Formation of Stable Homomeric and Transient Heteromeric Bone Morphogenetic Protein (BMP) Receptor Complexes Regulates Smad Protein Signaling

Abstract

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. Bone morphogenetic proteins (BMPs) 2 are members of the transforming growth factor-␤ (TGF-␤) cytokine superfamily (1-3). They play critical roles in numerous biological processes, including development, differentiation, and tissue regeneration in embryonic and mature tissues (4 -8), and have been implicated in disorders such as primary pulmonary hypertension (9, 10), brachydactyly-type dysostosis (11-13), juvenile polyposis syndrome (14 -16), and cancer (17). BMPs signal via two receptor Ser/Thr kinases, type I (BMPRIa and -Ib) and type II (BMPRII; as well as the type II activin receptors ActRII and ActRIIb) (5, 18 -20). Unlike the related TGF-␤ receptors where type I (T␤RI) fails to bind ligand and type II (T␤RII) is the high affinity receptor, BMPRII binds BMP-2 only weakly, whereas BMPRI binds the ligand with high affinity (20 -24).Using immunofluorescence co-patching and co-immunoprecipitation studies, we have demonstrated that even prior to ligand binding the BMP receptors exist at the cell surface as a mixed population largely comprising monomers but also containing homodimers and heteromeric complexes (the latter are termed preformed complexes (PFCs)) (18,20,25,26). Following ligand binding to PFCs (25), BMPRII phosphorylates BMPRI, which proceeds to phosphorylate Smad1/5/8; they then bind to Smad4 and translocate to the nucleus where they regulate the expression of specific target genes (2, 27). BMPs can also signal via non-Smad pathways, which appear to be initiated mainly by ligand-induced BMP receptor heterocomplexes, reflecting a different oligomerization mode (5,19,25,26,28,29).The crystal structures of BMP-2 in complex with the ectodomain (ECD) of BMPRIa (23) and of BMP-7 with the ECD of the ActRII (30, 31) show a dimeric ligand in complex with two receptors (homodimers). Recent studies (24,32) reported the structures of the ternary heteromeric complexes of BMP-2 with the ECDs of BMPRIa and ActRII or ActRIIb. Interestingly, these heterocomplexes differ from the ternary complex of TGF-␤3 with the ECDs of T␤RI and T␤RII (33) because only the TGF-␤ receptor heterocomplex displays a direct contact between T␤RI and T␤RII that contributes to the interactions (20).Although structural studies have shown that th...

SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

“…4, a-c), which is in accordance with our recent patch/FRAP studies (25). The restricted mobility of BMPRI ensures stabilization of this complex before and, more pronounced, after BMP-2 stimulation.…”

“…4, a-c, and supplemental material). This demonstrates very dynamic and transient binding between the BMP receptors and agrees with our recent studies (25). The unliganded heteromeric receptor complex has a life span of about 2 s before it separates.…”

Relay of retrograde synaptogenic signals through axonal transport of BMP receptors

“…6D-F; supplementary material Movie 8). This suggests that the vesicles containing both receptors are a mix of two populations, one in which the receptors form a complex and a second population that contains independent receptors, perhaps forming transient complexes (Marom et al, 2011).…”

“…Ligand binding results in stabilization of a heterocomplex of type I and type II BMP receptors (Marom et al, 2011). If such a complex is formed and relays the signal from NMJ to cell body, it is likely that the receptors colocalize in transport vesicles along the axon.…”

“…If, as expected from the biochemistry of BMP receptor activation (Marom et al, 2011;Moustakas and Heldin, 2009), the Immunofluorescence study of pMad carried out with the PS1-P antiserum (Persson et al, 1998). Scale bars: 10 mm.…”