Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes.
BACKGROUND
BMP-2 (bone morphogenetic protein-2) signals via two types of transmembrane serine/threonine kinase receptors (BRI and BRII), which form heteromeric complexes prior to and after ligand binding. Within a BMP-bound receptor complex, BRII transphosphorylates and activates BRI-a for further signaling. We investigated which signaling pathway is initiated by BMP-2 via preformed receptor complexes versus BMP-2-induced signaling receptor complexes.


METHODS
Immunofluorescence co-patching was used to study the oligomerization of receptors at the surface of live cells. Binding and chemical cross-linking of iodinated BMP-2 followed by immunoprecipitation was used to show association of receptors in the presence of ligand. Western blots with use of anti-phospho-Smad1 antibodies and reporter gene assays with use of SBE-lux were employed to show activation of the Smad pathway. Phosphorylation of p38-MAPK was shown by Western blots. Induction of alkaline phosphatase was determined by staining the cells. The cluster density of receptors was determined with use of image correlation spectroscopy.


CONCLUSIONS
We showed that the Smad pathway is induced by preformed receptor complexes, whereas BMP-2-induced signaling complexes result in the activation of p38-MAPK. We also found evidence that the clustering of BRI-a at the membrane is altered in the presence of BRII, suggesting that it associates with existing clusters of BRII to initiate efficient Smad signaling. These data clearly demonstrate that it is critical to fully understand receptor oligomerization in order to estimate signaling outcome for distinct receptor and ligand mutants.
Hassel
Sylke
S
Schmitt
Simone
S
Hartung
Anke
A
Roth
Martin
M
Nohe
Anja
A
Petersen
Nils
N
Ehrlich
Marcelo
M
Henis
Yoav I
YI
Sebald
Walter
W
Knaus
Petra
P
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D052004:Bone Morphogenetic Protein Receptors; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020239:Receptor Cross-Talk / Q000502:physiology; D017978:Receptors, Growth Factor / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins; D051898:Smad1 Protein; D013057:Spectrum Analysis; D015534:Trans-Activators / Q000502:physiology*; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D017978:Receptors, Growth Factor; C497399:SMAD1 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D052004:Bone Morphogenetic Protein Receptors