY
D041721:3T3-L1 Cells; D042683:Angiopoietin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042702:Angiopoietin-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D010766:Phosphorylation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D042787:Receptor, TIE-2 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D011994:Recombinant Proteins; D042787:Receptor, TIE-2; D003545:Cysteine