BMPs are a family of related molecules within the TGF-β superfamily. They regulate a broad spectrum of processes ranging from cell proliferation, lineage determination, differentiation, to cell death (Hogan, 1996
). As in the related TGF-β receptor system, BMP signaling requires both type I and type II BMP receptors (Kawabata et al., 1995
; Liu et al., 1995
; Nohno et al., 1995
; Rosenzweig et al., 1995
; Hoodless et al., 1996
), suggesting the relevance of heteromeric and homomeric interactions among the BMP receptors for their functional responses. TGF-β signaling depends on homocomplex and heterocomplex formation between the TGF-β receptors, and these interactions have been studied extensively (Wrana et al., 1992
; Moustakas et al., 1993
; Chen and Derynck, 1994
; Henis et al., 1994
; Gilboa et al., 1998
; Huse et al., 1999
; Wells et al., 1999
). However, the oligomeric state of the BMP receptors was not thoroughly studied and was inferred to be similar to that of the TGF-β receptors. This may not be the case, as indicated by several observations. First, there are two BMP type I receptors versus one type I receptor for TGF-β, enabling a larger repertoire of interactions among BMP receptors. Second, the two systems differ in the basic characteristics of ligand binding: TβR-II binds ligand on its own, whereas BR-II does not, and the type I BMP receptors (but not TβR-I) bind ligand in the absence of the type II receptor (Wrana et al., 1994
; Liu et al., 1995
; Rosenzweig et al., 1995
; Massague, 1998
). These differences emphasize the need for direct studies on the oligomeric state of the BMP receptors. In the current work, we used several independent methods to investigate this issue. Our findings demonstrate that the oligomerization pattern of the BMP receptors differs from that of the TGF-β receptors, especially in homomeric complex formation, and is more flexible and susceptible to modulation by ligand.
The current studies demonstrate for the first time the formation of homomeric BMP receptor oligomers. These complexes were detected by coimmunoprecipitation studies and, on the cell surface, by both ligand–cross-linking and immunofluorescence-copatching experiments (Figures , , and , respectively). The latter studies enable us to evaluate the extent of receptor oligomerization. The percentage of copatching of two differently tagged forms of each receptor (BR-II, BR-Ia, and BR-Ib) was ~20–25% in the absence of ligand, increasing to 45–50% for BR-Ia and BR-Ib (but not for BR-II) upon binding of BMP-2 (Figures and ). The simplest interpretation is that only a minor fraction of each BMP receptor type resides in homo-oligomers before ligand binding and that the ligand shifts the equilibrium strongly toward the homodimeric form. As discussed in RESULTS, the percentage of copatching underestimates the percentage of a given receptor that is in homodimers by one-third. Thus, assuming that the homomeric complexes detected are dimeric, these results suggest that 30% of each BMP receptor is in homodimers, increasing to ~75% in the presence of ligand for the two type I receptors. This is in contrast to the type I and type II TGF-β receptors, which are essentially all in homodimers before ligand binding and whose dimerization is therefore ligand independent (Henis et al., 1994
; Gilboa et al., 1998
). The validity of these observations is reinforced by the lack of effect of BMP-2 on BR-II homo-oligomerization (Figures and ), in accord with the inefficient binding of the ligand to this receptor when singly expressed (Figure B).
The homo-oligomerization of the BMP receptors and its dependence on ligand in the case of the type I receptors may have functional relevance, because homo-oligomerization of both type I and type II TGF-β receptors was found to play important roles in TGF-β signal transduction. Thus, homodimerization of TβR-II was shown to be involved via intermolecular autophosphorylation in both positive and negative regulation of TGF-β signaling (Luo and Lodish, 1997
), and homodimerization of TβR-I appears to be important for functional interactions between TβR-I subunits in the ligand-induced heterocomplex (Luo and Lodish, 1996
; Weis-Garcia and Massague, 1996
). The fact that BMP-2 can dramatically increase the homo-oligomerization of BR-Ia and BR-Ib raises the possibility that the homomeric interactions within these complexes may be distinct and serve to regulate functional responses. Although we did not detect signaling of type I BMP receptors when they were transfected into C2C12 cells without BR-II (Figure ), it should be noted that the luciferase reporter construct used to measure transcriptional activation reflects activity via the Smad pathway, and it is still possible that type I BMP receptor complexes signal via another pathway. Furthermore, even in the absence of such signaling, formation of homomeric or of BR-Ia/BR-Ib complexes may modulate the pool of type I BMP receptors available for heterocomplex formation with BR-II and regulate signaling in this manner. The ability of the ligand to modulate the homomeric interactions among the BMP type I receptors (which may also vary between different ligands) allows an additional level of regulation, which is absent in the closely related TGF-β receptor system. This additional variability, manifested by multiple ligands, two type I receptors, and ligand-induced homo-oligomerization, might underlie at least part of the multiple biological activities of the BMP receptors.
Hetero-oligomeric complexes between BR-II and BR-Ia or BR-Ib were clearly detected by the various methods (coimmunoprecipitation, ligand cross-linking, and copatching) used in the current studies (Figures – and ). Quantitation of the copatching studies performed on live cells (Figure ) indicated that 30 and 40% of BR-Ia and BR-Ib, respectively, resided in complexes with BR-II in the absence of ligand, increasing to 50–60% upon ligand binding. These values of ligand-independent complexes are significantly higher than those observed for type I/type II heterocomplex formation among TGF-β receptors (Wells et al., 1999
). These findings demonstrate that type I/type II BMP receptors have an intrinsic affinity for each other that is markedly elevated after the binding of BMP-2. It should be noted that although the outcome of ligand binding increases heterocomplex formation both in the BMP and in the TGF-β receptor systems, the ligand-binding patterns are opposite: BMP-2 binds to BR-II very weakly unless it is coexpressed with a type I BMP receptor, whereas TGF-β1 requires TβR-II to bind to TβR-I. Thus, it is plausible that BMP-2 binds first to its type I receptors, recruiting BR-II into the signaling complex. Alternatively, a higher affinity of BMP-2 to preformed BMP receptor heterocomplexes, as proposed for TGF-β2 binding to TβR-II/TβR-I (Rodriguez et al., 1995
), could shift the equilibrium toward them and facilitate their formation.
It is important to note that BR-II/BR-Ia and BR-II/BR-Ib heterocomplexes were detected not only in transiently expressing COS7 cells but also in the naturally expressing cell line C3H10T1/2, which is responsive to BMP (Ahrens et al., 1993
; Wang et al., 1993
; Asahina et al., 1996
). The heterocomplexes were detected both in the absence of ligand by coimmunoprecipitation (Figure A) and at the cell surface after ligand cross-linking (Figure B). These findings demonstrate that the oligomerization measured in COS cells is not attributable to overexpression of the transfected receptors, which is required for fluorescence imaging in the copatching experiments. This idea is further supported by the similar copatching results obtained on cells expressing as low as 15,000–20,000 receptors at the surface (evaluated by quantitative measurement of the cell-surface fluorescence intensity, using the protocol described by us previously [Henis et al., 1994
], which although higher is still of the same order of magnitude as the level on naturally expressing cells).
The significant subpopulation (~30%) of type I and type II BMP receptors that reside in heterocomplexes before ligand binding raises questions as to how spurious, ligand-independent signaling by such complexes is attenuated. A simple possibility is that heterocomplex formation per se is not sufficient for activation and that a ligand-mediated conformational change altering the relative orientation of the subunits within the complex is needed for activation. Such a mechanism was proposed for the activation of preformed high-affinity EGF receptor dimers by EGF (Gadella and Jovin, 1995
) and more recently for the erythropoietin receptor, whose extracellular domain was shown to be dimeric in its unliganded form (Livnah et al., 1999
) and to undergo a ligand-induced conformational change for its activation (Remy et al., 1999
). Retention of a preformed heteromeric complex in an inactive conformation may also be aided by the binding of inhibitory proteins that are released after ligand binding, as proposed for the binding of the immunophilin FKBP12 to the type I TGF-β receptor (Chen et al., 1997
; Huse et al., 1999
). Because FKBP12 was also shown to interact with BR-Ia (Wang et al., 1996
), it may play a similar role in the BMP receptor system. Other proteins that interact with type I BMP receptors, such as BRAM1 (Kurozumi et al., 1998
) or XIAP (Yamaguchi et al., 1999
), are also potential candidates that may be involved in suppression of ligand-independent signaling. However, the significant enhancement in the basal transcriptional activation of the reporter gene construct in cells cotransfected with BR-II together with BR-Ia or BR-Ib in the absence of ligand (Figure ) clearly suggests that preformed BMP receptor heterocomplexes are endowed with some signaling capability.
It is notable that BMP-2 augmented heterocomplex formation not only between type II and type I BMP receptors but also between BR-Ia and BR-Ib (from 25 to 50%; Figures and ). This raises the intriguing possibility that BR-Ia/BR-Ib heterocomplexes may be functionally distinct from the homomeric BR-Ia and BR-Ib complexes, either by themselves or (more likely) when they further complex with BR-II. This is in-line with the distinct expression patterns of BR-Ib and BR-Ia during differentiation and maturation of skeletal tissues (Dewulf et al., 1995
; Rosen et al., 1996
; Zou and Niswander, 1996
; Zou et al., 1997
) and with recent reports on synergistic signaling by two BMP type I receptors in Drosophila
dorsal–ventral patterning (Neul and Ferguson, 1998
; Nguyen et al., 1998
In conclusion, we have shown that oligomerization of the BMP receptors at the cell surface follows a different mode than that of the TGF-β receptors. The multiplicity of ligand-independent heterocomplexes and the induction of homo-oligomers of the type I receptors by BMP-2 are two major differences between the two related systems. Both systems use multiple ligands and downstream-signaling molecules to exert their various effects and display a measurable level of preformed complexes that is significantly enhanced by ligand binding. However, the existence of two type I BMP receptors that can interact with the type II receptor and among themselves along with the ability of the ligand to augment their homodimerization grants the BMP system a degree of flexibility that does not exist for the TGF-β receptors. Further studies are needed to elucidate the role of the various complexes in conveying the multiple effects of the BMP ligands.