Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.
Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFβ receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.
Institut für Pharmazie und Molekulare Biotechnologie, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
Hamed
H
Alborzinia
Hannah
H
Schmidt-Glenewinkel
Iryna
I
Ilkavets
Katja
K
Breitkopf-Heinlein
Xinlai
X
Cheng
Peter
P
Hortschansky
Steven
S
Dooley
Stefan
S
Wölfl
D001665:Binding Sites; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D006023:Glycoproteins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007700:Kinetics; D018613:Microscopy, Confocal; D000099176:Noggin Protein
D055396:Bone Morphogenetic Protein 2; D002352:Carrier Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D000099176:Noggin Protein; C525718:BMP2 protein, human; C486399:GREM1 protein, human; C054234:chordin